Switch in post-translational modifications upon Influenza virus infection

INTERNATIONAL DOCTORAL SCHOOL OF THE USC Rocio Seoane Abelenda PhD Thesis Switch in post-translational modifications upon Influenza virus infection Santiago de Compostela, 2022 Doctoral Programme in Molecular Medicine DOCTORAL THESIS SWITCH IN POSTTRANSLATIONAL MODIFICATIONS UPON INFLUENZA VIRUS INFECTION Rocío Seoane Abelenda INTERNATIONAL PHD SCHOOL OF THE UNIVERSITY OF SANTIAGO DE COMPOSTELA PHD PROGRAMME IN MOLECULAR MEDICINE SANTIAGO DE COMPOSTELA 2022 Dna. Rocío Seoane Abelenda Título da tese: Switch in post-translational modifications upon Influenza virus infection Presento a miña tese, seguindo o procedemento axeitado ao Regulamento, e declaro que: 1) A tese abarca os resultados da elaboración do meu traballo. 2) De ser o caso, na tese faise referencia ás colaboracións que tivo este traballo. 3) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. 4) A tese é a versión definitiva presentada para a súa defensa e coincide a versión impresa coa presentada en formato electrónico E comprométome a presentar o Compromiso Documental de Supervisión no caso de que o orixinal non estea na Escola. En Santiago de Compostela, 15 de Abril de 2022. Sinatura electrónica D./Dna. María del Carmen Rivas Vázquez En condición de: Directora Título da tese: Switch in post-translational modifications upon Influenza virus infection INFORMA: Que a presente tese, correspóndese co traballo realizado por D/Dna Rocío Seoane Abelenda, baixo a miña dirección/titorización, e a utorizo a súa presentación, considerando que reúne os r equisitos esixidos no R egulamento de Estudos de Doutoramento da USC, e que como director/titor desta non incorre nas causas de abstención establecidas na Lei 40/2015. En Santiago de Compostela, 15 de Abril de 2022 Sinatura electrónica INDEX 1. ABBREVIATIONS ......................................................................................... 23 2. SUMMARY .................................................................................................. 31 2.1 RESUMO ........................................................................................................... 33 2.2 SUMMARY ......................................................................................................... 42 3. INTRODUCTION .......................................................................................... 45 3.1 INFLUENZA VIRUS ................................................................................................ 47 3.1.1 Influenza virus, pathogenesis, and transmission.................................... 47 3.1.2 Influenza virus structure ......................................................................... 49 3.1.3 Influenza virus cell cycle ......................................................................... 50 3.1.4 Influenza A virus-host cell interaction .................................................... 55 3.2 POST-TRANSLATIONAL MODIFICATION SIGNALING PATHWAYS AND IAV INFECTION ........... 62 3.2.1 Phosphorylation ..................................................................................... 63 3.2.2 Glycosylation .......................................................................................... 66 3.2.3 Acetylation ............................................................................................. 66 3.2.4 Ubiquitination ........................................................................................ 68 3.2.5 NEDDylation ........................................................................................... 70 3.2.6 SUMOylation .......................................................................................... 72 3.2.7 UFMylation ............................................................................................. 73 3.2.8 Hypusination .......................................................................................... 74 4. OBJECTIVES ................................................................................................. 77 5. MATERIAL AND METHODS .......................................................................... 81 5.1 CELL CULTURE .................................................................................................... 83 5.2 TRANSFECTION ................................................................................................... 83 5.3 SIRNA TRANSFECTION ......................................................................................... 83 5.4 CELLULAR TREATMENTS ........................................................................................ 84 5.5 PLASMIDS .......................................................................................................... 84 5.6 ANTIBODIES ....................................................................................................... 85 5.7 BACTERIAL TRANSFORMATION ............................................................................... 85 5.8 SITE-DIRECTED MUTAGENESIS ................................................................................ 86 5.9 CLONING ........................................................................................................... 86 5.10 WESTERN BLOT ................................................................................................ 87 5.11 VIRAL INFECTION............................................................................................... 87 5.12 RESCUE OF RECOMBINANT INFLUENZA VIRUS .......................................................... 88 5.13 PLAQUE ASSAY ................................................................................................. 88 5.14 METABOLIC LABELING ........................................................................................ 88 5.15 IN VITRO TRANSLATION ....................................................................................... 89 5.16 IN VITRO SUMOYLATION AND DESUMOYLATION ASSAYS ....................................... 89 5.17 IN VITRO NEDDYLATION ASSAY ............................................................................ 89 5.18 PREDICTION OF SUMOYLATION SITES ................................................................... 89 5.19 HISTIDINE TAG PURIFICATION ............................................................................... 90 5.20 SUBES ............................................................................................................ 90 5.21 IMMUNOPRECIPITATION OF HA-TAGGED PROTEINS .................................................. 90 5.22 LUCIFERASE ASSAY ............................................................................................. 91 5.23 IMMUNOPRECIPITATION IN DENATURING CONDITIONS .............................................. 91 5.24 RNA EXTRACTION AND QUANTITATIVE RT-PCR ...................................................... 92 5.25 IMMUNOFLUORESCENCE ASSAY ............................................................................ 92 5.26 SUBCELLULAR FRACTIONATION ............................................................................. 93 5.27 DSRNA BINDING ASSAY ...................................................................................... 93 5.28 MASS SPECTROMETRY ANALYSIS .......................................................................... 93 5.29 YEAST STRAINS AND VIABILITY ASSAYS .................................................................... 94 5.30 POLYSOME PROFILE ANALYSIS .............................................................................. 94 5.31 IMAGE PROCESSING AND FIGURES ......................................................................... 94 5.32 STATISTICAL ANALYSIS ........................................................................................ 94 6. RESULTS ..................................................................................................... 95 6.1 INFLUENZA VIRUS AND EIF5A ................................................................................ 97 6.1.1 Influenza A virus (IAV) infection induces hypusination ........................... 97 6.1.2 Both infection with different RNA viruses and stimulation with dsRNA induce hypusination ........................................................................................ 98 6.1.3 Activation of eIF5A is modulated by NF-kB ............................................ 99 6.1.4 Inhibition of eIF5A hypusination impairs the replication of IAV ........... 101 6.1.5 Inhibition of eIF5A hypusination impairs the replication of VSV .......... 104 6.1.6 Inhibition of eIF5A hypusination impairs the replication of mayaro virus, una virus, chikungunya virus, punta toro virus and zika virus ....................... 105 6.1.7 Inhibition of eIF5A activates transcription of both type I Interferon (IFN) and type I IFN-stimulated genes .................................................................... 109 6.1.8 Identification of human p85β as a potential target of eIF5A ............... 110 6.1.9 eIF5A1 is modified by SUMO2 in vitro .................................................. 113 6.1.10 eIF5A1 is modified by SUMO2 in vivo ................................................. 115 6.1.11 eIF5A2 is modified by SUMO2 ............................................................ 116 6.1.12 eIF5A1 is modified by SUMO2 in a hypusine-independent manner.... 117 6.1.13 SUMO2 can be conjugated to different lysine residues in eIF5A1 ...... 119 6.1.14 Mutation of the SUMOylation sites in eIF5A1 reduces its stability .... 123 6.1.15 Mutation of the SUMOylation sites in eIF5A1 modulates its subcellular localization .................................................................................................... 125 6.1.16 Modulation of eIF5A1 SUMOylation by stress ................................... 126 6.1.17 Conjugation of SUMO to lysine residues K68, K85, and K126 on eIF5A is essential for yeast survival ............................................................................ 135 6.1.18 Mutation of the SUMOylation sites in eIF5A1 favors its interaction with STUB1 ............................................................................................................ 139 6.1.19 eIF5A is modified by NEDD8 ............................................................... 140 6.1.20 Evaluation of potential NEDDylation sites in eIF5A. ........................... 143 6.1.21 Inhibition of NEDDylation stabilizes eIF5A ......................................... 145 6.1.22 STUB1 promotes eIF5A1 NEDDylation ............................................... 146 6.2 INFLUENZA VIRUS AND NEDD8 ........................................................................... 147 6.2.1 Influenza virus infection induces global reprogramming of host NEDDylation .................................................................................................. 147 6.2.2 Differentially NEDDylated proteins after IAV infection ........................ 147 6.2.3 Validation of IAV-modulated NEDD8 targets ....................................... 149 6.2.4 Influenza virus NS1 protein is modified by NEDD8 ............................... 153 6.2.5 NS1 is modified by NEDD8 at lysines K108, K110, K126, and K131 ...... 155 6.2.6 NS1 NEDDylation facilitates the replication of IAV .............................. 159 6.2.7 NEDDylation of NS1 is necessary for both interaction of NS1 with dsRNA and inhibition of IFN signaling. ..................................................................... 160 6.3 INFLUENZA VIRUS AND UFM1 ............................................................................. 162 6.3.1 Influenza virus infection triggers UFMylation ...................................... 162 6.3.2 UFMylation affects IAV replication ...................................................... 162 6.3.3 UFM1 is regulated by SUMO ................................................................ 164 6.3.4 UFM1 is modified by NEDD8 ................................................................ 166 6.3.5 IAV modulates the post-translational modification of UFM1 by both NEDD8 and SUMO2 ....................................................................................... 166 7. DISCUSSION .............................................................................................. 169 8. CONCLUSIONS .......................................................................................... 181 9. TABLES ...................................................................................................... 185 10. BIBLIOGRAPHY .......................................................................................... 195 FIGURE INDEX FIGURE 1. ANTIGENIC SHIFT AND ANTIGENIC DRIFT IN IAV ..................................................... 48 FIGURE 2. LIFE CYCLE OF INFLUENZA A VIRUS ...................................................................... 51 FIGURE 3. FUNCTIONS OF NS1 IN THE LIFE CYCLE OF INFLUENZA VIRUS ..................................... 53 FIGURE 4. ACTIVATION OF THE RIG-I PATHWAY BY IAV ........................................................ 56 FIGURE 5. ROLE OF PTM MODIFICATIONS IN NS1 ACTIVITY ................................................... 64 FIGURE 6. NEDDYLATION PATHWAY ................................................................................. 72 FIGURE 7. SCHEMATIC REPRESENTATION OF EIF5A HYPUSINATION .......................................... 76 FIGURE 8.IAV INDUCES EIF5A ACTIVATION ........................................................................ 98 FIGURE 9. BOTH STOMATITIS VESICULAR VIRUS (VSV) INFECTION AND DSRNA STIMULI INDUCE EIF5A ACTIVATION ............................................................................................... 99 FIGURE 10. MODULATION OF THE HYPUSINATION PATHWAY BY NF-ΚB .................................. 100 FIGURE 11. EFFECT OF EIF5A INHIBITION ON INFLUENZA VIRUS REPLICATION. ......................... 103 FIGURE 12. INHIBITION OF EIF5A IMPAIRED VSV REPLICATION ............................................. 107 FIGURE 13. INHIBITION OF EIF5A HYPUSINATION REDUCES ALPHAVIRUS, FLAVIVIRUS AND PHLEBOVIRUS REPLICATION .................................................................................. 108 FIGURE 14. INHIBITION OF EIF5A INDUCES BOTH ER STRESS AND TRANSCRIPTIONAL TRANSACTIVATION OF IFN AND IFN-STIMULATED GENES............................................ 112 FIGURE 15. HUMAN P85Β IS A POTENTIAL TARGET OF EIF5A ............................................... 113 FIGURE 16. IN VITRO SUMOYLATION OF EIF5A1 .............................................................. 115 FIGURE 17. SUMO2 MODIFICATION OF EIF5A1 IN CELLS ................................................... 116 FIGURE 18. EIF5A2 IS MODIFIED BY SUMO2 ................................................................... 118 FIGURE 19. SUMOYLATION OF THE NON HYPUSINABLE MUTANT OF EIF5A1, EIF5A1-K50R .... 120 FIGURE 20. IDENTIFICATION OF SUMO CONJUGATION SITES IN EIF5A1 ................................. 122 FIGURE 21. SUMOYLATION OF EIF5A1 MODULATES ITS STABILITY ....................................... 124 FIGURE 22. SUMOYLATION OF EIF5A1 MODULATES ITS SUBCELLULAR LOCALIZATION .............. 126 FIGURE 23. PROTEASOME INHIBITION INDUCES EIF5A1 SUMOYLATION ................................ 128 FIGURE 24. SUMOYLATION OF EIF5A IN RESPONSE TO UV LIGHT IRRADIATION, ADRIAMYCIN, OR HYPOXIC CONDITIONS .......................................................................................... 130 FIGURE 25. MODULATION OF EIF5A1 SUMOYLATION BY VIRAL INFECTION. .......................... 131 FIGURE 26. INDUCTION OF EIF5A1 SUMOYLATION BY HEAT SHOCK STRESS ........................... 133 FIGURE 27. SUMO ACCEPTOR SITES IN EIF5A1 ARE REGULATED BY HEAT SHOCK STRESS ........... 134 FIGURE 28. SUMOYLATION OF EIF5A AT LYSINE RESIDUES K68, K85 AND K126 IS ESSENTIAL FOR COMPLEMENTING HYP2 FUNCTION IN YEAST ........................................................... 137 FIGURE 29. SUMOYLATION IS NECESSARY FOR EIF5A-MEDIATED TRANSLATION REPRESSION AFTER HEAT SHOCK STRESS ............................................................................................ 138 FIGURE 30. MUTATION OF THE SUMOYLATION SITES IN EIF5A1 POTENTIATES ITS INTERACTION WITH STUB1 .................................................................................................... 140 FIGURE 31. EIF5A IS MODIFIED BY NEDD8 ...................................................................... 142 FIGURE 32. INTERPLAY BETWEEN NEDD8 AND SUMO2 ON EIF5A1.................................... 144 FIGURE 33. NEDDYLATION MODULATES THE STABILITY OF EIF5A1 ....................................... 146 FIGURE 34. STUB1 PROMOTES EIF5A1 NEDDYLATION ..................................................... 146 FIGURE 35. IAV INDUCES GLOBAL REPROGRAMING OF HOST NEDDYLATION .......................... 148 FIGURE 36. DIFFERENTIALY NEDDYLATED PROTEINS AFTER IAV INFECTION ............................ 150 FIGURE 37. NEDDYLATION OF RPS27A, H2B AND VDAC1 AFTER INFLUENZA VIRUS INFECTION 153 FIGURE 38. NS1 IS MODIFIED BY NEDD8 ........................................................................ 156 FIGURE 39. NS1 IS MODIFIED BY NEDD8 AT LYSINES K108, K110, K126 AND K131 ............. 159 FIGURE 40. NEDDYLATION OF NS1 FACILITATES IAV REPLICATION ....................................... 159 FIGURE 41. THE NS1 NEDDYLATION MUTANT FAILS TO INHIBIT IFN SIGNALING ..................... 161 FIGURE 42. IAV INFECTION MODULATES UFMYLATION ...................................................... 163 FIGURE 43. EFFECT OF UFM1 DOWNMODULATION ON IAV REPLICATION .............................. 164 FIGURE 44. UFM1 IS MODIFIED BY SUMO2 ................................................................... 166 FIGURE 45. UFM1 IS MODIFIED BY NEDD8 .................................................................... 167 FIGURE 46. IAV MODULATES UFM1 SUMOYLATION AND NEDDYLATION ............................ 168 1. ABBREVIATIONS 2. SUMMARY Summary 33 2.1 RESUMO O virus influenza A (IAV) é o axente causal da gripe. Os síntomas da gripe, de manifestarse, adoitan ser de carácter leve incluíndo febre, tose, mocos e dor muscular. Sen embargo, en ciertos pacientes, especialmente en maiores, cativos e pacientes con patoloxías previas, pódese producir unha complicación da enfermidade, sobre todo se o virus consegue replicarse no tracto respiratorio inferior. Esta doenza afecta a millóns de persoas de todo o mundo causando máis de 600000 mortes anuais. A gripe é unha enfermidade estacionaria que aparece, nas zonas temperadas, principalmente no inverno. Este virus está asociado históricamente a varias pandemias, pero cabe remarcar a pandemia da gripe do 1918 coñecida como pandemia da “gripe española”. Calcúlase que causou preto de 50 millóns de mortes e ata o momento é a pandemia máis grave da que se ten constancia. O virus da gripe pertence á familia Orthomixoviridae e a súa información xenética está contida en 8 fragmentos de ARN. A presenza dun xenoma segmentado, xunto co feito de que a polimerasa encargada de crear as copias do ARN é unha polimerasa que introduce gran cantidade de erros, fan que o xenoma do virus cambie constantemente e mesmo se produza un intercambio de fragmentos xénicos entre IAV de distinta orixe que estean coinfectando unha célula. É por iso que non existe una vacina universal contra o virus e segue a causar un problema grave de saúde en todo o mundo. Xa que a vacina contra a gripe é anual e está especificamente dirixida contra ás cepas do virus que están en circulación nese momento, considerase unha prioridade a xeración dunha vacina universal e a obtención de tratamentos máis eficaces contra o virus. Para acadar estes dous obxetivos, é fundamental coñecer todos os detalles moleculares da relación entre o virus e a célula infectada. O virus da gripe, como todos os virus, depende en gran medida da maquinaria celular, que é capaz de inhibir, activar ou secuestrar no seu propio beneficio. Moitas das vías de sinalización celular son activadas en resposta á infección viral para impedir a replicación do virus nas ROCÍO SEOANE ABELENDA 34 células infectadas, avisar ás células veciñas da presenza dun patóxeno, ou promover a morte celular para así evitar que o virus complete o seu ciclo. Por outra banda, os virus teñen proteínas capaces de modular moitas destas vías de tal maneira que son capaces de activalas ou inactivalas en momentos precisos durante o proceso de infección co fin de completar con éxito a súa replicación. O coñecemento de todas as estratexias empregadas polo virus para favorecer a súa replicación é esencial para gañar a batalla contra o virus. Para ter éxito contra o virus a célula necesita poder responder rápidamente á infección. A célula conségueo grazas entre outros, á regulación das proteínas mediante modificacións post-traducionais (PTM). Estas modificacións afectan a proteínas xa existentes na célula e polo tanto permiten unha resposta rápida ó estrés causado pola infección viral. No caso do virus da gripe sabemos que en resposta á infección celular co virus hai un aumento global de distintos tipos de modificacions post-traducionais coma as causadas polas proteínas da familia das ubiquitinas. Estas modificacions poden afectar a proteínas celulares e virais. Ademais, varias modificacións poden afectar a unha mesma proteína nun determinado momento ou poden competir entre elas, e a maioría destas modificacións son reversibles, o que pode levar a incrementar de maneira importante a versatilidade das proteínas. Isto é especialmente relevante no caso das proteínas víricas, xa que permite a estes patóxenos con poucas proteínas aumentar a súa versatilidade para contrarrestar a resposta antiviral da célula. Por estes motivos, neste traballo puxémonos como obxetivo estudar a función que as modificacións post-traducionais teñen na relación entre o virus da gripe e a célula.. En primeiro lugar estudamos a posible relación entre o virus da gripe e a hipusinación. A hipusinación é unha modificación posttraducional que afecta soamente a unha proteína, o factor de tradución eIF5A. De feito considérase que é necesario que eIF5A estea hipusinado para que a proteína estea activa. O proceso de hipusinación é un proceso enzimático no que unha porción da espermidina, que é Summary 35 unha poliamina, se conxuga á lisina 50 de eIF5A mediante a acción do enzima DHS. A continuación, tras un paso de hidroxilación mediado polo enzima DOHH, finalízase a formación da hipusina necesaria para a actividade de eIF5A. eIF5A é un factor de elongamento e iniciación evolutivamente conservado. Unha das funcións máis relevantes deste factor é a liberación da pausa ribosomal que ocorre no proceso de tradución daquelas proteínas que posúen secuencias difíciles de traducir, como por exemplo rexións que posúan varias prolinas consecutivas. Altos niveis deste factor foron atopados en moitos tipos de tumores e adoitan correlacionar cun peor prognóstico da enfermidade. Ademais, fallos na regulación de eIF5A ou da súa modificación por hipusinación foron atopados en enfermidades neuronais e tamén en enfermidades cardiovasculares. Neste traballo demostramos que a infección polo virus da gripe, así como a infección polo virus da estomatite vesicular ou simplemente o tratamento das células cun axente sintético que simula o ARN de dobre cadea que se produce coma consecuencia da infección viral, son estímulos capaces de activar eIF5A. Ademáis propoñemos que esta activación está mediada pola vía do NF-κB. A activación desta vía induciría un aumento dos niveis de ODC, un enzima implicado no metabolismo das poliaminas, que favorece a formación da hipusina. Actualmente existe un fármaco capaz de inhibir a activación de eIF5A (capaz de inhibir polo tanto a hipusinación da proteína). Anteriormente demostrouse que este fármaco, GC7, é capaz de inhibir a replicación de diversos virus como o ébola ou o VIH. Neste traballo describimos que tanto o tratamento das células con este fármaco, coma a reducción nos niveis de eIF5A, teñen un efecto negativo sobre a replicación de virus de ARN tan diversos como o virus da gripe, o virus da estomatite vesicular, ou os virus emerxentes zika, una, chikungunya, punta toro ou mayaro. Ademais postulamos que o mecanismo polo que o fai é a través da ruta NF-κB, xa que a inhibición de eIF5A promove un estado antiviral na célula que disminúe na presenza de inhibidores da vía NF-κB. Ademais propoñemos á proteína p85β humana, unha proteína chave na ruta PI3K/AKT e que ten un papel moi importante na replicación do virus, ROCÍO SEOANE ABELENDA 36 coma unha diana de eIF5A posto que o GC7 modula os niveis da mesma. Polo tanto, eIF5A xoga un papel importante na replicación dunha gran variedade de virus de ARN. Sen embargo, o mecanismo polo cal se regula esta proteína é algo que non se coñece con profundidade máis alá da súa hipusinación. Aquí demostramos que a proteína da familia da ubiquitina SUMO é capaz de modificar eIF5A tanto in vitro como en células humanas. Tamén demostramos que esta modificación non depende da hipusinación da proteína e identificamos 5 lisinas implicadas na modificacion por SUMO. A conxugación con SUMO regula a localización subcelular de eIF5A. Así, cando estas lisinas están mutadas, eIF5A localízase maiormente no núcleo celular, a diferencia da localización no núcleo e no citoplasma celular da proteína eIF5A silvestre. Ademáis, a proteína mutada nos sitios de SUMOilación é moito menos estable ca proteína silvestre e interacciona máis forte coa ligasa da ubiquitina STUB1. Por outra banda, estudamos como afectan diversos tipos de estrés á SUMOilación de eIF5A e vemos que esta modificación parece xogar un papel moi relevante en resposta a estrés. Así, mentras que o tratamento das células con radiación ultravioleta ou con hipoxia diminúe a SUMOilación da proteína, a infección viral, o estrés proteotóxico e o estrés térmico incrementan considerablemente esta modificación. Dada a pouca estabilidade do mutante de eIF5A que non se pode SUMOilar, estudamos a funcionalidade desta noutro modelo máis sinxelo, en lévedos. Demostramos que en lévedos o mutante en SUMOilación é estable pero non consegue rescatar o crecemento de un lévedo no que impedimos a expresión da proteína eIF5A endóxena, mentres que a introdución da proteina silvestre sí que é capaz de rescatala. Ademáis podemos observar que en lévedos sometidos a estrés térmico, a parada da tradución mediada por eIF5A está comprometida cando expresan o mutante en SUMOilación. Estos resultados reforzan a teoría de que a SUMOilación de eIF5A xoga un papel moi relevante en condicións de estrés. Summary 37 Estudos previos de proteómica identificaron a eIF5A como candidata a ser SUMOilada pero tamén a identificaron como posible substrato de NEDD8, outro modificador de proteínas da familia da ubiquitina. Neste traballo demostramos que eIF5A se NEDDila e, a pesares de non identificar as lisinas que funcionan como aceptoras de NEDD8, demostramos que existe unha interrelación entre a SUMoilación e a NEDDilación para controlar eIF5A. Desta forma, demostramos que os mutantes en SUMOilación se NEDDilan máis, que o tratamento das células con inhibidor de SUMO aumenta a NEDDilación da proteína e que o tratamento cun inhibidor de NEDDilación aumenta a súa SUMOilación. Ademais, demostramos que a NEDDilación de eIF5A regula a súa estabilidade favorecendo a súa degradación, coma indican os resultados acadados co inhibidor de NEDDilación MLN4924. Finalmente, propoñemos como posible ligasa de NEDD8 a STUB1. A identificación de ligasas é un punto moi relevante unha vez identificado un substrato de NEDD8 ou de calquera outro membro da familia das ubiquitinas. O proceso de NEDDilación, ao igual que o de ubiquitinación ou SUMOilación entre outros, é un proceso ATP-dependente que require da actuación de tres enzimas, o E1, E2 e E3. Xeralmente os enzimas E1 e E2 son específicos para cada unha das modificacións e son comúns para a maioría de sustratos, polo tanto hai un número reducido delas, mentres que no caso das E3, as ligasas, existe multitude delas descritas e son as que marcan en certa medida a especificidade da reacción. Unha vez demostrada e caracterizada a relevancia da hipusinación na infección co virus da gripe, continuamos co estudio de outras modificacións post-traducionais no contexto da infección co virus da gripe. Anteriormente demostrouse que o tratamento das células co inhibidor de NEDDilación inhibe a replicación do virus da gripe e que a infección co virus da gripe aumenta os niveis de NEDDilación das cullin, unha familia de ligasas de ubiquitina que está considerada como o principal substrato de NEDD8. Nestre traballo ampliamos esa información mediante o estudo das proteínas NEDDiladas en células ROCÍO SEOANE ABELENDA 38 infectadas co virus da gripe e comparándolas coas proteínas NEDDiladas nas células non infectadas. Identificamos certos substratos, algúns que xa aforan identificados como substratos de NEDDilación que son regulados pola infección. Tamén identificamos novos substratos de NEDD8 como pode ser a proteína VDAC1. Esta proteína, involucrada no funcionamento da mitocondria, modifícase por NEDD8 na súa lisina K110, e esta modificación parece ser relevante para a infección co virus da gripe posto que sobreexpresar a proteína mutada afecta negativamente á replicación do virus mentres que a sobreexpresión da proteína silvestre non ten ningún efecto. Ademais, ata o momento poucas proteínas virais foron propostas como substratos de NEDD8. Dúas delas son as proteínas M1 e PB2 do virus da gripe. Segundo a literatura, a NEDDilación destas proteínas induce a súa degradación, e polo tanto unha inhibición da NEDDilación afectaría positivamente á replicación do virus. Sen embargo, o efecto do inhibidor da NEDDilación demostrou ter efectos negativos para o virus, e por iso hipotetizamos que podería haber outras proteínas virais modificadas por NEDD8. NS1 é a proteína do virus da gripe máis relevante na inhibición do interferón e dos mecanismos antivirais desatados na célula infectada. Neste traballo demostramos que NS1 se modifica por NEDD8 in vitro e in vivo. Ademais, analizamos a NEDDilación durante a infección, nun contexto non que non soamente sobreexpresamos a proteína NS1 senón que todas as proteínas virais están presentes, e observamos que efectivamente a NEDDilación de NS1 ocorre de maneira fisiolóxica en células infectadas. Para analizar o rol da NEDDilación sobre NS1 tentamos xerar mutantes en diferentes lisinas pero sen conseguir unha baixada clara na súa NEDDilación. Por ese motivo, mutamos todas as lisinas presentes en NS1 e reintroducímolas de novo unha a unha para ver cal delas permitía recuperar a NEDDilación. Mediante este método identificamos 4 lisinas de NS1 como as aceptoras de NEDD8. A continuación demostramos que unha proteína coas 4 lisinas mutadas non se pode NEDDilar. Este resultado foi ademais comprobado no Summary 39 contexto da infección viral. Para iso xeramos un virus recombinante no que a proteína NS1 ten as 4 lisinas importantes para a NEDDilación mutadas e analizamos a súa NEDDilación. Desta maneira demostramos que as lisinas 108, 110, 126 e 131 de NS1 son as aceptoras de NEDD8. Por outra banda, estudamos o efecto da falta de NEDDilación de NS1 sobre a replicación viral. Para iso comparamos o crecemento do virus silvestre co do virus recombinante que expresa una proteína NS1 que non se pode NEDDilar, e observamos que a inhibición da NEDDilación de NS1 afecta negativamente á replicación do virus. Este dato está en liña co xa descrito papel negativo do inhibidor de NEDDilación sobre a replicación viral. Finalmente, para coñecer máis especificamente o papel da NEDDilación sobre NS1 estudamos algunhas características da proteína viral. En primeiro lugar estudamos a súa estabilidade, posto que as proteínas M1 e PB2, anteriormente descritas como dianas de NEDD8, ven afectada a súa estabilidade por NEDD8. No caso de NS1 non detectamos ningunha variación na vida media da proteína mutante en comparación coa silvestre, descartando polo tanto un papel desta modificación na estabilidade da proteína viral. Unha das características máis importantes de NS1 e a súa habilidade para inhibir a síntese e a sinalización do interferón. Levando a cabo un ensaio de luciferasa vemos que as células que foron previamente transfectadas con NS1 silvestre son capaces de inhibir a actividade dun xene reporteiro do interferón, ISG54-luc, mentres que as células transfectadas coa versión mutante de NS1 non son capaces de inhibir esta sinalización. Un dos mecanismos polos cales NS1 inhibe o interferón é a unión ao ARN de dobre cadea. O ARN de dobre cadea non se presenta de maneira abundante nas células non infectadas, e xeralmente prodúcese durante o proceso de infección do virus. É por iso que a célula ten mecanismos para detectar este ARN e desencadear unha resposta antiviral. A unión de NS1 a esta molécula impide que sexa detectada pola célula e así atenuar a resposta antiviral. Neste estudo demostramos que o mutante de NS1 que non se pode NEDDilar presenta unha unión a ARN de dobre cadea máis débil que a proteína silvestre. O mesmo sucede cando analizamos a unión de NS1 silvestre a ARN bicatenario en presenza do ROCÍO SEOANE ABELENDA 40 inhibidor de NEDDilación. Con isto concluímos que a proteína NS1 do virus influenza A se NEDDila e que a súa NEDDilación é relevante para o seu papel inhibidor da resposta antiviral, posto que se une con pouca afinidade ao ARN de dobre cadea e non é quen de inhibir o interferón. Ademais da agora proposta NEDDilación, NS1 demostrouse que pode ser modificada por outras modificación post-traducionais como SUMOilación, ISGilación, acetilación ou fosforilación. Ademáis algúns dos residuos descritos poden ser modificados por máis dun mecanismo. Isto pon de relevancia o papel tan importante de NS1 na replicación do virus e o nivel tan alto de complexidade deste proceso. Así, todas estas modificacions permiten que unha proteína relativamente pequena, de aproximadamente 26 kDa, sexa capaz de realizar multitude de funcións e dunha maneira dinámica gracias a súa capacidade de interaccionar con diversos modificadores de proteínas. Finalmente, decidimos estudar o posible papel da UFMilación no contexto da infección polo virus da gripe. UFM1 é un modificador de proteínas similar á ubiquitina. O seu descubrimento é relativamente recente polo que descoñecemos moitos aspectos desta modificación como poden ser os mecanismos que o activan, a súa relevancia e moitas das súas dianas. As súas dianas máis salientables son a proteína ribosomal RPL26 e a proteína supresora de tumores p53. Ademais UFM1 e a UFMilación parecen ter un papel moi importante na resposta a estrés do retículo endoplasmico. A maioría dos enzimas implicados na ruta de UFMilación localízanse nas inmediacións do retículo endoplasmático e unha alteración do mesmo provoca un aumento de UFM1 ou das enzimas relacionadas. Dado que existe unha interconexión entre a infección co virus da gripe e o estrés do retículo endoplásmico, decidimos evaluar se UFM1 podía tamén estar alterado trala infección. Poidemos observar que existe un aumento na conxugación de UFM1 aos seus substratos tras a infección có virus da gripe e que este aumento non vai paralelo cunha maior expresión do xene que codifica para UFM1 ou para dous dos enzimas implicados na UFMilación, o que Introduction 47 3.1 INFLUENZA VIRUS 3.1.1 Influenza virus, pathogenesis, and transmission Influenza viruses are enveloped negative-sense single-stranded ribonucleic acid (RNA) viruses that belong to the family Orthomixoviridae (Fields et al., 2013; Palese and Shaw, 2006). This family comprises 4 genera: Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus and Deltainfluenzavirus, each containing only a single specie: Influenza A virus (IAV), Influenza B virus (IBV), Influenza C virus (ICV) and Influenza D virus (IDV), respectively. IBV, ICV and IDV are present almost exclusively in humans. Influenza A virus has a broad host range, although the natural reservoir are wild birds (Webster et al., 1992). The genome of influenza virus is segmented into 7-8 fragments that encode the different proteins of the virus. The segmented nature of the viral genome allows for an exchange of genome segments when two different influenza viruses co-infect a cell, by a process called reassortment, a major mechanism of influenza virus evolution (Fields et al., 2013). A reassortment of two or more influenza viruses within the same cell or a direct jump from an animal to humans can result in an antigenic shift, defined as the generation of a new virus with antigenic properties extremely different from those of the circulating strains (Bouvier and Palese, 2008; Krammer et al., 2018; Lowen, 2017; Palese and Shaw, 2006). In addition, the influenza RNA- dependent RNA polymerase (RdRp) lacks proofreading activity and exhibits high mutation rates (Steinhauer et al., 1992). Accumulation of point mutations in the genes encoding antigens that are recognized by the immune system (antigenic drift) can reduce or inhibit the binding of neutralizing antibodies, and consequently, the virus may evade the immune system and cause severe disease (Hay et al., 2001; Kim et al., 2018; Nachbagauer and Krammer, 2017; Webster and Govorkova, 2014) (Figure 1). ROCÍO SEOANE ABELENDA 48 Figure 1. Antigenic shift and antigenic drift in IAV. Schematic representation of the process of antigenic shift, by which two or more different strains of IAV combine to form a virus with different antigenic properties, and of antigenic drift, by which mutations in the genes encoding surface proteins produce viruses with new antigenic determinants. (Figure designed with Biorender). Influenza virus is the causative agent of seasonal influenza, an acute respiratory disease that causes severe illness and death in highrisk populations. Influenza virus typically causes an acute infection of the upper respiratory tract (URT). However, in severe cases, it can infect the lower respiratory tract (LRT) triggering severe pulmonary inflammation and inducing acute lung injury. Infection with influenza virus results in relevant host cell death, which can intensify inflammation and compromise the integrity of the epithelial cell barrier leading to respiratory failure (Fields et al., 2013). The symptoms of influenza include fever, headache, cough, muscle and body pain, runny nose, and fatigue, and usually appear within one to four days after infection. The disease typically resolves after 1-2 Introduction 49 weeks without the need for medical attention or further complications, but it can take a severe and even fatal course, especially in immunocompromised individuals, infants and the elderly (Fields et al., 2013). Influenza is a highly contagious airborne disease. Influenza A virus is believed to transmit through aerosols or respiratory droplets (Lakdawala and Subbarao, 2012; Lindsley et al., 2016, 2015; Milton et al., 2013). Influenza infections in humans exhibit a clear seasonal cycle in temperate regions where outbreaks usually occur in the winter season. However, in tropic areas, a flu outbreak may occur throughout the year. Reports from the World Health Organization (WHO) estimate that influenza virus infection results in around 1 billion illnesses and 290,000-650,000 deaths annually, although the overall impact of influenza varies from year to year (Iuliano et al., 2018; Thompson et al., 2009). So far, the 1918 pandemic, caused by influenza A H1N1, has remained as the most devastating pandemic in the recorded history. It infected almost 50% of the world’s population and killed an estimated 20-50 million people (Crosby, 2003; Garcia-Sastre and Whitley, 2006; Johnson and Mueller, 2002; Taubenberger et al., 2001). 3.1.2 Influenza virus structure Influenza virus is an enveloped virus with a size of 80-120 nm (Fujiyoshi et al., 1994; Noda, 2012). They are highly pleiomorphic, exhibiting spherical, filamentous, elliptical or even irregular shapes (Chu et al., 1949; Kilbourne and Murphy, 1960; Stevenson and Biddle, 1966). The envelope of influenza virus is derived from the host plasma membrane and contains multiple copies of the viral proteins hemagglutinin (HA), ion channel (M2), and neuraminidase (NA) (Fields et al., 2013; Nayak et al., 2009; Scheiffele et al., 1999). Right underneath the membrane, the matrix protein 1 (M1) forms an ROCÍO SEOANE ABELENDA 50 oligomeric layer critical for particle stability and pH-dependent RNA genome release. The membrane of influenza viruses contains embedded the glycoproteins HA and NA, which are the major antigenic components of the virus, and they are essential for virion binding to cellular receptors (Wiley and Skehel, 1987) and for the release of the progeny virus after completing the viral cycle. So far, there are at least 18 known HA subtypes and 11 known NA subtypes (Kosik and Yewdell, 2019), and the combination of HA and NA determines the influenza A subtype (e.g., H1N1, responsible for the 2009 influenza pandemic, or the highly pathogenic H5N1 (Gamblin and Skehel, 2010; Park and Ryu, 2018; Sullivan et al., 2010). Influenza virus genome is divided into 7 (ICV and IDV) or 8 segments (IAV and IBV) that encode the different proteins of the virus. The terminal regions of each segment are associated with the polymerase complex consisting of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA), and is covered by multiple copies of the viral nucleoprotein (NP), forming the viral ribonucleoprotein (vRNP) complex (Arranz et al., 2012; Klumpp, 1997; Ye et al., 2006). 3.1.3 Influenza virus cell cycle 3.1.3.1 Viral entry In humans, influenza virus replication takes place mainly in epithelial cells of the respiratory tract. The first step of influenza virus entry is the recognition of the terminal alpha sialic acid (the host cell receptor molecule) by HA (Dou et al., 2018; Sakai et al., 2017; Weis et al., 1988). Human IAV binds preferentially to sialic acid linked by an α2,6 linkage to the rest of the oligosaccharide (Fields et al., 2013). When HA binds to a non-optimal receptor, NA protein removes this interaction (Sakai et al., 2017). The interaction of HA with the receptors triggers the endocytosis of the virus. The endosome containing the virus is then translocated near the nucleus. There, acidification of the endosome induces a conformational change in HA that exposes its Introduction 51 fusion peptide (Cross et al., 2009; Martin and Heleniust, 1991; Pinto et al., 1992; Skehel and Wiley, 2000). As a consequence, the viral membrane fuses with the endosomal membrane and the vRNPs dissociate from M1 allowing them to interact with importin α/β and enter the nucleus (Boulo et al., 2007; Cros et al., 2005; Martin and Helenius, 1991; O’Neill et al., 1995) (Figure 2). Figure 2. Life cycle of Influenza A virus. Schematic diagram of the life cycle of influenza A virus in the host cell. Influenza virus binds to the host receptors and upon binding the virus becomes endocytosed. Then, viral and endosome membranes fuse, and the viral genomes are released in the cytoplasm. The vRNPs are transported to the nucleus where transcription and replication of viral RNA occurs. The mRNAs are transported to the cytoplasm where the translation of viral proteins occurs through host machinery. Some of these proteins enter the nucleus where they assemble with viral RNAs. Progeny vRNPs are exported from the nucleus towards the cytoplasmic membrane with viral components to be packaged into new infectious particles. Finally, the virus buds off from the host cell. (Figure designed with biorender). ROCÍO SEOANE ABELENDA 52 3.1.3.2 Transcription and replication of the viral RNA (vRNA) First, vRNA is transcribed into positive-strand viral messenger RNAs (mRNAs). Synthesis of viral mRNAs depends on short, capped primers derived from host RNA polymerase II transcripts. During a process known as cap-snatching, PB2 binds to the cap structure of cellular RNAs through a cap-binding domain and the PA protein subsequently cleaves the host’s pre-mRNA 10-15 nucleotides downstream of the cap structure (Dias et al., 2009; Engelhardt et al., 2005; Martínez-Alonso et al., 2016; Plotch et al., 1981). This capped oligonucleotide primer is then used by the RNA-dependent RNA polymerase (RdRp) to initiate transcription. After elongation, the poly (A) tail is generated through stuttering of the polymerase on a short Poly-U sequence proximal to the template 5’ end (Poon et al., 1999). This process is different from cellular polyadenylation, where the polyadenylation signal is cleaved by the cleavage and polyadenylation specific factor (CPSF) and then a poly(A) tail is added to the mRNA. Interestingly, the viral non-structural protein 1 (NS1) is able to bind CPSF and inhibit its cleavage activity, impairing host mRNA maturation and inducing host gene shutoff (Nemeroff et al., 1998; Shimizu et al., 1999) (Figure 3). In addition, NS1 prevents the binding of the mRNA export receptor complex NXF1-NXT1 (nuclear RNA export factor 1-nuclear transport factor 2-related export protein 1) to nucleoporins, causing an accumulation of mRNA in the nucleus and preventing host gene expression (Satterly et al., 2007; Zhang et al., 2019) (Figure 3). Another mechanism by which NS1 contributes to IAV replication is the increase in the rate of translation of viral mRNAs but not of cellular mRNAs (de la Luna et al., 1995). NS1 binds to translation initiation factor eIF4G1 (eukaryotic translation initiation factor 4 Gamma) and poly(A)-binding protein 1 (PABP1) and enhances the initiation of translation of viral mRNAs (Aragón et al., 2000; Arias- Mireles et al., 2018; Burgui et al., 2003) (Figure 3). Introduction 53 Influenza A virus has developed several strategies to optimize the coding potential of its segmented genome (Dubois et al., 2014). Thus, in addition to the classical HA, NA, PB1, PB2, PA, M1, NP, and NS1 proteins, PB1-F2, PB1-N40, PA-N155, and PA-N182 are generated by using alternative translation initiation sites (W. Chen et al., 2001; Muramoto et al., 2013; Wise et al., 2009), PA-X is generated by a ribosomal frameshift (Jagger et al., 2012), and M2 and NEP are generated by alternative splicing (Lamb and Choppin, 1981, 1979). Noteworthy, NS1 interacts with components of the splicing machinery and modulates both cellular and viral mRNA splicing (Fortes et al., 1995). NS1 negatively modulates host splicing processes but promotes splicing of the viral mRNAs (Dubois et al., 2014; Fortes et al., 1995; Qiu et al., 1995; Zhang et al., 2019) (Figure 3). Figure 3. Functions of NS1 in the life cycle of influenza virus. NS1 interferes with three steps in RIG-I activation: binds Trim25 to prevent the ubiquitination of RIG-I, directly interacts with the second CARD of RIG-I, and interacts with dsRNA preventing the activation of RIG-I. ROCÍO SEOANE ABELENDA 54 Interaction with dsRNA also inhibits the OAS/RNase pathway. Binding of NS1 to PKR prevents PKR activation, and interaction between NS1 and p85β activates the PI3K pathway. NS1 also hijacks the host translation machinery to increase the rate of translation of viral mRNAs but not of cellular mRNAs. NS1 prevents polyadenylation of host mRNAs, the nuclear export and the splicing of host pre-mRNA. (Figure designed with Biorender). The transcribed mRNAs are translated into the novo viral proteins in cytosolic ribosomes (PB1, PB2, PA, NS1, NEP, and M1) or in endoplasmic reticulum-associated ribosomes (HA, NA, and M2) (York and Fodor, 2013). Then, the newly synthesized proteins can enter the nucleus and assist in viral genome replication and mRNA transcription, or suppress the host antiviral response (Figure 2). The replication of IAV genome involves two steps: first, the RdRp initiates RNA synthesis in a prime-independent manner generating a complementary RNA (cRNA) that binds to viral proteins generating complementary ribonucleoproteins (cRNPs). Then, the heterotrimeric polymerase assembles and binds to the newly formed cRNPs to transcribe new vRNA copies (Figure 2). 3.1.3.3 Assembly and budding After assembly of progeny vRNPs in the nucleus, these are exported from the nucleus to the cytoplasm. This process is mediated by the cellular exportin CRM1 (Elton et al., 2001; Watanabe et al., 2001), and requires the participation of several viral proteins. The two major proteins that mediate this process are M1 and NEP, especially the latter, which is able to bind chromosomal maintenance 1 (CRM1) and hydrolyze RanGTP (Boulo et al., 2007; Bui et al., 2000; Neumann, 2000; O’Neill, 1998). Once in the cytoplasm, vRNPs, as well as viral proteins HA, NA, and M2, are transported to the apical plasma membrane. The mechanism that explains the packing of all the eight vRNPs into the virion is still not very well understood. Evidence suggests that each of the viral segments contains specific signals that would orchestrate the proper incorporation into the virion (Li et al., 2021; Liang et al., 2008; Marsh et al., 2008, 2007). Budding of IAV requires the alteration of membrane curvature, a complex multiple-step Introduction 55 mechanism mediated by HA and NA proteins (Chen et al., 2007; Chlanda et al., 2015). After budding, NA catalyzes the hydrolysis of sialic acid residue from glycoproteins and glycolipids, preventing the binding of HA to the cell surface and facilitating the release of the virus (Dou et al., 2018; Palese et al., 1974) (Figure 2). N-mediated cleavage of sialic acid also contributes to the viral movement through mucus and respiratory epithelial cells (Cohen et al., 2013; Yang et al., 2014). 3.1.4 Influenza A virus-host cell interaction Viral infections trigger the induction of signaling cascades in the infected cells to control virus replication, and some viruses are able to hijack cellular pathways to promote their propagation. Infection with influenza A virus modulates different essential cellular signaling pathways such as the TLR/RIG-I, PI3K/AKT, MAPK, PKR, and posttranslational modification signaling pathways. 3.1.4.1 TRL/RIG-I pathway and influenza A virus infection Innate immunity is the first line of defense against invading viruses. When host pattern recognition receptors (PRRs) such as Tolllike receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RIG-I, MDA5 and LGP2) recognize pathogen-associated molecular patterns (PAMPs), they trigger the activation of signaling pathways leading to the production of type-I interferon (IFN) and otherproinflammatory cytokines. RIG-I senses the 5’-triphosphate of viral single-stranded or short double-stranded RNA as a pathogen-associated molecular pattern (Hornung et al., 2006) (Figure 4). Structurally, RIG- I and MDA5 have two N-terminal caspase activation and recruitment domains (CARD), an RNA helicase domain, and a C-terminal regulatory domain (Kolakofsky et al., 2012). Under normal conditions, RIG-I is in an inactive, closed conformation. The PAMP recognition provokes a conformational change, the exposure of the N-terminal CARDs of RIG-I, and its ubiquitination by tripartite interaction motif 25 (TRIM25) and RING (really interesting new gene) finger protein ROCÍO SEOANE ABELENDA 56 135 (RNF135/RIPLET) (Gack et al., 2007; Oshiumi et al., 2010). Ubiquitinated CARDs allow the interaction of RIG-I with the mitochondrial antiviral-signaling protein (MAVS) and the consequent activation of the downstream signaling for phosphorylation and nuclear translocation of interferon regulatory factor 3 and 7 (IRF3 and IRF7), the phosphorylation of NF-κB by the IKKalpha/beta/gamma complex, and type I interferon (IFN) production (Chattopadhyay and Sen, 2017; García-Sastre, 2017; Rehwinkel and Gack, 2020; Wu and Hur, 2015). Figure 4. Activation of the RIG-I pathway by IAV. 5’-triphosphate of vRNAs are recognised as a pathogen-associated molecular pattern that induces a conformational change in RIG-I, that can be then be ubiquitinated and consequently activate MAVS, which trigger the signaling cascades that promote the phosphorylation of IRF3 and IRF7, as well as the nuclear translocation of NF-κB, all of them promoting the translation of type I IFN as well as proinflammatory cytokines. (Figure designed with Biorender). Influenza A virus has developed many strategies to counteract the TLR/RIG-I pathway. For example, the virus performs its transcription inside the nucleus, which keeps its genome inaccessible for the cytoplasmic PAMP sensors, such as RIG-I (Dou et al., 2018; Fields et Introduction 63 proteins increases their already multifunctional nature or can act as a mechanism to sequester the modifiers and therefore interfere with the host response. However, the functions of many of these modifications are still unknown (Kumar et al., 2020). IAV infection induces global changes in PTM signaling pathways and the virus employs PTM to modulate different steps through the viral replication cycle. In addition, IAV infection induces the modification of viral proteins by different PTMs such as phosphorylation, SUMOylation, NEDDylation, Ubiquitination, ISGylation, glycosylation, acetylation, ADP-ribosylation, and palmitoylation. Some of the PTMs that occur in response to IAV infection contribute to virus replication. Other PTMs are part of the cellular antiviral response (Dawson and Mehle, 2018; Hu et al., 2020). One example is the NS1 protein, the most relevant protein of IAV involved in the attenuation of the host-immune response, and in many other aspects of the replication of the virus. This protein harbors many different posttranslational modifications, some contribute to improve NS1 activities, and others are part of the host cellular antiviral response to block NS1 functions (Ji et al., 2021; Lamotte and Tafforeau, 2021) (Figure 5). 3.2.1 Phosphorylation Phosphorylation is the most studied PTM and consists on a reversible enzymatic process where a phosphate group from adenosine triphosphate (ATP) is transferred to a target protein. This modification can occur in different amino acids although serine (Ser), tyrosine (Tyr), and threonine (Thr) are the amino acids most commonly modified by phosphorylation (McKay and Johnson, 2010; Ramazi and Zahiri, 2021). Phosphorylation has a pivotal role in most of the cellular processes, especially in response to stress (Hunter, 1995; Karve and Cheema, 2011), and alterations in phosphorylation/dephosphorylation processes have been related to many pathological conditions such as metabolic or neurological disease and cancer (Anderson et al., 2006; ROCÍO SEOANE ABELENDA 64 Harsha and Pandey, 2010; Haystead et al., 1989; Hernandez‐Aya and Gonzalez‐Angulo, 2011; Levine and Puzio-Kuter, 2010; Maclaine and Hupp, 2011; Zhao et al., 2009). Figure 5. Role of PTM modifications in NS1 activity. Phosphorylation of S42, T49 and T80 attenuates the IFN inhibition whereas phosphorylation of S83 and Y83 favors the RIG-I activation. Acetylation of K108 is relevant for nuclear translocation of NS1. ISGylation of K41 impairs its nuclear import. (Figure designed with Biorender). As mentioned above, IAV infection induces the activation of kinase cascades during the course of the infection. Some of these pathways such as the Ras/MEK/ERK signaling pathway, NF-κB signaling, PI3K/AKT, and PKC signaling cascade are essential for IAV replication and their inhibition has been proposed as a potential strategy to control IAV. Furthermore, several IAV proteins are regulated by phosphorylation and at least 36 phosphorylation sites in IAV proteins have been identified (Hutchinson et al., 2012). NP protein is phosphorylated at different residues and this modification has different consequences. Phosphorylation of NP at the N-terminal nuclear Introduction 65 localization signal inhibits its interaction with the importin machinery whereas its phosphorylation at the internal region inhibits the interaction with nuclear export factors (Cui et al., 2019). In addition to modulate NP subcellular localization, phosphorylation also modulates protein-protein interaction. Phosphorylation of NP protein at the homotypic interface modulates NP oligomerization and inhibition of phosphorylation causes defects in RNP assembly, function, and viral replication (Cui et al., 2019). Other IAV proteins that are regulated by phosphorylation are the polymerase subunits PB1, PB2, and PA, although the functionality of these modifications is unknown. M1 is another phosphorylation substrate. M1 phosphorylation promotes its nuclear import and mutants with altered M1 phosphorylation exhibit replication defects (Wang et al., 2013). However, sometimes phosphorylation of viral proteins are cellular strategies to disarm viral countermeasures. One example is the NS1 protein whose activity can be negatively modulated by phosphorylation. NS1 is phosphorylated in different residues (Hutchinson et al., 2012), and some of these modifications disrupt protein-RNA and protein-protein interactions. Thus, phosphorylation of influenza virus A/Udorn/72 (Ud) NS1 at serine 42 has been shown to decrease the RNA binding capacity of the protein and to attenuate viral replication (Hsiang et al., 2012). Similarly, phosphorylation of NS1 at threonine 49 also impairs the binding of NS1 to dsRNA, TRIM25 and RIG-I, affecting significantly its IFN- antagonism activity. In addition, phosphorylation of NS1 at threonine 80 has been reported to difficult the growth of influenza virus A/WSN/1933 probably due to the reduced translation of viral mRNAs (Zheng et al., 2017). NS1 can be phosphorylated at other residues such as serine 48 (Hsiang et al., 2012) or threonine 215 (Hale et al., 2009), but with no significant consequences on viral replication. In addition, NS1 phosphorylation can be also beneficial for IAV replication as shown by Chen (Cheng et al., 2019). These authors demonstrated that phosphorylation of NS1 at both serine 83 and tyrosine 73 in A/swine/Shanghai/3/2014 favors the RIG-I-mediated inhibition of IFN (Figure 5). ROCÍO SEOANE ABELENDA 66 3.2.2 Glycosylation Glycosylation is a PTM that involves the transference of a sugar moiety to a target molecule (proteins or lipids) by a covalent bond. Protein glycosylation can occur in a wide variety of amino acids but especially in side chains of serine (Ser), threonine (Thr), aspargine (Asn) and tryptophan (Trp) (Ohtsubo and Marth, 2006). There are different classes of protein glycosylation, the major ones representing the N-linked (the sugar moiety is attached to the oxygen molecule of a threonine or serine) and the O-linked (the sugar is bound to a nitrogen atom or the asparagine residue) glycosylation. Most of the target proteins for this modification are membrane proteins and this modification regulates protein solubility, folding, thermostability, catalytic efficiency, antigenicity, etc. Glycosylation plays a major role in cell adhesion, cellular proliferation, differentiation, migration, invasion, and cell cycle control, among others, being critical for physiological and pathological functions (Goulabchand et al., 2014; Karve and Cheema, 2011; Lauc et al., 2013; Ohtsubo and Marth, 2006). Glycosylation of viral envelope proteins is the most well-known example of glycosylation in the virus and it has been identified as a mechanism of immune evasion. In IAV infected cells, modulation of N-linked glycosylation of HA has been shown to protect the protein from antibody recognition and neutralization (Skehel et al., 1984). This process is exploited by the virus to increase viral spread virulence and fitness (Kosik et al., 2018; Zhao et al., 2017). In addition, glycosylation of HA has been also reported to modulate the affinity of HA for sialic acid receptor (de Vries et al., 2010). 3.2.3 Acetylation Acetylation is the transfer of an acetyl group from acetyl-CoA to lysine residues (Lys) of a target protein. It is one of the most common Introduction 67 PTMs in histones. It plays a critical role in global regulation of gene expression. Acetylation has been demonstrated to regulate different stages of virus cycle including virus entry, replication, fusion, transport, and release by modulating cellular and viral proteins. One example is the p53 protein, whose acetylation during infection induces the transactivation of pro-apoptotic and IFN-stimulated genes (Muñoz- Fontela et al., 2011). However, acetylation of cellular proteins can also favor virus replication. Thus, C-terminal deacetylation of RIG-I has been demonstrated to enhance RNA sensing (Choi et al., 2016). Moreover, HDAC6-mediated deacetylation of microtubules has been shown to prevent the traffic of IAV to the membrane (Husain and Cheung, 2014). Many different influenza virus proteins are also targeted by acetylation such as PB1, PA, and NP (Hutchinson et al., 2012). Acetylation of some residues may have a positive impact on IAV(Chen et al., 2019). Thus, deacetylation of PA by HDAC6 promotes its degradation suppressing RNA polymerase activity, and consequently, restricting RNA transcription and replication (Chen et al., 2019). In contrast, acetylation of NP protein on several internal lysine residues abolishes the stabilization of replication intermediates induced by the viral protein, and thereby inhibits virus replication (Giese et al., 2017; Hatakeyama et al., 2018). Interestingly, NP can be modified by both acetyl groups and ubiquitin at the same lysine residue, suggesting a competition for occupancy between these two modifiers. Acetylation of IAV NS1 plays a positive role in virus replication by increasing the activity of the protein and enhancing NS1 mediated IFN antagonism. Thus, inhibition of K108 acetylation in NS1 from influenza virus A/WSN/1933 attenuates viral replication due to an impaired IFN inhibition and the retention of the viral protein in the cytoplasm (Ma et al., 2020) (Figure 5). ROCÍO SEOANE ABELENDA 68 3.2.4 Ubiquitination Ubiquitin (UB) is an ubiquitous small protein (76 amino acids), (Goldstein et al., 1975) highly conserved among species that in humans is encoded by four different genes, UBB, UBC, RPS27A, and UBA52. Ubiquitin belongs to a family of ubiquitin-like (UBL) proteins that are covalently attached to target proteins to modify their function. All UBL proteins possess a beta grasp-fold (β-GF) composed of a five-stranded beta-sheet that partially wraps around a central alpha-helix. The ATP- dependent degradation of ubiquitin-protein conjugates was described for the first time in 1978 (Ciehanover et al., 1978) and since then an uncountable number of proteins have been identified as ubiquitin substrates. Ubiquitin is attached to substrates by a 3-step ATP- dependent enzymatic cascade. First, ubiquitin-specific proteases process the immature UB, which can then be activated by forming a thioester bond with E1 ubiquitin-activating enzyme through its carboxyl terminus. Then, the activated UB is transferred to an E2 ubiquitin conjugating enzyme. Finally, a variety of E3 ubiquitin ligases transfer UB from E2 to substrates to facilitate formation of an isopeptide bond between the C-terminal carboxyl of UB and the eamino group of a substrate lysine sidechain of free N-terminal amino group (Ciechanover and Schwartz, 1998). This process could be reversed by the action of a specialized family of proteases, the deubiquitinating enzymes, which remove conjugated ubiquitin molecules from target substrates (Wilkinson, 1997). Importantly, although under non-stress conditions, each UBL has unique and dedicated E1 and E2 enzymes, under stress conditions or a reduction in the levels of free ubiquitin, the ubiquitin E1 enzyme Ube1 can mediate conjugation of the ubiquitin-like protein NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) (Leidecker et al., 2012). Moreover, it has been found that ubiquitin E3 enzymes can also function as NEDD8 E3 enzymes (Noguchi et al., 2011; Xirodimas et al., 2004). Proteomic studies identified thousands of ubiquitinated proteins and ubiquitination has emerged as a key post-translational Introduction 69 modification for the maintenance of homeostasis. When a single ubiquitin molecule is attached to a substrate lysine residue is called monoubiquitination. But ubiquitin itself can be modified by different PTM including ubiquitination, increasing the complexity. Although the most common function attributed to UB modification is the proteasome-mediated degradation (usually linked to K48 polyubiquitin chains), ubiquitination can also modulate the subcellular localization, protein-protein interaction, or activity of the target proteins (Flotho and Melchior, 2013; Mayer et al., 1991). Ubiquitination plays an important role in virus infection. In the case of IAV, it has been demonstrated that components of the ubiquitin proteasome system (UPS) are upregulated in IAV infected cells, and that proteasome activity is required for entry and replication of the virus (Kroeker et al., 2013). IAV employs different strategies to modulate ubiquitination in order to promote replication. Thus, whereas IAV HA protein induces the ubiquitination and degradation of the Interferon-α/β receptor 1 IFNAR1) in order to attenuate IFN signaling (Xia et al., 2015), NS1 inhibits the E3 ubiquitin ligase Trim25, a key mediator in antiviral RIG-I signaling pathway (Gack et al., 2009). In addition, IAV proteins themselves can be directly modified by UB. Thus, the antiviral protein ZAPL can induce the ubiquitination and degradation of PA and PB2 (Liu et al., 2015). In addition, different proteins belonging to the RING family of E3 ubiquitin ligases can ubiquitinate NP and PB1 targeting them for degradation (Fu et al., 2015; Liao et al., 2010; Patil et al., 2018). But conjugation of ubiquitin to IAV proteins not necessarily leads to protein degradation. Conjugation of monoubiquitin to lysine K184 in NP has been reported to regulate its interaction with genomic RNA to facilitate replication (Lin et al., 2017). Ubiquitination of M2 plays a role in particle assembly and release (Liu et al., 2021; Su et al., 2018), and ubiquitination of M1 facilitates the release of the incoming virion (Mahesutihan et al., 2018). Furthermore, ubiquitin proteins have been detected incorporated in IAV virions although its biological significance is still unclear (Hutchinson et al., 2014). ROCÍO SEOANE ABELENDA 70 3.2.5 NEDDylation Among the members of the UBL protein family, NEDD8 is the closest in sequence to ubiquitin, with around 60% sequence identity (Kumar et al., 1993; Whitby et al., 1998). This protein is ubiquitously expressed in all cells in the body and is essential for viability, growth and development. NEDD8 precursor is cleaved by the NEDD8-specific protease 1 NEDP1 (Gan-Erdene et al., 2003) to expose the C-terminal di-glycine domain. The mature NEDD8 is then activated by the E1 NAE (NEDD8-activating enzyme) in an ATP-dependent manner and transferred between the active cysteine residue of the NEDD8 activating enzyme (NAE) to an E2-conjugating enzyme, UBE2M or EBE2F. Finally, NEDD8 E3 ligases catalyze the transfer of NEDD8 from the E2 onto the target protein. Importantly, so far, all the reported NEDD8 E3 enzymes like MDM2 (murine double minute 2) or tripartite interaction motif 40 (TRIM40) can also function as ubiquitin E3 enzymes. Conjugation of NEDD8 is reversed by the action of NEDP1, that deconjugates NEDD8 from the substrate (Abidi and Xirodimas, 2015; Lu and Yeh, 2013; Soucy et al., 2010) (Figure 6). However, and as mentioned above, this scenario corresponds to canonical NEDDylation, under homeostasis conditions. Upon stress conditions or a reduction in free ubiquitin levels, NEDD8 can be additionally activated by the ubiquitin activating enzyme E1, UBA1, and the global NEDDylation pattern changes. Thus, some proteins or even specific lysine residues in a target protein are only NEDDylated upon stress conditions (Lobato-Gil et al., 2021). The most well-known targets for NEDDylation are the members of the cullin protein family (Pan et al., 2004). Cullin proteins are molecular scaffolds for ubiquitin ligases. The members of the cullin protein family assemble multi-subunit Cullin- RING E3 ubiquitin ligases (CRL) complexes constituting the largest family of E3 ubiquitin ligases. The core CRL consists of four components: a cullin protein that serves as the scaffold, a RING finger protein that binds to an E2 ubiquitin, a substrate receptor that recognizes the target protein, and adaptor proteins that bridge the substrate receptor Introduction 71 to the cullin (Sarikas et al., 2011). NEDDylation of cullins occurs in their C-terminal domain and leads to a conformational change that facilitates the interaction of the RING finger protein with the ubiquitin substrate (Jones et al., 2008; Ohh et al., 2002; Pan et al., 2004). Thus, cullin NEDDylation results in the activation of cullin-RING ligases and, concomitantly, in the ubiquitination of their targets. In addition to cullins, an increasing list of proteins has been reported to be NEDDylated, such as the tumour suppressor p53, MDM2, most of the components of the NEDDylation machinery, and a relevant list of ribosomal proteins (Abida et al., 2007; Enchev et al., 2015; Jones et al., 2008; Xirodimas, 2008; Xirodimas et al., 2008, 2004). Different evidence support a relationship between deregulation of the NEDDylation pathway and pathogenic processes. Cancer is one of the pathological conditions associated with an alteration in NEDDylation. Components of the NEDDylation machinery have been found overexpressed or hyperactivated in different types of cancer (Abidi and Xirodimas, 2015; Chairatvit and Ngamkitidechakul, 2007; Gai et al., 2021; Salon et al., 2007; Soucy et al., 2010), and NEDDylation is considered as a therapeutic target for many of them (Soucy et al., 2009). The NEDDylation inhibitor MLN4924, also known as pevonedistat, is a potent and selective inhibitor of NAE that has been shown to induce autophagy, apoptosis and senescence, impairing tumor growth in cancer cell lines and xenografts (Luo et al., 2012; Soucy et al., 2009). Apart from its role in tumor progression, NEDD8 and NEDDylation inhibition have been shown relevant in other pathological conditions such as neurological disorders (Dil Kuazi et al., 2003; Mori et al., 2005) or in viral infections. Treatment with MLN4924 has been reported to inhibit the replication of different viral agents such as hepatitis B virus, cytomegalovirus, or influenza virus (Abounouh et al., 2022; Flores- Martínez et al., 2021; Sun et al., 2018). IAV has been shown to promote the NEDDylation of cullins, and inhibition of NEDDylation has been demonstrated to decrease IAV replication (Sun et al., 2018). However, overexpression of NEDD8 and ROCÍO SEOANE ABELENDA 72 the E3 HDM2 has also been found to inhibit IAV replication (Zhang et al., 2017), suggesting that NEDD8 plays different roles in the IAV-host cell interplay. So far, there is little information about the potential modification of IAV proteins by NEDD8, and only two proteins of the virus, PB2 and M1, have been identified as NEDD8 substrates. Interestingly, NEDDylation of both proteins has been reported to negatively modulate IAV replication. Thus, conjugation of NEDD8 to PB2 or M1 destabilizes both proteins and inhibits virus replication (Li et al., 2020; Zhang et al., 2017). Figure 6. NEDDylation pathway. Schematic representation of the NEDDylation pathway. After maturation, NEDD8 is activated by forming a thioester linkage with NAE in an ATP dependent manner. Activated NEDD8 is transferred to the active cysteine site of E2 (UBE2M/UBE2F)). Finally, E3 catalyzes the transfer of NEDD8 from the E2 onto the target protein.The protease NEDP1 can revert this modification. (Figure designed with Biorender). 3.2.6 SUMOylation SUMOylation plays a key role in influenza biology. IAV infection results in global changes in SUMOylation and most of the IAV proteins are SUMO substrates. SUMO conjugation to IAV NP protein is essential for its nuclear retention and virus expressing a SUMOylationdeficient NP mutant exhibits defects in replication (Han et al., 2014). SUMOylation of the IAV M1 protein has been shown to facilitate its Objectives 79 Viruses adapt to the host cellular environment and manipulate cellular machinery and functions to perform virus replications tasks. A better understanding of virus-host cell interaction is essential to pinpoint critical virus and host factors to control virus pathogenesis. Posttranslational modifications modulate protein functions and are critical for cellular responses to virus infection. PTM can be hijacked by viruses to increase the multifunctionality of their proteins, controlling key steps during virus replication. With the aim of understanding the role of different post-translational modifications in Influenza A virus infection, we propose the following objectives: 1.To determine the role of hypusination on Influenza A virus infection 1.1 To investigate the potential effects of Influenza A virus infection on eIF5A hypusination 1.2 To study the role of hypusination on Influenza A virus infection 2. To determine the role of NEDDylation on Influenza A virus infection 2.1 To investigate the potential effects of Influenza A virus infection on global NEDDylation 2.2 To study the NEDDylation of the viral protein NS1 3. To determine the role of UFMylation on Influenza A virus infection 3.1 To investigate the potential effects of Influenza A virus on global UFMylation 3.2 To study the role of UFMylation on Influenza A virus infection 5. MATERIAL AND METHODS Material and Methods 83 5.1 CELL CULTURE The cell lines used in this studio were: A549 (human lung adenocarcinoma), SW480 (human colorectal cancer), A375 (human melanoma), HEK-293T (human embryonic kidney), HeLa (human cervical adenocarcinoma), MDCK (Madin-Darby canine kidney), Vero (african green monkey kidney), and BSC40 (african green monkey kidney). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) (#35-079-CV, Corning), 10 U/mL penicillin/streptomycin (#P4333, Sigma-Aldrich), and 2 mM L-Glutamine (#G7513, Sigma-Aldrich), and in a humidified incubator at 37°C and 5% CO2 (carbon dioxide). Cells were disgregated with Trypsin-EDTA (ethylenediaminetetraacetic acid) (#59418, Sigma-Aldrich). 5.2 TRANSFECTION Cells were transfected using polyethilenimine (PEI) (#23966, CliniSciences), unless otherwise indicated, following manufacturer’s instructions. Briefly, DNA or PEI were diluted in serum and antibioticfree DMEM in a ratio of 6:1 PEI:DNA. After 5 min, both dilutions were mixed and incubated at room temperature for 20 min. Then, the mixture was added to the cells (70-80% confluency) dropwise. Cells were analyzed 24-48 h after transfection. Lipofectamine 2000 (#11668019, Thermofisher) was used to transfect RNA. Lipofectamine 2000 or RNA (Poly I:C (tlrl-picW, Invivogen)) were diluted in antibiotic and serumfree medium in a ratio of 3:1. 5 min later, the dilutions were mixed and incubated for 20-30 min at room temperature. The mixture was added to the cells dropwise. 5.3 SIRNA TRANSFECTION 70-80% confluent cells were treated with siRNAs targeting eIF5A (#J- 015739-06-0020, Horizon) or with non-targeting control siRNAs (#D- 001810-01-20, Horizon). For a 24-well, siRNAs (5 pmol) were diluted in 25 µL of antibiotic and serum-free DMEM and 1.5 µL of ROCÍO SEOANE ABELENDA 84 Lipofectamine RNAiMAX (#13778-150, Invitrogen) were also diluted in the same volume of antibiotic and serum-free DMEM. Then, both preparations were mixed and incubated at room temperature for 15 min. After the incubation time, the mixture was added to the cells dropwise. 5.4 CELLULAR TREATMENTS GC7 (#259545, Millipore) was added at a concentration of 20 µM unless otherwise indicated. MG132 (Z-Leu-Leu-al (#C2211, Sigma- Aldrich), was used at a final concentration of 20 µM). MLN4924 (#505477, Sigma-Aldrich) was added to a final concentration of 0.1 µM, and ML752 (#HY-108702, MedChemExpress was used at a final concentration of 0.1 µM. ML752 was kindly provided by Dr. Emilio Lecona (CBM, UAM). Cycloheximide was added at a final concentration of 50 µg/mL). BAY 11-7082 (#1956870, Sigma-Aldrich) was used at a final concentration of 2 mM and IKK16 (#sc-204009, Santa Cruz Biotechnology), was used at a final concentration of 10 mM. 5.5 PLASMIDS The plasmids used in this work were: pcDNA 3.1 empty vector (Invitrogen) pcDNA-His6-NEDD8, pcDNA-His6-SUMO2, and pcDNA-ubc9-V5 were kindly provided by Dr. Manuel S Rodriguez (CNRS) (Rodriguez et al., 2001, 1999; Vertegaal et al., 2006). NS1-WT (PR8), pdZ-PA, pdZ-NA, pdZ-PB1, pdZ-PB2, pdZ-NS-split, pdZ-NP, pdZ-HA, and pdZ M1 were kindly provided by Dr. Adolfo Garcia-Sastre (Mount Sinai) (Martínez-Sobrido and García-Sastre, 2010). pCMV-Flag- STUB1 (#HG12496-NF, Sino Biological). PGK-H2BeGFP (#21210, Addgene). 3xFlagCMV-7-1/heIF5A, kindly provided by Dr. Myung Hee Park (NIDR, NIH) (Park et al., 2006). prK5-HA-UFM1 and UFM1 sg-RNA were kindly provided by Dr. Rosa Barrio (CICbioGUNE). pcDNA-HA-eIF5A1, pcDNA-HA-NS1, HA-UFM-ΔG, pdZ-NS Split 4KR, pcDNA-HA-RPS27A, pcDNA-VDAC1 were generated by Material and Methods 85 cloning. NS1-K0, the different mutants of NS1, pcDNA-VDAC1- K110R, HA-UFM-ΔG-K41R, and the mutants of HA-eIF5A1 were generated by site directed mutagenesis using the primers listed in Table 1. 5.6 ANTIBODIES The primary antibodies used were: anti-HA (#901503, Biolegend), antieIF5A1 (#ab32443, Abcam), anti-phospho-eIF2a (#9721 Cell Signaling), anti-GAPDH (#sc-32233, Santa Cruz Biotechnology), anti- VSV G (a generous gift of Dr I Ventoso, CBMSO, Madrid), anti-actin (sc-4778, Santa Cruz Biotechnology), anti-influenza A virus M1 protein (#GTX127356, Gene Tex), anti-influenza A virus NS1 protein (#GTX125990, Gene Tex), anti-Alphavirus E1 and NSP1 (Llamas- González et al., 2019) , anti-PTV (mouse ascitic fluid kindly provided by Dr. Scott Weaver from World Reference Center for Emerging Arboviruses (WRCEVA) at University of Texas Medical Branch (UTNM), USA), anti-Hypusine antibody (#ABS1064-I, Millipore), anti-GFP (#902605, Biolegend), anti-Flag (OctA-probe) (#sc-166355, Santa Cruz Biotechnology), anti-NEDD8 (#ab81264, Abcam), anti-6x- His Tag (#MA1-21315, Thermo Fisher), anti-β-tubulin (#2146, Cell Signaling), anti-β-actin (#sc-47778, Santa Cruz Biotechnology), anti- UFM1 (#ab109305, Abcam), anti-p85β (#ab180967, Abcam), anti- Histone3 (#4499T, Cell Signaling), anti-STUB1 (#PA5-29024, Invitrogen), and anti-p65 (#3034, Cell Signaling). The secondary antibodies for Wester blot analysis were anti-rabbit (#a16035, Invitrogen) or anti-mouse (#a16072, Invitrogen) antibodies conjugated to horseradish peroxidase (HRP). 5.7 BACTERIAL TRANSFORMATION Competent Escherichia coli DH5α are thawed on ice. Then, the DNA of interest is added to the bacteria. After 30 min of incubation on ice, the bacteria are heat shocked for 1 min at 42º C and cooled immediately on ice for 2-5 min. Then, 350 µL of Luria Bertani (LB) growth media with no antibiotics are added to the bacteria and the mixture is incubated ROCÍO SEOANE ABELENDA 86 for 1 h at 37º C with shaking (750 rpm). Finally, the culture is pelleted at 4000g for 5 min and 100 µL of the resuspended pellet are plated in an LB agar plate supplemented with the antibiotic of interest. 5.8 SITE-DIRECTED MUTAGENESIS All the mutants used in this work were generated by site-directed mutagenesis using Phusion High Fidelity DNA polymerase and 100- 300 ng of DNA. The primers used are listed in table 1. After the amplification, the template DNA was digested with DpnI (Thermo Fisher) for 2 h at 37º C and 1/5 of the total PCR volume was transformed into Escherichia coli DH5α and plated in an LB plate with the appropriate antibiotic. Colonies were then grown overnight in LB supplemented with antibiotic and the following day plasmidic DNA was purified using a commercial kit. The presence of the mutation and confirmation of no additional mutations were confirmed by Sanger sequencing. 5.9 CLONING The gene of interest was amplified from a plasmid or from complementary DNA (cDNA) using Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase (#F-549S, Thermo Fisher) with the primers listed in Table 1. These primers include a region complementary to the 3’ or 5’ ends of the sequence targeted for amplification (the forward (F) and reverse (R) primers) together with sequences recognized by the restriction enzymes that will be used to cut the vector. After the amplification of the insert, vector and inserts were digested with the restriction enzymes for 30 minutes at 37º C. The resulting DNA was then loaded in an agarose gel and the bands corresponding with the vector or the insert were cut and the DNA was extracted from the agarose gel using a kit (#K0691, Thermofisher). Both purified vector and insert were incubated together in a ligation reaction in a ratio of 3:1 insert-vector using T4 DNA Ligase (#15224- 025, Invitrogen). 20 min after incubation, 1/4 of the reaction was used Material and Methods 87 to transform competent DH5α. The cloning was confirmed by digestion and Sanger sequencing. 5.10 WESTERN BLOT Proteins were collected in Laemmli sample buffer and boiled for 5 minutes at 100º C. Then, proteins were separated by size using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a 0.45 μm nitrocellulose membrane. Membranes were then blocked by incubating in 5 % non-fat dry milk in tween trisbuffered saline (TBST). Blocked membranes were incubated with the primary antibody at 4º C overnight in a shaker. The following day, the membranes were washed in Tris buffered saline Tween20 (TBST) (3 washes, 10 minutes each) and incubated with the secondary antibody for 1 hour, at room temperature in a shaker. Finally, the membranes were washed in TBST (3 washes, 10 minutes each), incubated in ECL for 2 min and exposed to an X-ray film. 5.11 VIRAL INFECTION Infections were carried out with the viruses: vesicular stomatitis virus (VSV) of indiana strain, recombinant VSV expressing GFP (rVSV- GFP), the mouse-adapted influenza A/PR/1934 (PR8) virus, mayaro virus (MAYV, AVR0565 strain, San Martin, Peru), una virus (UNAV, BT-1495-3, Bocas del Toro, Panama), chikungunya virus (CHIKV, Panama_256137_2014 strain, Panama), zika virus (ZIKV, 259249 strain, Panama), and punta toro virus (PTV, Adames strain, Panama). All arboviruses were a kindly gift of Dr. Scott Weaver from WRCEVA, UTMB, USA. For cellular infection, MAYV, UNAV, CHIKV, ZIKV, PTV and VSV were added to the cells resuspended in serum-free medium, whereas PR8 was resuspended in 0.3% bovine serum albumin (BSA) in phosphate buffer saline (PBS). One hour after incubation with the virus solution, the unbound virus was discarded and fresh medium was added to the cells. In the case of influenza virus, TPCK- treated trypsin (2.0 µg/mL) was added to the medium. ROCÍO SEOANE ABELENDA 88 5.12 RESCUE OF RECOMBINANT INFLUENZA VIRUS A co-culture of HEK-293T and MDCK cells in a ratio of 3:1 was mixed with a transfection mixture of the pdZ plasmids encoding all eight segments of the viral genome . The co-culture is then transferred to a culture plate and cells are incubated for 30 h in incubator. Then, the incubation medium was replaced by DMEM supplemented with 1 µg/mL of TPCK-treated trypsin. 48 h later, supernatants were collected and virus yields were measured by plaque assay. 5.13 PLAQUE ASSAY BSC40 (VSV), MDCK (IAV) or Vero cells (MAYV, UNAV, CHIKV, ZIKV, and PTV) were seeded in 12-well culture plates until a monolayer is formed. Then, the cells were washed with PBS and the serial dilutions of the virus, prepared in DMEM (for VSV, MAYV, UNAV, CHIKV, ZIKV, and PTV), or PBS-BSA 0.3% (for IAV), were added to the monolayer. After one h of incubation, rocking the plate every 5-10 min, the virus was aspirated from each well and 1 mL of the agar solution was added to the cells. Once the agar was solidified, the plates were incubated at 37º C and 5º CO2 for 1-2 days. The agar solution was prepared by mixing 25 mL of oxoid agar 1.8 % and 25 mL of DMEM 2X. For influenza virus, TPCK-treated trypsin was added to the medium. 5.14 METABOLIC LABELING Cells were infected at a multiplicity of infection (moi) of 5, washed twice with methionine-free DMEM and labeled by incubation in methionine-free DMEM supplemented with 35S-methionine (100 µCi/mL) for the times indicated in the figure. Whole protein extracts were analyzed by SDS-polyacrylamide gel electrophoresis, fixed and dried. The radioactively labeled proteins were visualized by autoradiography. 6. RESULTS Results 97 6.1 INFLUENZA VIRUS AND EIF5A 6.1.1 Influenza A virus (IAV) infection induces hypusination Several reports demonstrated that infection with different viruses such as human cytomegalovirus (HCMV) (Isom, 1979), herpes simplex virus type 1 (HSV-1) (Greco et al., 2005), or Epstein-Barr virus (EBV) (Shi et al., 2013) can alter the polyamine pathway at different levels. However, whether influenza virus infection modulates the polyamine pathway is still unknown. Therefore, we decided to study whether IAV can alter the hypusination levels, which is one of the downstream targets of the polyamine pathway. To study the putative modulation of eIF5A activation by IAV, we infected A549 cells with influenza virus A/PR8/1934 (PR8) strain at an moi of 5 and, at the indicated times after infection, cells were collected and protein extracts were analyzed by Western blot with anti-hypusine antibody. We observed an increase in the levels of hypusination from 0 to 12 h after IAV infection that decreased at longer times after infection (Figure 8). These results indicated that IAV infection promoted the activation of eIF5A. ROCÍO SEOANE ABELENDA 98 Figure 8.IAV induces eIF5A activation. A549 cells were infected with influenza virus A/PR8/1934 and at different times after infection protein extracts were analyzed by Western blot with the indicated antibodies (upper panel). Hypusinated eIF5A and eIF5A protein intensity bands from three biological replicates were quantified using Image J software. The hypusinated eIF5A/ total eIF5A ratios were plotted. Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *,p<0.05, **,p<0.01, ***,p<0.005. 6.1.2 Both infection with different RNA viruses and stimulation with dsRNA induce hypusination Here we showed that infection with IAV infection induces hypusination. However, whether infection with other viruses triggers hypusination is unknown. To investigate this possibility, we infected A549 cells with vesicular stomatitis virus (VSV) and, at different times after infection, cells were collected and the protein extracts were analyzed by Western blot. Incubation with anti-hypusine antibody revealed an increase in hypusination levels from 0 to 5 h after VSV infection that decreased after longer times (Figure 9A), indicating that infection with VSV activates eIF5A. Then, we decided to test whether treatment with synthetic analogue double-stranded RNA (dsRNA) (Poly I:C) can also trigger the activation of eIF5A. For that, we transfected A549 or A375 cells with Poly I:C and, at different times after treatment, protein extracts were collected and analyzed by Western blot with anti-hypusine antibody. We observed that transfection with Poly I:C induced an increase in hypusination levels in both A549 (Figure 9B) and A375 (Figure 9C) cells. Altogether these results indicated that dsRNA promotes the activation of eIF5A. Results 99 Figure 9. Both stomatitis vesicular virus (VSV) infection and dsRNA stimuli induce eIF5A activation. A549 cells were infected with VSV (A, upper panel) or transfected with Poly I:C (B, left panel) and, at different times after treatment, protein extracts were analyzed by Western blot with the indicated antibodies. Hypusinated eIF5A and eIF5A protein intensity bands from three biological replicates were quantified using Image J software. The hypusinated eIF5A/ total eIF5A ratios in cells infected with VSV (A, lower panel) or transfected with Poly I:C (B, right panel) were plotted. The hypusinated eIF5A/ total eIF5A ratios were plotted. Data represent the mean and error bars of three biological replicates. C, A375 cells were transfected with Poly I:C and, at different times after treatment, protein extracts were analyzed by Western blot with the indicated antibodies. Statistical analysis was assessed by a Student’s t-test. *,p<0. 05, **,p<0.01, ***,p<0.005. 6.1.3 Activation of eIF5A is modulated by NF-kB In order to identify the mechanisms underlying the induction of hypusination in response to virus infection, we evaluated the transcription of different genes involved in the polyamine or hypusination pathways in cells infected with VSV or IAV using RT- qPCR. A significant increase in mRNAs for ornithine decarboxylase (ODC) and spermidine/spermine-N(1)-acetyltransferase (SSAT) were detected in both VSV and IAV infected cells (Figure 10A and Figure 10B). Different reports demonstrated that ODC and SSAT are targets of NF-κB (Casero and Pegg, 2009; Choi et al., 2006; Tacchini, 2004). Therefore, we hypothesized that the activation of eIF5A by virus ROCÍO SEOANE ABELENDA 100 infection could be mediated by the NF-κB pathway. In order to test this hypothesis, we first analyzed the hypusine levels in A549 cells treated with the NF-κB inhibitor BAY 11-7082 or after p65 depletion, by Western blot analysis. The levels of hypusine in cells treated with the NF-κB inhibitor or depleted of p65 were lower than in control cells Figure 10. Modulation of the hypusination pathway by NF-κB. A, A549 cells were infected with VSV (A) or IAV (B) and, at the indicated times after infection, RNA was analyzed by Q RT-PCR. Data represents the normalized fold change of three replicates of infected cells in comparison with the mock-infected cells. C, A549 cells were treated or not with the NF-kB inhibitor BAY 11-7082 and at 12 h after treatment, protein extracts were analyzed by Western blot with anti-hypusine antibody (left panel). Hypusinated eIF5A and eIF5A protein intensity Results 101 bands from three biological replicates were quantified using Image J software. The hypusinated eIF5A/ total eIF5A ratios in cells treated with BAY 11-7082 were plotted (right panel). D, A549 cells were transduced with sh-p65 and after 48 h transduced cells were selected by incubating with puromycin for 3 days. Then, protein extracts were analyzed by Western blot with antihypusine antibody (left panel). Hypusinated eIF5A and eIF5A protein intensity bands from three biological replicates were quantified using Image J software. The hypusinated eIF5A/ total eIF5A ratios in the cells with downmodulated p65 were plotted (right panel). Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *,p<0.05, **,p<0.01, ***,p<0.005. D, A549 cells were treated or not with the NF-kB inhibitor BAY 11-7082 or IKK16 for x h and then, cells were transfected with Poly I:C. At the indicated times after treatment, protein extracts were analyzed by Western blot with anti-hypusine antibody. (Figure 10C and 10D), indicating that the NF-κB pathway modulates hypusination in unstimulated cells. Finally, we analyzed the hypusine levels in cells treated or not with the NF-κB inhibitors IKK16 or BAY 11-7082 and then transfected with Poly I:C for the indicated times. The increase in hypusine levels observed after poly I:C treatment in cells untreated with NF-κB inhibitors, was not detected in those cells treated with IKK16 or BAY 11-7082 (Figure 10E). Altogether these results suggest that NF-κB is a positive regulator of eIF5A hypusination under both basal and dsRNA-stimulated conditions. 6.1.4 Inhibition of eIF5A hypusination impairs the replication of IAV It has been previously demonstrated that the activation of eIF5A is essential for the replication of ebola virus, marburg virus, and human immunodeficiency virus type 1 (HIV-1) (Hauber et al., 2005; Olsen et al., 2016; Ruhl et al., 1993; Schroeder et al., 2014). In addition, here we demonstrated that IAV induces hypusination. Therefore, we hypothesized that eIF5A can play a role on IAV. To evaluate this hypothesis, we first studied whether the treatment with the spermidine analogue and competitive deoxyhypusine synthase (DHS) inhibitor N1- guanyl-1,7-diamine-heptane (GC7), which inhibits the activation of eIF5A, had an impact on IAV replication. A549 cells were incubated with GC7 20μM or dimethylsulfoxide (DMSO) overnight and then the ROCÍO SEOANE ABELENDA 102 cells were infected with IAV at an moi of 5. At 24 h after infection cellular supernatants were collected for titration of infectious virus by plaque assay. Treatment with GC7 resulted in a significant decrease in the viral titer (Figure 11A). Then, A549 cells treated with DMSO or GC7 20μM overnight were infected with IAV at an moi of 5 and, at the indicated times after infection, protein extracts were collected and analyzed by Western blot. We observed a clear decrease in the M1 protein levels in those cells incubated with GC7 in comparison with the DMSO-treated cells (Figure 11B). To analyze whether GC7 treatment also decreased the IAV RNA transcription, we infected cells previously treated or not with GC7 and, at the indicated times after infection, cells were collected, total RNA was isolated, and mRNA of IAV transcripts was quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). We observed a significant reduction in IAV transcripts in the cells treated with GC7 (Figure 11C). Finally, we evaluated the impact of eIF5A depletion, by using small interfering RNA (siRNA) targeting eIF5A (sieIF5A) or negative control siRNAs (siControl), on both the synthesis of IAV proteins and IAV titers. We detected a clear reduction in the levels of the IAV NS1 protein in the cells depleted from eIF5A in comparison with the siC transfected cells (Figure 11D). A significant decrease in the viral titer was also observed in those cells depleted from eIF5A compared to control cells (Figure 11E). Altogether these results demonstrated that activation of eIF5A is essential for efficient IAV replication. Results 103 Figure 11. Effect of eIF5A inhibition on influenza virus replication. A, Effect of hypusine inhibition on IAV replication. A549 cells treated with GC7 or DMSO were infected with IAV and 24 h after infection viral titers in the supernatants were analyzed by plaque assay. Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *,p<0.05, **,p<0.01, ***,p<0.005. B, Consequences of GC7 treatment on IAV protein synthesis. A549 cells treated with GC7 or DMSO were infected with IAV and, at ROCÍO SEOANE ABELENDA 104 the indicated times after infection viral synthesis of viral proteins were analyzed by Western blot. C, Consequences of hypusine inhibition on IAV gene transcription. A549 cells treated with DMSO or GC7 were infected with IAV and at the indicated times after infection total RNA was purified and mRNA levels of viral genes were analyzed using qRT-PCR. Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *,p<0.05, **,p<0.01, ***,p<0.005. D, Effect of eIF5A downmodulation on NS1 IAV protein expression. A549 cells were transfected with siRNA against eIF5A (sieIF5A) or siRNA control (siC) and 72 h after transfection, cells were infected with IAV and at different times after infection the synthesis of viral proteins was analyzed by Western blot. F, Effect of eIF5A downmodulation on IAV replication. A549 cells transfected with siRNA against eIF5A (sieIF5A) or siRNA control (siC) were infected with IAV and 24 h after infection the viral titers in the supernatants were quantified by plaque assay. Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *,p<0.05, **,p<0.01, ***,p<0.005. 6.1.5 Inhibition of eIF5A hypusination impairs the replication of VSV Here we showed that eIF5A activation is essential for IAV replication. We then decided to evaluate whether eIF5A is also essential for the replication of VSV. First, A549 treated with GC7 or DMSO were infected with VSV at an moi of 1 and 24 h after infection, cellular supernatants were collected and analyzed by plaque assay. A significant reduction in the viral titer (up to 2-log decrease) was detected in the supernatant from GC7 treated cells (Figure 12A), indicating that hypusination is essential for efficient VSV replication. Then, A549 treated with GC7 or DMSO were infected with a recombinant VSV expressing green fluorescent protein (GFP) at an moi of 1 or 0.1 and 12 h after infection cells were fixed, analyzed by fluorescence microscopy, photographed, and then collected for fluorescence-activated cell sorting analysis. A picture of the infected cells can be seen in figure 12B (upper panel). The percentage of GFP-positive cells in GC7 treated cells was significantly lower than in DMSO treated cells (Figure 12B, lower panel). Metabolic labeling with 35S-methionine of A549 cells treated or not with GC7 and infected with VSV at an moi of 5 was then carried out. For that, cells were washed and incubated with methionine-free DMEM for 1 hour. Then, cells were infected with VSV and 35S- Results 111 ROCÍO SEOANE ABELENDA 112 Figure 14. Inhibition of eIF5A induces both ER stress and transcriptional transactivation of IFN and IFN-stimulated genes. A549 cells were treated with GC7 and at different times after treatment protein extracts were analyzed by Western blot with anti-phosphorylated ERK (A) or anti-phosphorylated eIF2α (B) antibodies. C, A549 cells were treated with increasing doses of GC7 and 24 h after treatment protein extracts were analyzed by Western blot with antiphosphorylated eIF2α antibody. D, A549 cells were transfected with smart-pool small interfering RNAs (siRNAs) against eIF5A1 (sieIF5A) or non-targeting siRNAs (siC). 72 h after transfection, cells were transfected with Poly I:C and at different times after transfection protein extracts were analyzed by Western blot with anti-phosphorylated eIF2α antibody. Total RNA purified from A549 cells treated with GC7 for 24 h (E) or transfected with siC or sieIF5A for 72 h (G) were analyzed by qRT-PCR. and represented as fold induction over untreated cells. F, Total A549 cells were treated with IKK16 for 16 h and then treated with GC7 or DMSO for 24 h. Total RNA was purified and was quantified by real-time RT-PCR and represented as fold induction over untreated cells. Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *,p<0.05, **,p<0.01, ***,p<0.005. To test whether eIF5a modulates the levels of p85β, we carried out a Western blot analysis of the human A549 or SW480 cells treated with GC7 or DMSO using anti-hypusine antibody. We observed a clear downmodulation in p85β protein levels after GC7 treatment (Figure 15B). We then analyzed the transcription of p85β in A549 cells treated with GC7 or DMSO by qRT-PCR. We did not observe significant different differences in the levels of the mRNA for p85β (Figure 15C). Altogether these results suggest that p85β can be a target of eIF5A. We then decided to study whether mouse p85β protein levels are also modulated by eIF5A. HEK-293 cells were transfected with a plasmid encoding HA-tagged mouse p85β and 24h after transfection cells were treated with DMSO or GC7, and 24 h after treatment we analyzed the protein extracts by Western blot with anti-HA antibody. We did not observe differences in the levels of HA-p85β protein between DMSO and GC7 treated cells (Figure 15D). Additional experiments are required in order to confirm the regulation of human p85β by eIF5A in the context of virus infection. Results 113 Figure 15. Human p85β is a potential target of eIF5A. A, In silico analysis of the amino acid sequence of human and mouse p85β, B, Human A549 or SW480 cells were treated with DMSO or GC7 and 24 h after treatment protein extracts were analyzed by Western blot with anti-p85β antibody. C, A549 cells were treated with DMSO or GC7 and 24 h after treatment mRNA levels of p85β were analyzed by qRT-PCR. D, HEK-293 cells were transfected with a plasmid encoding HA-tagged mouse p85b and 24 h after transfection cells were treated with DMSO or GC7. 24 h after treatment protein extracts were analyzed by Western blot with anti-HA antibody. 6.1.9 eIF5A1 is modified by SUMO2 in vitro The expression of eIF5A has been found altered in different pathological conditions (Chen et al., 2003, 2009; Fujimura et al., 2014; Guan et al., 2004; He et al., 2011; Luo et al., 2009; Meng et al., 2015; Xie et al., 2008; Yang et al., 2009), and a correlation between the protein and the mRNA expression levels has not been always found. Therefore, it has been proposed that eIF5A can be regulated by additional mechanisms. We hypothesized that eIF5A may be post- ROCÍO SEOANE ABELENDA 114 translationally regulated by ubiquitin-like proteins. Firstly, we tested whether eIF5A1 can be modified by SUMO in vitro. For that, we used 35S-methionine labeled in vitro translated HA-eIF5A1. The protein was incubated in a SUMOylation reaction with recombinant SUMO2 protein, the E1 SUMO-activating enzyme 1 (Sae1), and the E2 Ubc9. 3 h later, the reaction was stopped, the products were run in an SDSPAGE and the dried gel was then exposed to a X-ray film. We observed a band of around 20 kDa molecular weight corresponding to unmodified HA-eIF5A1, and the appearance of a band of the expected eIF5A1-SUMO2 molecular weight only when SUMO2 was added to the reaction, indicating that eIF5A1 can be modified by SUMO2 in vitro (Figure 16A). To further demonstrate that the higher molecular weight band correspond with SUMOylated eIF5A1, we carried out a deSUMOylation assay. eIF5A1 subjected to an in vitro SUMOylation assay was incubated with SUMO specific peptidase 1 (SENP1), an enzyme that catalyzes the de-conjugation of SUMO to the target proteins, and the samples were then processed as described above. As shown in Figure 16B, the band with the expected eIF5A1-SUMO2 molecular weight disappeared after incubation with SENP1, confirming that eIF5A1 can be modified by SUMO2 in vitro. Results 115 Figure 16. In vitro SUMOylation of eIF5A1. A, In vitro SUMOylation assay with SUMO2 using 35S-methionine labeled in vitro translated HA-eIF5A1 protein as a substrate. Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates the SUMO2-conjugated eIF5A1 protein. B, In vitro deSUMOylation assay using recombinant SENP1. Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates the SUMO2-conjugated eIF5A1 protein. 6.1.10 eIF5A1 is modified by SUMO2 in vivo To determine whether eIF5A1 can be modified by SUMO2 in cells, we transfected HEK-293T cells with HA-eIF5A1 together with pcDNA or pCDNA-His6-SUMO2 and pCDNA-Ubc9-V5. 36 h after transfection, cells were collected, lysed under denaturing conditions, and subjected to histidine-purification assays. Analysis of the purified histidinetagged proteins by Western blot with anti-HA antibody showed a band corresponding with the unmodified HA-eIF5A1 protein in both lanes. In addition, we observed a strong band of around 40 kDa as well as a fainter 60 kDa molecular weight band in the cells co-transfected with the SUMO machinery, indicating that eIF5A1 is modified by SUMO2 in cells (Figure 17A). Similar results were observed when the experiment was carried out using Flag-eIF5A1 plasmid (Figure 17B). We then studied the SUMOylation of eIF5A1 in endogenous conditions. SUMOylation is a reversible and very dynamic process, and the levels of modified substrate at a given time is limited. Therefore, we used SUMO-traps (SUBES) to capture endogenous SUMOylated proteins. HEK-293T cells were lysed in the presence of ubiquitin-like isopeptidases inhibitors. Lysates were clarified by centrifugation and the supernatants were incubated with GST-agarose beads or GSTSUBES-agarose beads for 2 h at 4º C. Then, the beads were washed and the bound proteins were eluted using Laemmli buffer. Samples were then analyzed by Western blot with anti-eIF5A1 or anti-SUMO2 antibodies. Western blot analysis using anti-eIF5A1 antibody revealed the presence of two bands of around 35 and 55 kDa only in the sample incubated with SUBES (Figure 17C). Altogether these results demonstrate that eIF5A1 is modified by SUMO2 in cells. ROCÍO SEOANE ABELENDA 116 Figure 17. SUMO2 modification of eIF5A1 in cells. A, HEK-293T cells were co-transfected with a plasmid encoding HA- eIF5A1 together with pcDNA, Ubc9 and His6-SUMO2 and 36 h after transfection whole protein extracts or histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrows indicate SUMO2-conjugated eIF5A1 protein. B, HEK-293T cells were co-transfected with a plasmid encoding Flag-eIF5A1 together with pcDNA or Ubc9 and His6-SUMO2 and 36 h after transfection whole protein extracts or histidine-tagged purified proteins were analyzed by Western blot with anti-Flag antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrows indicate SUMO2-conjugated eIF5A1 protein. C, Analysis of the endogenous SUMOylation of eIF5A1. SUMOylated proteins in HEK-293 cells were captured using SUBES and analyzed by Western blot using anti-eIF5A1 antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrows indicate SUMO2-conjugated eIF5A1 protein. 6.1.11 eIF5A2 is modified by SUMO2 eIF5A1 and eIF5A2 share large sequence homology, and most of the lysine residues are conserved between the two isoforms (Figure 18A). Therefore, we speculated that eIF5A2 may be also modulated by SUMO2. To test this hypothesis, we carried out an in vitro and in vivo SUMOylation assays using 35S-methionine labeled in vitro translated Flag-eIF5A2 protein and Flag-eIFA2 expression plasmid, respectively. Results 117 We observed that eIF5A2 was modified by SUMO2 in vitro (Figure 18B) and in vivo (Figure 18C). 35S-methionine labeled in vitro translated Flag-eIF5A2 protein was incubated in a SUMOylation reaction with recombinant SUMO2 protein, the E1 SUMO-activating enzyme 1 (Sae1), and the E2 Ubc9. 3 h later, the reaction was stopped, the products were run in an SDS-PAGE and the dried gel was then exposed to a X-ray film. We observed a band of around 20 kDa molecular weight corresponding to unmodified Flag-eIF5A2, and the appearance of a band of the expected eIF5A1-SUMO2 molecular weight only when SUMO2 was added to the reaction, indicating that eIF5A1 can be modified by SUMO2 in vitro (Figure 18B). To test the result in mammalian cells, HEK-293T were transfected cells with FlageIF5A2 together with pcDNA or pCDNA-His6-SUMO2 and pCDNA- Ubc9-V5. 36 h after transfection, cells were collected, lysed under denaturing conditions, and subjected to histidine-purification assays. Analysis of the purified histidine-tagged proteins by Western blot with anti-Flag antibody shows a band corresponding with eIF5A2-SUMO2 (Figure 18C). 6.1.12 eIF5A1 is modified by SUMO2 in a hypusineindependent manner eIF5A1 protein is modified by hypusine at lysine K50 readily after translation. Therefore, we decided to analyze whether lysine K50 is required for eIF5A1 SUMOylation. We first analyzed whether a mutant of eIF5A1 in lysine 50 (eIF5A-K50R) was still SUMOylated. We cotransfected HEK-293T cells with HA-eIF5A1-K50R together with pcDNA or pCDNA-His6-SUMO2 and pCDNA-Ubc9-V5. 36 h after transfection, cells were collected, lysed under denaturing conditions and subjected to histidine-purification assays. ROCÍO SEOANE ABELENDA 118 Figure 18. eIF5A2 is modified by SUMO2. A, Alignment of the amino acid sequences of the two isoforms of eIF5A using CLUSTAL O(1.2.4). B, In vitro SUMOylation assay with SUMO2 using 35S-methionine labeled in vitro translated eIF5A2 protein. Arrowhead indicates unconjugated eIF5A2 protein. Arrow indicates SUMO2-conjugated eIF5A2 protein. B, HEK- 293T cells were transfected with a plasmid encoding Flag-eIF5A2 together with pcDNA or Ubc9 and His6-SUMO2 and 36 h after transfection whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-Flag antibody. Arrowhead indicates unconjugated eIF5A2 protein. Arrow indicates SUMO2-conjugated eIF5A2 protein. Analysis of the purified proteins by Western blot with anti-HA antibody revealed a band of around 40 kDa molecular weight corresponding with the SUMO2 modified eIF5A1-K50R protein only in the cells cotransfected with the SUMOylation machinery (Figure 19A), indicating that the eIF5A-K50R hypusination mutant is modified by SUMO2 in cells. We then analyzed the potential SUMO2 modification of eIF5A1- K50R in vitro. 35S-methionine labeled in vitro translated eIF5A1-WT or eIF5A-K50R proteins were subjected to an in vitro SUMOylation Results 119 assay in the presence of SUMO2. The resulting products were analyzed by SDS-PAGE and the dried gel was exposed to a X-ray film. Incubation with the SUMOylation machinery led to the appearance of a 40 kDa molecular weight band corresponding with SUMO2 modified eIF5A1 or eIF5A1-K50R protein (Figure 19B). Altogether these results indicate that eIF5A1 is SUMOylated in a hypusination-independent manner. 6.1.13 SUMO2 can be conjugated to different lysine residues in eIF5A1 To understand the relevance of SUMO conjugation to a specific target protein, a straightforward approach is to identify the lysine residue(s) involved in SUMO conjugation and then to analyze the properties of the SUMOylation mutant in comparison with the wild type protein. Proteomic data previously published pointed to lysine residues K27, K34, K39, K67, K85, and K126 as probably SUMOylation sites in eIF5A1 (Hendriks and Vertegaal, 2016). In addition, using GPS-SUMO software (Zhao et al., 2014), we identified three 3 highly scored potential SUMOylation sites (K126, K68, and K85). Next, we generated mutants of eIF5A1 in the lysine residues with higher probability to be SUMOylation sites, and then we evaluated their SUMOylation in cells. ROCÍO SEOANE ABELENDA 120 Figure 19. SUMOylation of the non hypusinable mutant of eIF5A1, eIF5A1-K50R. A, HEK-293T cells were co-transfected with a plasmid encoding HA-eIF5A1-K50R together with pcDNA or Ubc9 and His6-SUMO2 and 36 h after transfection whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates SUMO2-conjugated eIF5A1 protein. B, In vitro SUMOylation assay with SUMO2 using 35S-methionine labeled in vitro translated eIF5A1-K50R or eIF5A-WT protein as a substrate. Arrowhead indicates unconjugated eIF5A1-K50R or eIF5A1-WT protein. Arrow indicates SUMO2-conjugated eIF5A1 or eIF5A-K50R protein. Results 127 pcDNA or Ubc9 and His6-SUMO2 and 36 h after transfection cells were treated with DMSO or MG132 for 4 h. Then, cells were lysed and histidine-tagged proteins were purified under denaturing conditions. Western blot analysis of the purified proteins with antibody against HA revealed the appearance of a band or around 40 kDa, corresponding with eIF5A1-SUMO2 protein in the cells co-transfected with the SUMOylation machinery and treated with DMSO (Figure 23A). Higher levels of the 40 kDa protein and additional higher molecular weight bands corresponding with eIF5A-SUMO2 protein were observed in those cells treated with MG132 (Figure 23A). It has been previously reported that inhibition of the proteasome induces the accumulation of SUMO2/3 conjugates in vivo (Schimmel et al., 2008; Uzunova et al., 2007) and part of these conjugates can represent ubiquitin associated SUMO2/3 conjugates. Therefore, we speculated that MG132 may induce the formation of heterologous SUMO2/3-ubiquitin chains. To explore this hypothesis, we then generated a mutant of SUMO2 in all the lysine residues (SUMO2-K0) and then we evaluated the SUMOylation of eIF5A1 in cells transfected with HA-eIF5A1 and pcDNA or Ubc9 and His6-SUMO2-K0 and treated or not with MG132. Western blot analysis of the histidine-tagged proteins purified under denaturing conditions using anti-HA antibody revealed the appearance of two bands of around 40 kDa and 80 kDa molecular weight in the untreated cells co-transfected with the SUMOylation machinery (Figure 23B). A clear increase in the levels of the 40 kDa band as well additional bands were detected in those cells treated with MG132 (Figure 23B). Altogether these results indicated that inhibition of proteasome promoted the SUMOylation of eIF5A1 independently of the formation of SUMO2 chains or of hybrid chains composed of SUMO2 and ubiquitin. ROCÍO SEOANE ABELENDA 128 Figure 23. Proteasome inhibition induces eIF5A1 SUMOylation. A, HEK-293T cells were transfected with a plasmid encoding HA-eIF5A1 together with pcDNA orUbc9 and His6- SUMO2, and 36 h after transfection cells were treated with MG132 for 4 hours. Whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. B, HEK-293T cells were transfected with a plasmid encoding HA- eIF5A1 together with pcDNA, Ubc9 and His6-SUMO2 or Ubc9 and His6-SUMO2-K0 and 36 h after Results 129 transfection cells were treated with MG132 for 4 hours. Whole protein extracts and histidinetagged purified proteins were analyzed by Western blot with anti-HA antibody. The regulation of protein translation allows cellular adaptation during stress conditions by adjusting the proteome without the necessity of changes in RNA synthesis. Although eIF5A has been reported to only marginally affect protein synthesis under normal conditions, it is essential for the onset of stress-induced translational repression. In addition, SUMOylation status of different substrate proteins has been reported to play a crucial role in cellular responses to stress. We then decided to study whether the SUMOylation of eIF5A1 is modulated in response to other types of stress. First, we analyzed the effect of UV light on eIF5A1 SUMOylation. HEK-293T cells were transfected with HA-eIF5A1 and pcDNA or Ubc9 and His6-SUMO2 and 36 h after transfection cells were subjected to irradiation of UV light and cells were collected at two times after irradiation. Western blot analysis of the histidine-tagged proteins purified under denaturing conditions using anti-HA antibody revealed that the 40 kDa band corresponding with eIF5A1-SUMO2 protein detected in the untreated cells disappeared after UV light irradiation (Figure 24A). Then, we analyzed the potential modulation of eIF5A1 SUMOylation in response to hypoxic or adriamycin treatment. HEK-293T cells were transfected as described above and at 36 h after transfection cells were subjected to hypoxic conditions or treated with adriamycin. Finally, the cells were recovered and the histidine-tagged proteins were purified under denaturing conditions. Analysis by Western blot of the purified proteins revealed similar levels of eIF5A-SUMO2 protein in untreated cells and in cells subjected to hypoxic stress (Figure 24B). We also observed a reduction in the levels of the eIF5A-SUMO2 band in those cells treated with adriamycin (Figure 24B). Altogether these results indicated that treatment with DNA-damage agents downmodulated eIF5A SUMOylation. ROCÍO SEOANE ABELENDA 130 Figure 24. SUMOylation of eIF5A in response to UV light irradiation, adriamycin, or hypoxic conditions. A, HEK-293T cells were transfected with a plasmid encoding HA-eIF5A1 together with pcDNA or Ubc9 and His6-SUMO2, and 36 h after transfection cells were subjected to UV light irradiation. Whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates SUMO2-conjugated eIF5A1. B, HEK-293T cells were transfected with a plasmid encoding HA-eIF5A1 together with pcDNA or Ubc9 and His6- SUMO2, and 36 h after transfection the cells were subjected to hypoxic conditions or treated with adriamycin. Whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates SUMO2-conjugated eIF5A1. Results 131 Next, we decided to evaluate whether virus infection had an impact on eIF5A SUMOylation. For that, we carried out similar experiments to the above described, but instead of treating the cells with cytotoxic agents, we infected the cells with VSV (Figure 25A) or IAV (Figure 25B) for 8 h. Western blot analysis with anti-HA antibody of the histidine-tagged purified proteins showed an increase in the levels of the eIF5A-SUMO2 protein in the infected cells, indicating that virus infection induces eIF5A SUMOylation. Figure 25. Modulation of eIF5A1 SUMOylation by viral infection. HEK-293T cells were were co-transfected with a plasmid encoding HA-eIF5A1 together with pcDNA or Ubc9 and His6-SUMO2. 36 h after transfection, cells were infected with VSV (A) or IAV (B) for 8 h. Whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates SUMO2-conjugated eIF5A1. Finally, we evaluated the potential modulation of eIF5A1 SUMOylation in response to heat shock. Cells were transfected as described above and 48 h after transfection, cells were subjected or not to heat shock stress. Whole cell extracts and histidine-tagged purified proteins were then analyzed by Western blot with anti-HA antibody. ROCÍO SEOANE ABELENDA 132 We observed a clear increase in the intensity of the 40 kDa SUMOylation band in addition to the appearance of additional SUMOylation bands in those cells subjected to heat shock stress (Figure 26A), indicating that heat stress induces the SUMOylation of eIF5A1. It has been reported that eIF5A is required for stress granules assembly in cells subjected to adverse environmental conditions and for the rapid onset of stress-induced translational repression. We then evaluated the dynamics of eIF5A-SUMO2 conjugation after heat shock. Cells were transfected as above described and at 36 h after transfection, we subjected the cells to heat shock. Cells were processed immediately after heat shock or they were incubated at 37º C for 30 min or 2 h. Then, we collected the cells and analyzed them as above indicated. Analysis by Western blot of the histidine-tagged purified proteins using anti-HA antibody showed increased levels of eIF5A-SUMO2 protein immediately and 30 minutes post-heat shock, and a complete return to basal SUMOylation levels was observed at 2 h of recovery at 37º C (Figure 26B). Altogether these results indicated that eIF5A SUMOylation is modulated in response to different types of stress. In agreement with our results, different proteomic data previously reported have identified eIF5A as a SUMO target which SUMOylation increases following proteasome inhibition or heat shock stress (Golebiowski et al., 2009; Hendriks et al., 2014). These results also suggested that SUMOylation sites in eIF5A may be modulated by stress conditions. To test this hypothesis, we evaluated the SUMOylation of the eIF5A-3KA mutant in cells subjected to heat shock. HEK-293T cells were co-transfected with HA-eIF5A1-3KA and pcDNA or Ubc9 and His6-SUMO2 and 36 h after transfection, the cells were incubated at 43º C or left at 37º C for 2 h. Then, cells were collected and the histidine-tagged proteins were purified under denaturing conditions. Western blot analysis of the purified proteins using anti-HA antibody did not reveal a band corresponding to eIF5A-3KR-SUMO2 protein in the untreated cells (Figure 27). Results 133 Figure 26. Induction of eIF5A1 SUMOylation by heat shock stress. A, HEK-293T cells were co-transfected with a plasmid encoding HA-eIF5A1 together with pcDNA or Ubc9 and His6-SUMO2. 36 h after transfection, the cells were incubated at 43º C for 2 h. Whole protein extracts and histidine-tagged purified proteins were then analyzed by Western blot with anti- HA antibody. Arrowhead indicates unconjugated eIF5A1 protein.. B, HEK-293T cells were were transfected with a plasmid encoding HA-eIF5A1 together with pcDNA or Ubc9 and His6- SUMO2. 36 h after transfection, the cells were incubated at 43º C for 2 hours. Cells were analyzed immediately or incubated at 37º C for 30 min of 2 h. Whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates unconjugated eIF5A1 protein. ROCÍO SEOANE ABELENDA 134 However, we detected a 40 kDa band corresponding to the SUMOylated eIF5A-3KR protein in the cells subjected to heat shock, indicating that the SUMO acceptor sites on eIF5A are regulated by stress. Figure 27. SUMO acceptor sites in eIF5A1 are regulated by heat shock stress. HEK-293T cells were co-transfected with a plasmid encoding HA-eIF5A1-3KA together with pcDNA or Ubc9 and His6-SUMO2. 36 h after transfection, the cells were incubated at 43º C for 2 hours. Whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates the SUMO2-conjugated eIF5A1-3KA. Results 135 6.1.17 Conjugation of SUMO to lysine residues K68, K85, and K126 on eIF5A is essential for yeast survival The reduced stability of eIF5A mutants in the SUMOylation sites in mammalian cells and the induction of eIF5A SUMOylation in response to proteasome inhibition difficult to evaluate the functionality of the SUMOylation mutants in mammalian cells. eIF5A is essential for cell growth in yeast and human eIF5A has been demonstrated to complement the inviability of a yeast strain in which the yeast eIF5A genes were disrupted (Schnier et al., 1991b). Therefore, we decided to study the functionality of a mutant of eIF5A1 in the SUMOylation sites in yeast (Saccharomyces cerevisiae) in collaboration with Dr. Miguel González Blanco lab (CiMUS). First, we analyzed the potential SUMOylation of eIF5A-WT in yeast. For that, yeast cells containing an empty vector (His-Hyp2-3-empty) or yeast cells with histidine-tagged human eIF5A1-WT (His-Hyp2-3 WT) were grown at 25º C and histidine-tagged proteins were purified under denaturing conditions. Western blot analysis of purified proteins using anti-SMT3 antibody revealed the presence of a band of around 40 kDa, likely corresponding with SUMO modified eIF5A1 protein (Figure 28A), indicating that eIF5A1 protein is modified by SUMO in yeast. Then, thermosensitive yeast mutant strains hyp2-3 (C39Y, G118D) and hyp2-1 (P83S) were transformed with plasmids encoding human eIF5A1-WT, eIF5A1-3KA or the hypusination deficient eIF5A-K50R, under the control of the constitutive GPD promoter and assessed their viability at 37º C. Both the WT and mutants of eIF5A exhibited normal growth at 25º C (Figure 28B). However, only the WT strain as well as the hyp2 strain harbouring the eIF5A-WT protein could proliferate at the restrictive temperature (Figure 28B), despite we detected expression of all the proteins at both permissive and restrictive temperatures (Figure 28C). These results suggested that SUMOylation of human eIF5A1 is essential for complementing HYP2 function in yeast. To further probe this hypothesis, we decided to evaluate the SUMOylation of eIF5A- 3KA in yeast. Histidine-tagged proteins purified under denaturing ROCÍO SEOANE ABELENDA 136 conditions from yeast cells expressing histidine-tagged eIF5A-WT or eIF5A-3KA were analyzed by Western blot using anti-SMT3 antibody. We observed that the SUMOylation of eIF5A-3KA at 25º C was reduced in comparison with the WT protein (Figure 28D). Altogether these results suggest that SUMOylation of eIF5A at lysine residues K68, K85 and K126 is essential for complementing HYP2 function in yeast. Results 143 6.1.20 Evaluation of potential NEDDylation sites in eIF5A. In order to evaluate whether hypusination is required for NEDDylation of eIF5A, we evaluated the conjugation of the hypusination mutant HA-eIF5A-K50R to NEDD8 in cells. HEK-293T cells were co-transfected with HA-eIF5A-K50R and pcDNA or His6- NEDD8 and 48 h after transfection, histidine-tagged proteins were purified under denaturing conditions and analyzed by Western blot with anti-HA antibody. We observed the appearance of a band corresponding with NEDD8 conjugated to eIF5A-K50R in the cells expressing the NEDDylation machinery (Figure 32A), indicating that NEDDylation of eIF5A1 does not require its hypusination. Proteomic data pointed to lysine K27 as involved in NEDD8 conjugation. Therefore, we studied whether mutation of lysine K27 in eIF5A inhibits its SUMOylation. HEK-293T cells were co-transfected with HA- eIF5A-WT or HA-eIF5A-K27R and pcDNA or His6-NEDD8 and 48 h after transfection we evaluated the presence of NEDDylated eIF5A1 bands in the histidine-tagged purified extracts. The levels of the eIF5A1-WT-NEDD8 band were similar to the levels of the eIF5A- K27R-NEDD8 band (Figure 32B), indicating that other lysine residues in eIF5A can conjugate NEDD8. Finally, we decided to evaluate whether mutation of the lysine residues in eIF5A1 involved in SUMO conjugation altered its NEDDylation. HEK-293T cells were cotransfected with HA-eIF5A1-WT, HA-eIF5A1-3KA, or HA-eIF5A- 5KA together with pcDNA or His6-NEDD8 and 36 h after transfection cells were treated with MG132. Cell extracts were recovered and lysed under denaturing conditions and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. The levels of the bands corresponding with NEDD8 conjugated to the eIF5A SUMOylation mutants were higher than the levels of the eIF5A1-WT- NEDD8 protein (Figure 32C), suggesting an interplay between NEDDylation and SUMOylation. To further evaluate this interplay, we studied the effect of inhibiting SUMOylation on eIF5A1 NEDDylation as well as the effect of inhibiting NEDDylation on eIF5A ROCÍO SEOANE ABELENDA 144 SUMOylation. For that, cells were co-transfected with HA-eIF5A1 and pcDNA, His6-NEDD8 or Ubc9 and His6-SUMO2 and 36 h after transfection cells were treated with the SUMOylation inhibitor ML792 or the NEDDylation inhibitor MLN4924, as indicated. At 48 h after transfection we recovered the cells, purified the histidine-tagged proteins and analyzed them by Western blot with anti-HA antibody. We observed a small increase in the levels of eIF5A SUMOylation after MLN4924 treatment (Figure 32D) and an increase in NEDDylation in those cells treated with the SUMOylation inhibitor ML792 (Figure 32E), confirming the existence of an interplay between SUMO and NEDD8 conjugation to eIF5A. Figure 32. Interplay between NEDD8 and SUMO2 on eIF5A1. HEK-293T cells were transfected with HA-eIF5A1-50KR (A) or with HA-eIF5A1-WT or HA-eIF5A1-K27R (B) in the presence or absence of His6-NEDD8. 36 h after transfection whole cell extracts and histidine-tagged purified proteins were analyzed by Western blot using anti-HA antibody. C, Results 145 HEK-293T cells were co-transfected with HA-eIF5A1-WT, HA-eIF5A1-3KA or HA-eIF5A1- 5KA together with pcDNA or His6-NEDD8 and 24 h after transfection cells were treated with MG132. 16 h after treatment whole cell protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates NEDD8-conjugated eIF5A1 protein. D, HEK-293T cells were transfected with a plasmid encoding HA-eIF5A1-WT together with pcDNA or His6-SUMO2 and 24 h after transfection cells were treated or not with MLN4924. 16 h after treatment whole cell protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. E, HEK-293T cells were transfected with a plasmid encoding HA-eIF5A1- WT together with pcDNA or His6-NEDD8 and 24 h after transfection cells were treated or not with ML792 and 16 h after treatment whole protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody (right panel). Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates NEDD8-conjugated eIF5A1 protein. 6.1.21 Inhibition of NEDDylation stabilizes eIF5A To determine the impact of NEDDylation on eIF5A1, we evaluated the effect of the NEDDylation inhibitor MLN4924 on eIF5A1 stability. HEK-293T cells were transfected with HA-eIF5A1-WT, HA-eIF5A1- 3KA or HA-eIF5A1-5KA and 36 h after transfection, cells were treated with the NEDDylation inhibitor MLN4924 for 12 h. Then, cells were treated with cycloheximide and at different times after treatment the cells were collected and the protein extracts were analyzed by Western blot with anti-HA antibody. Treatment with MLN4924 significantly increased the stability of eIF5A1-WT as well as of the eIF5A-3KA and eIF5A-5KA mutants (Figure 33). ROCÍO SEOANE ABELENDA 146 Figure 33. NEDDylation modulates the stability of eIF5A1. A, HEK-293 cells were transfected with HA-eIF5A1-WT or the SUMOylation mutants, treated overnight with MLN4924 and then treated with CHX. At the indicated times after CHX treatment, protein extracts were analyzed by Western blot with the indicated antibodies (upper panels). Stability of the protein in presence or absence of MLN4924 from three independent experiments is shown (lower panels). Statistical analysis was assessed by a Student’s t-test. 6.1.22 STUB1 promotes eIF5A1 NEDDylation eIF5A can conjugate to different ubiquitin-like proteins, including Ubiquitin and NEDD8. It has been previously demonstrated that STUB1 is an E3 Ubiquitin ligase that mediates eIF5A ubiquitination and degradation (Shang et al., 2014). Interestingly, it has been reported that STUB1 can also work as an E3 NEDD8 ligase (Yoo et al., 2018). Therefore, we decided to evaluate the potential impact of STUB1 on NEDDylation. For that, we tested the NEDDylation of eIF5A1 in cells overexpressing STUB1. We observed that higher levels of STUB1 correlated with higher levels of eIF5A1 NEDDylation (Figure 34), indicating that STUB1 can promote eIF5A1 NEDDylation. Figure 34. STUB1 promotes eIF5A1 NEDDylation. HEK-293T cells were transfected with HA-eIF5A1 and His6-NEDD8 in the presence or absence of Flag-STUB1. 36 h after transfection, whole cell protein extracts and histidine-tagged purified proteins were analyzed by Western blot with anti-HA antibody. Arrowhead indicates unconjugated eIF5A1 protein. Arrow indicates NEDD8-conjugated eIF5A1 protein. Results 147 6.2 INFLUENZA VIRUS AND NEDD8 6.2.1 Influenza virus infection induces global reprogramming of host NEDDylation IAV infection has been previously shown to induce the NEDDylation of cullins (Sun et al., 2018), probably the major NEDD8 substrate, suggesting that infection with IAV may alter NEDDylation. To evaluate this hypothesis, we infected A549 cells with influenza virus A/PR8/1934 at an moi of 5 and cells were recovered at different times after infection. Western blot analysis of the protein extracts using anti- NEDD8 antibody revealed dynamic NEDDylation changes during IAV infection (Figure 35A). Observed changes in NEDDylation could be due to transcriptional alterations in NEDDylation machinery genes. Therefore, we decided to analyze the transcript levels of NEDD8 as well as of the two NEDD8-conjugating enzymes E2 at different times of IAV infection. However, as shown in figure 35B, we did not observe significant changes in the mRNA expression levels of the analyzed genes. 6.2.2 Differentially NEDDylated proteins after IAV infection The NEDDylation changes upon IAV infection as well as the previously reported upregulation in cullin NEDDylation in IAV infected cells (Sun et al., 2018), led us to study the NEDDylated proteome in IAV infected cells. Antibodies that recognize the Lys-ϵ- Gly-Gly (diGly) remnant on ubiquitinated proteins after trypsin digestion are used for the quantitation and identification of ubiquitinated proteins as well as for mapping of endogenously modified lysines by quantitative mass spectrometry (Xu et al., 2010). However, trypsin digestion generates a similar diGly remnant in NEDDylated, ubiquitinated and ISGylated proteins, therefore this approach cannot distinguish between these modifications (Wagner et al., 2011) (Figure 36A). To identify the NEDDylated proteome, and in collaboration with Dimitris Xirodimas (CNRS), we used a protocol developed by Vogl et al. based on the use of a NEDD8-R74K mutant together with the anti- ROCÍO SEOANE ABELENDA 148 diGly antibody, and using Lys-C protease, that cleaves at the carboxyl side of the lysine (Vogl et al., 2020). Figure 35. IAV induces global reprograming of host NEDDylation. A, A549 cells were infected with IAV (at an moi of 5), collected at the indicated times after infection and analyzed by Western blot using anti-NEDD8 antibody. B, A549 cells were infected with IAV as indicated above and at the indicated times after infection, the transcript levels of NEDD8, the two NEDD8-conjugating enzymes E2 and two different IAV genes were analyzed by Q RT-PCR and represented as fold induction over mock-infected cells. Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *, P<0.05; **, P<0.005, ***P<0.001. Results 149 Treatment with this protease will specifically generate diGly remnants only in NEDD8 modified proteins but not ISGylated or ubiquitylated proteins (Figure 37A) (Vogl et al., 2020). A549 were infected with lentivirus expressing His6-NEDD8-R74K protein and infected cells were then selected using puromycin. Immediately after selection, A549 cells stably expressing NEDD8-R74K were infected with IAV at an moi of 1. At 24 h after infection, cells were lysed under denaturing conditions and in collaboration with Dr. Jeffrey Johnson’s laboratory at Icahn School of Medicine at Mount Sinai, proteins were analyzed by mass spectrometry. We observed changes in the NEDDylation of 55 putative NEDD8 substrates. Gene ontology enrichment analysis of the differentially modulated proteins revealed an enrichment for proteins involved in nucleosome assembly and chromatin-related biological processes (Figure 36B), including well known NEDD8 substrates such as histone proteins (Figure 36C). We identified 14 NEDDylated protein candidates in mock-infected cells that were not detected in IAV infected cells (Figure 36D) and 9 NEDDylated protein candidates that were detected only in the infected cells (Figure 36D). 6.2.3 Validation of IAV-modulated NEDD8 targets In order to validate the mass spectrometry results, we decided to study the conjugation to NEDD8 of several NEDDylation candidates in cells infected or not with IAV. For that, HEK-293T cells were cotransfected with the plasmid encoding the protein of interest together with pcDNA or His6-NEDD8 expression plasmids. At 24 h after transfection, cells were infected with IAV at an moi of 1 and 24 h after infection cells were collected and histidine-tagged purified proteins were analyzed with the indicated antibodies. First, we evaluated the NEDDylation of two well-known NEDD8 substrates, H2B and RPS27A. In cells transfected with H2B or RPS27A and co-transfected with NEDD8 we detected the appearance of bands of the expected H2B- NEDD8 and RPS27A-NEDD8 molecular weight, respectively, ROCÍO SEOANE ABELENDA 150 Figure 36. Differentialy NEDDylated proteins after IAV infection. A, Representation of different strategies to generate diGly remnants. B, Gene ontology analysis of the differentially NEDDylated proteins detected by MS after IAV infection. C, List of proteins with Results 151 NEDDylation upregulated or downregulated after IAV infection. D, List of proteins whose NEDDylation is only detected in IAV or mock-infected cells. indicating that both proteins can be conjugated to NEDD8 (Figure 37A left panel and 37B). Infection with IAV clearly decreased the NEDDylation of the proteins (Figure 37A right panel and 37B), indicative of the negative modulation of their NEDDylation upon IAV infection. We then studied the potential NEDDylation of one of the novel NEDDylation candidates identified in this study, the voltagedependent anion channel 1 (VDAC1) protein, a regulator of mitochondrial function (and modulator of apoptosis, autophagy, and inflammation). Western-blot analysis of histidine-tagged purified proteins revealed the appearance of a band of the expected VDAC1- NEDD8 molecular weight in the cells transfected with His6-NEDD8, confirming that VDAC1 can be modified by NEDD8 (Figure 37C). An increase in the intensity of this band as well as additional bands were detected after IAV infection (Figure 37C), suggesting that VDAC1 NEDDylation increases upon IAV infection. The mass spectrometry data pointed to lysine residue K110 in VDAC1 as the acceptor site of NEDD8 in VDAC1. Therefore, we generated a mutant of VDAC1 in K110 (VDAC1-K110R). Then, we analyzed the NEDDylation of VDAC1-WT or VDAC1-K110R in HEK-293T cells transfected with His6-NEDD8. NEDDylation of VDAC1-K110R was reduced in comparison with the WT protein (Figure 37D), indicating that K110 in VDAC1 is involved in NEDD8 conjugation. Finally, we evaluated the potential impact of VDAC1 NEDDylation on IAV. For that, we transfected HEK-293T cells with VDAC1-WT or VDCA1-K110R expression plasmids. 36 h after transfection cells were infected with IAV, and at different times after infection, we analyzed the synthesis of the viral NS1 protein by Western blot analysis. Overexpression of VDAC1-WT did not alter the synthesis of NS1 in the IAV infected cells; however, we observed a clear reduction in the levels of the NS1 protein synthesized in the cells expressing VDAC1-K110R. These results suggested that NEDDylation of VDAC1 has a positive impact ROCÍO SEOANE ABELENDA 152 on virus replication (Figure 37E). However, an analysis of the impact of NEDDylation on VDAC1 properties and functions is required in order to confirm this data.