ultrastructural features of calicivirus infection

ULTRASTRUCTURAL FEATURES OF CALICIVIRUS INFECTION VÍTOR DANIEL GONÇALVES CARNEIRO Dissertação de Mestrado em Bioengenharia 2013 VÍTOR DANIEL GONÇALVES CARNEIRO ULTRASTRUCTURAL FEATURES OF CALICIVIRUS INFECTION Dissertação de Candidatura ao Grau de Mestre em Bioengenharia – Biotecnologia Molecular submetida ao Instituto de Ciências Biomédicas Abel Salazar Orientador: Doutor David Bhella Categoria: Professor Honorário Afiliação: Universidade de Glasgow Co-orientador: Doutora Maria São José Alexandre Categoria: Professor Catedrático Afiliação: Faculdade de Farmácia da Universidade do Porto ULTRASTRUCTURAL FEATURES OF CALICIVIRUS INFECTION GONÇALVES C., DANIEL1,2,3 1 – Abel Salazar Biomedical Sciences Institute 2 – Faculty of Engineering of the University of Porto 3 – Medical Research Council University of Glasgow Centre for Virus Research 4 – Faculty of Pharmacy of the University of Porto This dissertation was developed under supervision of Dr. David Bhella3 and co-supervision of Professor Maria São José Alexandre4 The author (Vítor Daniel Gonçalves Carneiro) The co-supervisor (Professor Maria São José Alexandre) PREFACE This thesis is organized into four chapters: a general introduction and three chapters where results obtained are presented and discussed. The general introduction gives an overview of the molecular biology of caliciviruses, in particular, feline calicivirus, with a brief description of its structure and relevant host-cell interactions. This section also reviews electron microscopy and threedimensional reconstruction techniques. Regarding the results, chapter two analyzes the structure of recombinant Sapporo virus-like particles and compares different three-dimensional reconstructions. Chapter three concerns the conformational changes that feline calicivirus undergoes in low pH environment. In the last chapter, structural features of the feline calicivirus RNA replication complexes in the context of the whole cell are discussed. The work herein described is the product of a six month internship carried out by the author at the Medical Research Council University of Glasgow Centre for Virus Research, in Glasgow, Scotland, United Kingdom, under the supervision of Dr. David Bhella. Ultrastructural features of calicivirus infection | 2013 12 | P a g e CONTENTS Abstract .......................................................................................................................... 15 Resumo .......................................................................................................................... 17  Chapter 1: General Introduction ................................................................................. 19 1. Caliciviridae ........................................................................................................... 21 Historical Overview ................................................................................................... 21 Five genera of the Caliciviridae ................................................................................. 22 2. Feline calicivirus Infection...................................................................................... 24 Pathology.................................................................................................................. 24 Epidemiology ............................................................................................................ 25 Transmission and Tropism ........................................................................................ 26 Genomic and Antigenic Diversity .............................................................................. 27 Diagnosis, Treatment and Prevention ....................................................................... 28 3. Feline calicivirus Genome Organization and Viral Proteins.................................... 30 Genome .................................................................................................................... 30 Nonstructural Proteins .............................................................................................. 32 Structural Proteins .................................................................................................... 34 4. FCV life cycle ........................................................................................................ 37 Host Cell Recognition and Cell Entry ........................................................................ 37 Intracellular Pathway................................................................................................. 38 Translation and Replication Strategy ........................................................................ 40 5. Cryo-Electron Microscopy and Icosahedral Reconstruction ................................... 42 Interaction between biological material and electrons ............................................... 42 Functional parts of a Transmission Electron Microscope .......................................... 43 Compensation mechanisms for low contrast images................................................. 44 Tomography ............................................................................................................. 45 Processing ................................................................................................................ 45 6. Aims of the Project ................................................................................................ 47  Chapter 2: A structural comparison of Sapovirus-like Particles with other caliciviruses using cryo-electron microscopy and three-dimensional image reconstruction ................................................................................................................ 49 1. Introduction ........................................................................................................... 51 2. Materials and Methods .......................................................................................... 51 Ultrastructural features of calicivirus infection | 2013 13 | P a g e 3. Results .................................................................................................................. 53 Recombinant Sapporo virus-like particles exhibit common features of caliciviruses. . 53 SV-like particles are morphologically closer to murine norovirus than to feline calicivirus. ................................................................................................................. 55 Application of different software approaches to 3D reconstruction can lead to different outcomes. ................................................................................................................. 56 4. Discussion ............................................................................................................. 59  Chapter 3: A study of Feline calicivirus conformational changes in low-pH environment ................................................................................................................... 61 1. Introduction ........................................................................................................... 63 2. Materials and Methods .......................................................................................... 63 3. Results .................................................................................................................. 65 FCV-fJAM-A microscopic structure is significantly affected by low pH environment .. 65 FCV propagation in FEA cells ................................................................................... 68 FCV purification ........................................................................................................ 70 4. Discussion ............................................................................................................. 73  Chapter 4: FCV RNA replication complexes are closely associated with endoplasmic reticulum .................................................................................................. 75 1. Introduction ........................................................................................................... 77 2. Materials and Methods .......................................................................................... 77 3. Results .................................................................................................................. 78 FCV infected cells show several characteristic features of positive-sense RNA virus infection. ................................................................................................................... 78 Golgi apparatus disappearance may be related to the formation of FCV-induced vesicles. .................................................................................................................... 80 Tomography and 3D image reconstruction of FCV-infected cells shows that ER is associated with vesicle membranes. ......................................................................... 81 FCV-induced vesicles are organized in a three-dimensional complex with connections between them. .......................................................................................................... 83 4. Discussion ............................................................................................................. 85 Conclusion ..................................................................................................................... 86 References ..................................................................................................................... 87 Ultrastructural features of calicivirus infection | 2013 14 | P a g e TABLE OF FIGURES  Chapter 1 Figure 1.1 – Schematic representation of the FCV genome and ORFs. Figure 1.2 – VP1 monomer and capsid assembly.  Chapter 2 Figure 2.1 – Cryo-EM visualization and three-dimensional reconstruction of SVLPs. Figure 2.2 – Comparison of SVLP reconstruction with other caliciviruses. Figure 2.3 – Comparison of SVLP reconstructions generated by EMAN and EM3DR2. Figure 2.4 – Application of B-factor correction to different reconstruction of SVLPs.  Chapter 3 Figure 3.1 – FCV-fJAM-A cryoEM micrographs 100,000X at low pH. Figure 3.2 – Undecorated FCV cryoEM micrographs and reconstruction. Figure 3.3 – FCV titration using FEA cells. Figure 3.4 – Evolution of FCV cytopathic effect in FEA cells using optical microscopy. Figure 3.5 – Negative-stained FCV after PEG precipitation in EM. Figure 3.6 – Negative-stained FCV after PEG precipitation and boric acid buffer dissolution.  Chapter 4 Figure 4.1 – FCV cytopathic effect in FEA cells in EM using epoxy resin embedding preparation. Figure 4.2 – Golgi apparatus in non-infected cells and tubular compartments in FCVinfected cells. Figure 4.3 – Tomography and 3D image reconstruction of FCV-infected cells shows that ER is associated with vesicle membranes. Figure 4.4 – FCV-induced vesicles are organized in a three-dimensional complex with connections between them. Ultrastructural features of calicivirus infection | 2013 15 | P a g e ABSTRACT The Caliciviridae is a family of small, nonenveloped, positive-stranded RNA viruses that cause a wide range of diseases in their hosts, including acute gastroenteritis in humans, and respiratory illness, systemic and haemorrhagic diseases in animals. This family is divided into five genera: Lagovirus, Nebovirus, Norovirus, Sapovirus, and Vesivirus. Norovirus and Sapovirus are the only genera to contain species that infect humans, and together are the worldwide leading cause of acute gastroenteritis. The study of calicivirus infection, however, has been hindered by the lack of an efficient in vitro culture system. Still, some species can be propagated in cell culture, including Feline calicivirus (FCV), which has been used for several decades as a tractable model to help to understand the infection mechanisms of caliciviruses. FCV belongs to the Vesivirus genus of Caliciviridae and is of major importance in small animal veterinary medicine; generally causing oral and upper respiratory tract disease in felids, although recently virulent strains have emerged that cause systemic disease with a high mortality rate. In recent years, its physiological receptor – feline junction adhesion molecule – was identified, enabling studies that elucidate the attachment and mechanism of entry of related viruses. Such studies may contribute to the development of a culture system for human caliciviruses, inasmuch as the attachment and entry step is the block in efficient human calicivirus growth in cell culture. Herein, we exploit cryo-electron microscopy and three-dimensional image reconstruction techniques to better assess structural insights of calicivirus infection. In order to evalute three-dimensional image reconstuction techniques, we compared recombinant Sapporo virus-like particles reconstructions (derived from a human sapovirus that belongs to the Sapovirus genus) with reconstructions of related caliciviruses. These reconstrcutions were calculated using different three-dimensional image reconstruction software, namely EMAN and BSFOT in combination with PFT2/EM3DR. Furthermore, we exploited cryo-electron microscopy and three-dimensional reconstruction to understand FCV cell entry, in particular, the effect of low pH environment in the endosome. We observed that FCV undergoes a dramatic conformational change at low pH that might relate to viral genome penetration. We also assessed FCV RNA replication in Feline Embryonic fibroblasts, which has been shown to occur in FCV-induced membrane-bound vesicles. We observed that such vesicles are related to the endoplasmic reticulum of the host cell and possess connecting regions between each vesicle. Ultrastructural features of calicivirus infection | 2013 16 | P a g e To conclude, the data presented here shows how FCV is able to interact with different structures of the host cell in order to sucessfully infect and replicate, highlighting the importance of low pH environment in endosomes and endoplasmic reticulum (ER) deformation in the early stages of FCV infection. Ultrastructural features of calicivirus infection | 2013 17 | P a g e RESUMO Caliciviridae é uma família de vírus de RNA de polaridade positiva e sem invólucro, sendo responsáveis por uma vasta gama de doenças do epitélio nos seus hospedeiros, incluindo gastroenterite aguda em humanos, e doenças respiratórias, hemorrágicas e sistémicas em animais. Esta família contém cinco géneros: Lagovirus, Nebovirus, Norovirus, Sapovirus, e Vesivirus. Os géneros Norovirus e Sapovirus são os únicos que contêm espécies que infetam seres humanos, sendo a principal causa de gastroenterite aguda. Contudo, o estudo da infeção por calicivirus tem sido dificultada pela falta de sistemas eficientes de cultura in vitro. Ainda assim, algumas espécies podem ser propagadas em cultura de tecidos, incluindo o calicivirus felino, o qual tem sido utilizado como um modelo para o estudo do mecanismo de infecção de calicivirus. Este vírus pertence ao género Vesivirus da família Caliciviridae e assume uma grande importância na veterinária de animais de pequeno porte; causa geralmente doenças orais e do tracto respiratório superior em felídeos, embora outras estirpes mais virulentas tenham emergido recentemente causando uma síndrome sistémica com elevada taxa de mortalidade. Recentemente, o recetor fisiológico do calicivirus felino foi identificado – a molécula de adesão das tight-junctions – possibilitando estudos que investiguem o mecanismo de adsorção e entrada do vírus na célula do hospedeiro. Estes estudos podem contribuir para o desenvolvimento da cultura de calicivirus humanos, uma vez que a adsorção e entrada do vírus da célula foram identificadas como o passo que impossibilita a propagação de calicivirus humanos em cultura. Neste documento, técnicas de criomicroscopia eletrónica e reconstrução tri-dimensional de imagens são exploradas para estudar as interações estruturais na infeção por calicivirus. Para avaliar diferentes técnicas tri-dimensionais de reconstrução de imagem, reconstruções de Sapporo “virus-like particles” recombinantes (geradas a partir de um sapovirus humano pertencente ao género Sapovirus) foram comparadas com recontruções de outros calicivirus. Estas reconstruções foram calculadas usando diferente software de reconstrução tri-dimensional, nomeadamente o programa EMAN e o BSFOT em combinação com PT2/EM3DR. A entrada celular do calicivirus felino também é estudada recorrendo a técnicas de microscopia, em particular o efeito do pH ácido dos endossomas na cápside viral. Foi observado que o calicivirus felino sofre uma dramática mudança conformacional a baixo pH que poderá estar relacionado com a penetração do genoma no citoplasma. Por último, reportamos um estudo sobre a replicação do calicivirus Ultrastructural features of calicivirus infection | 2013 18 | P a g e felino em fibroblastos embriónicos de origem felina, que ocorre em vesículas membranares induzidas pelo virus. Os resultados deste estudo mostram que a formação de vesículas está relacionada com o retículo endoplasmático das células do hospedeiro e possuem regiões de comunicação entre elas. Em suma, os resultados apresentados aqui mostram que o FCV é capaz de interagir com diferentes estruturas da célula, realçando a importância do baixo pH nos endossomas e a reorganização do retículo endoplasmático em estádios iniciais da infeção. Ultrastructural features of calicivirus infection | 2013 19 | P a g e CHAPTER 1: General Introduction General Introduction Ultrastructural features of calicivirus infection | 2013 20 | P a g e Ultrastructural features of calicivirus infection | 2013 21 | P a g e 1. Caliciviridae The Caliciviridae are a family of non-enveloped, positive-stranded RNA viruses that infect a wide range of animals causing diseases, such as gastroenteritis and respiratory illness (Clarke and Lambden 1997). Members of this family, commonly designated as caliciviruses, belong to Class IV of the Baltimore Scheme for animal virus genome classification (Baltimore 1971). The name of the family derives from the latin word for cup – calyx – in reference to the morphology of these viruses under electron microscopy (EM): the invaginations found in calicivirus capsids are often described as cup-like depressions (Zwillenberg and Burki 1966). Historical Overview For the past 80 years, calicivirus infections have been reported in a broad range of animals, mainly mammals but sometimes in birds and reptiles too. The vesicular exanthema of swine virus (VESV) was the first calicivirus to be identified and was found in pigs in California in 1932 (Hopkins 1958). These swine calicivirus infections had their source in some calicivirus species of marine mammals, in particular the San Miguel sea lion virus (SMSV) that were isolated in the northern Pacific Ocean (Smith, Skilling et al. 1998, Etherington, Ring et al. 2006). Feline calicivirus (FCV) was first isolated in 1957 from feline kidney cells (Fastier 1957). Fastier was trying to induce an infection of feline panleukopenia virus, when this new virus appeared to induce a different phenotype in cell culture. First, FCV was designated as kidney cell-degenerating virus and later Feline picornavirus, since at that time caliciviruses were considered a genera of Picornaviridae (Burki 1965). Due to the subsequently discovered differences between caliciviruses and picornaviruses, the Caliciviridae family was created in 1976 (Fenner 1976). In China, 1984, a calicivirus was discovered in rabbits, which caused a fatal disease. This was designated rabbit haemorrhagic disease virus (RHDV) and was later identified in other countries throughtout the world (SJ, HP et al. 1984, Abrantes, van der Loo et al. 2012). Only two caliciviruses are known to infect humans: human noroviruses and sapoviruses. Human norovirus was first identified in an acute gastroenteritis outbreak in Norwalk, Ohio, in 1968 (Adler and Zickl 1969). Since then, human norovirus has been described in numerous outbreaks in cruise ships and hotels as well as in instituitions such as schools, hospitals and care-homes. Sapporo virus was first isolated during a Ultrastructural features of calicivirus infection | 2013 28 | P a g e Diagnosis, Treatment and Prevention Diagnosis is generally accomplished through the combined findings of three approaches: (1) clinical presentation, (2) virus isolation in cell culture or ReverseTranscriptase-PCR (RT-PCR) assay and (3) serological assays (Pesavento, Chang et al. 2008). Other diseases, such as feline rhinotracheitis, can be confused with milder forms of FCV infection. Due to the low correlation between the presence of virus and the manifestation of clinical signs in FCV infected cats (e.g., the asymptomatic carrier phase), and the propensity of cats to shed following vaccination, caution should be taken in diagnosis (Sykes, Studdert et al. 1998, Ruch-Gallie, Veir et al. 2011). Thus, combining the clinical presentation with virus identification is essential to obtain a correct diagnosis. RTPCR assays are used to detect viral RNA in blood samples, oral swabs or lung tissue. Although this test is sensitive (depending on the set of primers used), RT-PCR can fail to detect FCV due to the high variability of its genome. Still, this test is the most effective for detection of variant strains of FCV (Radford, Addie et al. 2009). On the other hand, virus isolation in cell culture can be advantageous since this method is less sensitive to FCV variability, when compared to RT-PCR. This method depends on the ability of FCV to infect certain cell lines in culture. Virus is isolated from oral, conjunctival or nasal swabs. A major drawback of this method is that the number of viable virus particles found in a swab may sufficient to induce cytopathic effect in vitro. This may be because of poor sample preservation or or the presence of neutralizing antibodies that can prevent FCV replication in tissue culture (Marsilio, Di Martino et al. 2005, Radford, Addie et al. 2009). Finally, FCV presence can be detected through serological studies, in which virus antibodies are identified using enzyme-linked immunosorbent assay (ELISA) or virus neutralization. However, in vaccinated or pre-infected cats levels of FCV antibodies are generally high, which can lead to misdiagnosis (Gore, Lakshmanan et al. 2006). As previously described, FCV infection presents a wide range of symptoms, so treatment should be specific for the symptoms and strains involved in the infection. Acute upper respiratory tract disease is common to all FCV infections; in its treatment, electrolyte replenishment and rehydration are essential, as well as the use of anti-inflammatory drugs to reduce fever and pain. Antibiotics directed to the respiratory tract and oral cavity are commonly used, to prevent secondary infection (Radford, Addie et al. 2009). Antivirals for FCV infection have not been identified yet. Studies showed that certain compounds can inhibit FCV replication in vitro: ribavirin – which is toxic for cats and is no longer used (Povey 1978) - and feline interferon-ω – are shown to be effective only against certain Ultrastructural features of calicivirus infection | 2013 29 | P a g e strains (Taira, Suzuki et al. 2005, Ohe, Takahashi et al. 2008). In the treatment of stomatitis, corticosteroids, immunosuppressants or immunomodulatory drugs – such as clorambucil, thalidomide and cyclosporine - can be used (White, Rosychuk et al. 1992, Addie, Radford et al. 2003). Treatment for VS-FCV infection is not available. Some reports have noted that intensive care with steroids and interferon treatment may lead to clinical improvement (Hurley 2006 adapted from Radford, Addie et al. 2009). The control of FCV infection is highly dependent on vaccination. Different vaccines are commercially available; most are live-attenuated or inactivated adjuvanted and are administered intramuscularly or intranasally (Radford, Dawson et al. 2006). Considering strain variability and the adaptability of FCV, producing an efficient multi-strain vaccine has been challenging. Conventionally, the vaccines in use are developed from a single strain – most commonly strains FCV-F9 or FCV-255 (Bittle and Rubic 1976, Povey, Koonse et al. 1980). Recently developed vaccines are based upon two (Poulet, Brunet et al. 2005) or three strains (Masubuchi, Wakatsuki et al. 2010). It is believed that a vaccine based upon a greater number of strains can grant a better protection against circulating wild-type FCV (Addie, Poulet et al. 2008, Huang, Hess et al. 2010). Other vaccines, such as Leucofeligen, are tetravalent vaccines against several different viruses – FCV, Feline herpersvirus, Feline panleukopenia virus, and Feline leukaemia virus – and have been shown to provide vaccinated kittens with increased protection against virulent strains of FCV (Lesbros, Martin et al. 2013). Despite vaccination, infection levels have not been reduced (Gaskell, Gaskell et al. 1982, Coyne, Christley et al. 2012) –these vaccines are, however, considered effective at reducing the clinical signs of oral and respiratory disease (Radford, Dawson et al. 2006). Ultrastructural features of calicivirus infection | 2013 30 | P a g e 3. Feline calicivirus Genome Organization and Viral Proteins Genome FCV has a positive-sense RNA genome. RNA viruses have higher mutation rates – when compared to DNA viruses – granting them the ability to evolve more quickly, enhancing their chances of survival in the face of host defences (Domingo, MenendezArias et al. 1997, Moya, Holmes et al. 2004). This high mutation rate has been attributed to the fact that the polymerase assembles the nascent RNA molecule without any proofreading activity (Steinhauer, Domingo et al. 1992, Elena and Sanjuan 2005). Studies demonstrated that RNA viruses have an average mutation rate of 1 mutation per genome per replication (Drake 1993). As previously stated, calicivirus mutation rates are among the highest amongst RNA viruses (Nilsson, Hedlund et al. 2003, Coyne, Gaskell et al. 2007, Victoria, Miagostovich et al. 2009), contributing to the plasticity of its genome and wide variation of genotypes. The FCV genome is a linear, polyadenylated single-stranded positive sense RNA molecule that is approximately 7.7 kilo base pairs (kbp) in length (Carter, Milton et al. 1992). It comprises three open reading frames (ORFs): ORF1 (nucleotides 20 to 5308), ORF2 (5314 to 7317) and ORF3 (7317 to 7634) (Fig1.1) (Sosnovtsev, Garfield et al. 2002). Infected cells generally present two types of viral RNA: the genome and a subgenomic molecule of 2.4kbp. This subgenomic RNA corresponds to ORFs 2 and 3 (Neill and Mengeling 1988, Neill, Reardon et al. 1991). Figure 1.1 – Schematic representation of the FCV genome and ORFs. The dashed lines indicate the genome and subgenomic RNAs. VPg is depicted as a filled circle at the end of dashed lines. The three ORFs are represented as blue rectangles in association to their position in the genome. The numbers above indicate the nucleotide where each ORF begins and ends. Ultrastructural features of calicivirus infection | 2013 31 | P a g e Another feature of caliciviruses is that the capsid and non-structural proteins are encoded in distinti and seperate ORFs (Clarke and Lambden 1997). ORF1 encodes a polyprotein that is later cleaved into six proteins (Sosnovtsev, Garfield et al. 2002). Most of these proteins are non-structural proteins, which mean that they are not found in mature assembled virus particles. The only exception is a viral protein that is found linked to the genome (VPg). ORF1 also encodes a nucleotide triphosphatase (NTPase) and a proteinase polymerase (Pro-Pol) (Herbert, Brierley et al. 1997, Wei, Huhn et al. 2001, Sosnovtsev, Garfield et al. 2002). Three further proteins are synthesized – designated p5.6, p30, and p32 – their function, however, is still unknown. Structural proteins are encoded by ORF2 and 3. ORF2 encodes the precursor for the major viral capsid protein (VP1) with a leader capsid (LC) sequence (Carter 1989). ORF3 encodes a minor capsid protein (VP2), which is found in low quantity in mature virus particles (1 or 2 copies per virion) (Carter 1989). Gene overlapping occurs only at the ORF2/ORF3 interface, in which the termination codon of ORF2 overlaps the start codon of ORF3 by four nucleotides (Clarke and Lambden 1997). Ultrastructural features of calicivirus infection | 2013 32 | P a g e Nonstructural Proteins The FCV genome encodes both structural and non-structural proteins. Nonstructural proteins mediate virus replication in the host and are encoded in ORF1. Pro-pol is a precursor protein which, after cleavage, becomes the mature proteinase and the RNAdependent RNA polymerase (RdRp). RdRps play a critical role in virus replication and are found in all positive sense RNA viruses (O'Reilly and Kao 1998). The main function attributed to the viral proteinase is the cleavage of the polyprotein translated by the ORF1 and the cleavage of the precursor capsid protein (Sosnovtsev, Sosnovtseva et al. 1998, Sosnovtseva, Sosnovtsev et al. 1999). Kaiser et al. observed that the immature form of the pro-pol protein can form homo-oligomers and it interacts with VPg and the ORF2 (Kaiser, Chaudhry et al. 2006). A similar activity has been described for RHDV, which suggests that this phenomenon may play an important role in the replication of caliciviruses (Ng, Cherney et al. 2002). VPg is a ~14kDa protein which is covalently linked to the 5’ terminus of the FCV genome or subgenome (Burroughs and Brown 1978, Schaffer, Ehresmann et al. 1980). VPg was found to be required for translation initiation, acting as a cap for ribosomal recruitment (Sosnovtsev and Green 1995, Herbert, Brierley et al. 1997). More recent studies demonstrated that VPg interacts with the cap-binding protein eukaryotic initiation factor 4E (eIF4E). This protein is involved in the first step of translation binding to 5’capped mRNAs (Gingras, Raught et al. 1999). Its interaction with VPg suggests that caliciviruses use translation initiation factors to recruit the ribosome (Goodfellow, Chaudhry et al. 2005, Chaudhry, Nayak et al. 2006). Furthermore, VPg can be nucleotidylated by the viral propol, generating a template that may be involved in genome replication (Rohayem, Robel et al. 2006, Belliot, Sosnovtsev et al. 2008). This interaction though appears to be different among genera (Leen, Kwok et al. 2013). A study published by Kaiser et al. showed that VPg also interacts with VP1, suggesting a putative role in viral RNA packaging during virus particles assembly (Kaiser, Chaudhry et al. 2006). Three further non-structural proteins are encoded by ORF1: p5.6, p30, and p32 – designated according to their molecular weight, 5.6kDa, 30kDa, and 32kDa, respectively. The function of these three proteins is unknown; however some studies indicate that p30 and p32 may play a role in the formation/activity of FCV replication complexes. Green et al. in 2002, identified both p30 and p32 in enzymatically active replication complexes isolated from FCV-infected cells (Green, Mory et al. 2002). In this study, p30 and its precursor form (p30-VPg) are shown to accumulate in replication complexes over time. Ultrastructural features of calicivirus infection | 2013 33 | P a g e Additionally, owing to the similarity between polioviruses and caliciviruses, a parallel is established between p30 and its homologue in poliovirus (3A protein), which has been proposed to be anchored in membranes so that VPg is appropriately positioned for RNA replication (Datta and Dasgupta 1994, Lama, Paul et al. 1994, Towner, Ho et al. 1996). In 2010, Bailey et al. demonstrated that p30, p32 and NTPase proteins locate to the endoplasmic reticulum (ER), leading to ER membrane reorganization similar to the patterns observed in FCV-infected cells (Bailey, Kaiser et al. 2010). Collectively, these data suggest that p30 and p32 play a role in FCV RNA replication. Ultrastructural features of calicivirus infection | 2013 34 | P a g e Structural Proteins Structural proteins are those that are found in mature virions. In the case of FCV, there are only two desingated structural proteins: VP1, which is encoded by ORF2, and VP2, encoded by ORF3. ORF2 encodes a precursor capsid protein that is posttranslationally cleaved to release two products: VP1 and the ‘leader of the capsid’ (LC) protein. The expression of VP1 from a precursor protein is a unique feature of the Vesivirus genus (Fretz and Schaffer 1978, Carter 1989). Infectious virus particles can not be recovered when this cleavage is inhibited, suggesting that VP1 is only functional following removal of the LC (Sosnovtsev, Sosnovtseva et al. 1998). LC is a protein of 124 amino acids of which the function is unknown. A recent study demonstrated that cells transiently expressing wild-type LC protein, showed a cell-rounding phenotype typical of in vitro FCV infected cells (Abente, Sosnovtsev et al. 2013). The same study also found that LC binds to the host cellular protein annexin A2 – a protein associated with the cytoskeleton and cell motility (Gerke and Moss 2002) – suggesting that the LC-annexin A2 interaction may change the structure of the cell, to favour virus replication. Interestingly, LC was shown to promote the replication of Norwalk virus in cell culture, supporting the idea that LC is required for efficient replication of feline calicivirus (Chang, George et al. 2008). ORF2 is divided into six regions with respect to its degree of conservation among species (Neill 1992, Radford, Willoughby et al. 1999). These regions are designated from A to F: region A is the LC that has been described above, which shows a moderate degree of variability between species except for the cleavage site which is highly conserved (Glenn, Radford et al. 1999). Regions B, D and F have been reported to be the most conserved parts of VP1, suggesting that they are functionally important (Neill 1992). In particular, region B has an ATP/GTP binding motif with homology to picornavirus VP3 protein, which has been shown to be essential for virus survival (Tohya, Taniguchi et al. 1991, Seal, Ridpath et al. 1993, Seal 1994). VP1 has a molecular mass of 60kDa and is divided into three domains: an N-terminal arm (NTA), the shell (S) domain and the protruding (P) domain (Fig1.2A). The NTA faces the interior of the capsid; the S domain forms the capsid surface; and the P domain is projected outwards from the capsid floor. Additionally, the P domain is divided into two subdomains, designated P1 and P2. P1 is proximal to the S domain; while the P2 domain is distal to the capsid shell and on the outermost surface of the viral particle (Prasad, Matson et al. 1994, Prasad, Hardy et al. 1999, Chen, Neill et al. 2006). The P2 domain is therefore the region where most of the Ultrastructural features of calicivirus infection | 2013 35 | P a g e neutralizing epitopes are located (Matsuura, Tohya et al. 2001, Geissler, Schneider et al. 2002). The second protein found in FCV virions is VP2 which is encoded by ORF3. VP2 is a 12kDa protein and its function is unknown. Sosnovtsev and colleagues showed that ORF3 deletion is lethal to FCV. Furthermore several mutations were made in this region, that abolished virus replication (Sosnovtsev, Belliot et al. 2005). It has also been demonstrated that co-expressing VP1 and VP2 generates FCV VLPs that more closely resemble wild-type virions compared to the single expression of VP1, suggesting a role for VP2 in the correct assembly of FCV particles (Di Martino, Marsilio et al. 2007, Di Martino and Marsilio 2010). The calicivirus capsid consists of 180 copies of VP1, organized into 90 arch-like dimers which give caliciviruses their characteristic morphology (Prasad, Matson et al. 1994) (Fig1.2B). VP1 dimers are arranged according to a T=3 icosahedral symmetry, originating from three quasi-equivalent environments Consequently VP1 monomers fall into three distinctive equivalent conformations – designated A, B and C. VP1 dimers are distributed in two classes: AB dimers, arranged around the 5-fold axes, and CC dimers, located at the 2-fold axes (Prasad, Hardy et al. 1999). Figure 1.2 - VP1 monomer and capsid assembly. (A) X-ray structure of VP1 of FCV-5, ribbon representation. The N-Terminal Arm (green), Shell domain (blue), and P1 (red) and P2 (yellow) subdomains are indicated (Ossiboff et al. 2010). (B) T=3 icosahedral capsid formed from VP1 assembly, represented in three quasi-equivalent environments: A (blue), B (purple) and C (magenta). VP1 dimers: AB and CC. (Bhella et al. 2008) Ultrastructural features of calicivirus infection | 2013 36 | P a g e The viral capsid has a variety of functions and is fundamental for the life cycle of the virus. It is responsible for attachment and entry: delivering the viral genome across the host cell plasma membrane into the cytoplasm. The capsid must also specifically package progeny viral genomes to form new virions. The viral capsid must also prevent genome damage, protecting it from the harsh extracellular environment during transmission from host to host. Studies have shown that the FCV capsid is moderately stable, maintaining infectivity following exposure to harsh environmental conditions: in a pH range of 4 to 8.5 (Pesavento, Chang et al. 2008) and temperature range (Topping, Schnerr et al. 2009); additionally, as previously mentioned, FCV can survive in fomites for several days or weeks. Thus the viral capsid protein must be capable of forming a robust shell that is nonetheless sufficiently dynamic to package the viral genome and then uncoat upon arrival in specific compartments of the new host cell. Ultrastructural features of calicivirus infection | 2013 37 | P a g e 4. FCV life cycle Host Cell Recognition and Cell Entry Host cell recognition and cell entry are two critical steps in a virus’s life cycle. In the case of FCV infection, susceptibility has been demonstrated to be conferred on nonpermissive cells by exogenous receptor expression. Understanding the early stages of infection is therefore of fundamental importance a target for potential antiviral intervention. Two molecules have been described as receptors for FCV: fJAM-A and α-2,6 sialic acid. Despite α-2,6 sialic acid having the ability to enhance FCV binding at the cell surface, it can not mediate virus entry by itself; on the other hand, fJAM-A expression on nonpermissive cells renders them susceptible to infection (Makino, Shimojima et al. 2006, Stuart and Brown 2007). fJAM belongs to the family of immunoglobulin-like molecules and is important in the assembly and maintenance of tight junctions and the establishment of epithelial cell polarity (Martin-Padura, Lostaglio et al. 1998). Pesavento et al. have shown that fJAM-A has an extensive distribution in feline tissues, localized in epithelial and endothelial cell-cell junctions, and feline platelets (Pesavento, Stokol et al. 2011). During FCV infection, fJAM relocates to the cytosol, as a result of FCV binding and tight junction disruption; furthermore, this disruption induced by FCV is thought to promote the cell rounding phenotype in FCV infection. fJAM-A is a transmembrane protein composed of five domains: the N-terminal signal peptide; two immunoglubin-like domains designated D1 - positioned farthest from the membrane – and D2 – closer to the membrane; the Cterminal transmembrane domain; and the cytoplasmic domain (Kostrewa, Brockhaus et al. 2001). Ossiboff and colleagues identified D1 as the main domain for FCV interaction; the same study identifies several critical amino acids for FCV infection (Ossiboff and Parker 2007). Bhella et al. showed that the D1 domain of fJAM-A interacts with the P2 domain of VP1 of FCV, and the region involved in this interaction possesses several neutralizing epitopes (Bhella, Gatherer et al. 2008). The same laboratory showed later that interactions with FCV and fJAM-A lead to a group of conformational changes in the virus capsid that might represent the initial steps of virus uncoating (Bhella and Goodfellow 2011). In particular, this study showed that binding of the P domain of VP1 to fJAM-A leads to a loss of the icosahedral symmetry in the capsid. They observed two distinctive Ultrastructural features of calicivirus infection | 2013 44 | P a g e Compensation mechanisms for low contrast images The resultant image is a representation of the detected electrons. However, as biological material contains mainly light elements (Hydrogen, Carbon, Oxygen and Nitrogen), electrons are only slightly deflected, and the number of electrons before and after the specimen is the same – generating low contrast images. To bypass this situation, microscopy takes advantage of the properties of the deflected electrons: as these have different path lengths after interaction with the specimen they exhibit different phase from those that did not interact with the sample. This phase shift is made visible by converting phase variation into amplitude variations. Still, phase shifts induced by biological samples generate very low contrast images. So, phase plates can be used to shift the phase of scattered light even more and induce an interference image (Glaeser 2008, Henderson and McMullan 2013). Another consequence of inelastic scattering is that deflected electrons have lower energy, which means that these electrons have a longer wavelength. This wavelength gap between inelastic and elastic scattered electrons causes a chromatic aberration, corrupting the imaged with additional background and making it blurry. To correct this, energy filters are applied to the path of light after the specimen, deflecting electrons with different wavelength (Orlova and Saibil 2011). The usage of such filters substantially improves the contrast of the final image. One simple method for increasing contrast is the manipulation of defects of lenses, in particular spherical aberrations, and the level of focus. Spherical aberration is an optical and electromagnetic effect characterized by the increased refraction of the beam, where refracted rays do not meet in one focal point. When the effect of spherical aberration and image defocus is combined, image contrast can be enhanced, since this combination leads to a phase shift between scattered and unscattered electrons (Henderson and McMullan 2013). Ultrastructural features of calicivirus infection | 2013 45 | P a g e Tomography Tomography is a variation of microscopy distinguished by a series of images of the same region over a range of tilt angles. Such procedure retrieves the information of several focus planes within an object that can be recovered to generate a threedimensional reconstruction. One limitation of this technique is that tomograms (the result of a tilt series in tomography) will lack the information from a region in space: a wedge of ~50°, due to the extended path length of the beam through the samples. This effect is known as the missing wedge effect (Bartesaghi, Sprechmann et al. 2008). Moreover, during high tilt, inelastic scattering is increased, leading to a less coherent image. However, energy filters can be used to overcome this problem. Processing A drawback of improving contrast with spherical aberrations and defocus is that some features of the object have reversed contrast. Restoration of the image is partially possible; however, the operator should balance the contrast enhancement and the distortion of the object representation. In order to retrieve the true features of the object, the phase contrast transfer function (CTF) of the microscope must be corrected. CTF is a function intrinsic to a microscope and it modulates the amplitude and phase of the electron diffraction pattern formed on the back focal plane of the lens. CTF depends on several values, namely the spherical aberration coefficient, wavelength and defocus as illustrated in the following equation: ( ) [ ] Where, Cs is the spherical aberration coefficient, λ, wavelength, Δf, defocus values, and k, the spatial frequency. Correcting the CTF will eliminate its effects. CTF correction is accomplished by comparing and fitting the CTF curve to a model CTF (Zhou, Hardt et al. 1996). Micrographs contain variations in contrast that represent density variation of the object; however, it also includes noise. In single-particle reconstruction, each image represents a projection of the object at a certain point of view. After image recording and processing, images must be aligned to extract three-dimensional information of the object – which means that the position and orientation of each particle must be identified so it can be superimposed with similar images. This is accomplished by the identification of Ultrastructural features of calicivirus infection | 2013 46 | P a g e modifications (such as rotations or shifts) relative to a reference image. Usually, crosscorrelation function (CCF) is used to compare two images and it is represented as ( ) ∫ (  ) ( )  √∫ ( ) ( )  ∫ ( ) ( )  Where g1( ) and g2( ) are two functions,  is a vector in space, and  is the shift between images. Iteration is also important in this step since it gradually improves the reference image during refinement rounds (Orlova and Saibil 2011). Ultrastructural features of calicivirus infection | 2013 47 | P a g e 6. Aims of the Project Members of the Caliciviridade family are very important pathogens of human and animals. The lack of an efficient propagation system is a major impediment to the study of human caliciviruses. Additionally, some studies suggest that cell attachment and entry appears to be the main block to efficient human calicivirus growth in cell culture. Further investigation of the early stages of the calicivirus infection is required in order to better understand host selectivity and to develop new culture systems. FCV, a common surrogate model for human caliciviruses, has been extensively studied. The mechanism of entry, however, is not completely understood. Previous data showed that FCV capsid undergoes a major conformational change upon binding with its physiological receptor, suggesting the initial steps of uncoating. The extent of this conformational change may only be understood by taking into consideration the endosomal low pH envirornment required for FCV infection. Although FCV replication is known to occur in membrane vesicles characteristic of positive-sense RNA virus, the mechanism of formation of these vesicles is still unknown. Understanding how FCV hijacks cellular systems would contribute to the development of vaccines and antivirals used in the prevention and treatment of calicivirus infection. CryoEM, along with three-dimensional image reconstruction, have greatly contributed to the study of structural biology, and, in particular, to virus structure. However, the diversity and complexity of techniques involved in such procedures can generate misleading results. Further analysis of different reconstruction techniques will benefit the production of more reliable reconstructions. The focus of this project was to elucidate the mechanism of cell entry for FCV, in particular, (1) the role of endosomal pH in conformational changes to the capsid and also (2) the process of uncoating. Also, (3) to use cryoEM and three-dimensional image reconstruction to understand how FCV-induced replication complexes relate structurally to each other and to organalles in the cell. To better assess the reconstruction techniques used in this project, we compared reconstructions of sapovirus-like particles calculated throught different procedures. Ultrastructural features of calicivirus infection | 2013 48 | P a g e Ultrastructural features of calicivirus infection | 2013 49 | P a g e CHAPTER 2: A structural com parison of S apovirus -like Partic les with oth er cali civiruses us ing cry o-el ectron mi croscopy a nd thr eedim ensional im age reconstruc tion A structural comparison of sapovirus-like particles with other caliciviruses using cryo-electon microscopy and three-dimensional image reconstruction Ultrastructural features of calicivirus infection | 2013 50 | P a g e Ultrastructural features of calicivirus infection | 2013 51 | P a g e 1. Introduction Due to the lack of efficient culture systems for caliciviruses, the mechanism by which viruses interact with host cells is poorly understood. Two surrogate models have been intensively studied to understand calicivirus infection, FCV and MNV (Guix, Asanaka et al. 2007). Atomic resolution structures of both virions and recombinant virus-like particles have been solved for several caliciviruses using cryo-electron microscopy and Xray crystallography. In recent years, VP1 of SV has been successfully expressed in insect cells, leading to the formation of empty virus-like particles that appear to be similar in structure to the native SV virion (Hansman, Ishida et al. 2007, Hansman, Oka et al. 2008). The structure of SV capsid, however, has not been described in great detail yet. Herein, an icosahedral reconstruction of recombinant sapovirus-like particles generated by cryo-electron microscopy is described and compared with reconstructions of related caliciviruses. In addition, we compare reconstructions generated by different software. We show that SV-like particles exhibit the same general features of caliciviruses, with major differences present in the P2 domains, and resemble more closely the MNV structure rather than FCV. 2. Materials and Methods 1 Viruses. Two strains of SV were isolated: SV Mc114 strain (GenBank no. AY237422) – recovered from an infant with acute gastroenteritis in Chiang Mai, Thailand, in 2001 (Hansman, Katayama et al. 2004) – and SV C12 strain (GenBank no. AY603425) – isolated from an infant with gastroenteritis in Sakai, Japan, in 2001 (Katayama, Miyoshi et al. 2004). Cloning of viral cDNA to produce recombinant bacmids. SV consctructs used in recombinant VP1 (rVP1) expression contained (1) the VP1 gene from the predicted VP1 start AUG codon, (2) the VP2 gene and (3) polyadenylated sequences. Fragments were 1 Note: The production of recombinant sapovirus-like particles, cryo-electron microscopy and the three-dimensional reconstruction of one of the models presented here was carried out by David Taylor, from the National Institute for Physiological Sciences, Okazaki, Japan, and Grant Hansman, from the National Institute of Infectious Diseases, Tokyo, Japan. Such methods are only described here for the validity of the study. Ultrastructural features of calicivirus infection | 2013 52 | P a g e amplified by PCR and cloned according to the Baculovirus Expression system protocol (InvitrogenTM Life Technologies). The primers used for Mc114 strain were p+1Mc114 and attB2TX30SXN. For the amplification of the C12 strain, the primers used were the p+1C12 and attB2TX30SXN. After electroforesis, PCR fragments were cut out from the 0.8% agarose gel and purified. Fragments were cloned into a donor vector pDONR201 (InvitrogenTM Life Technologies) and subsequently transferred into a baculovirus transfer vector pDEST8 (InvitrogenTM Life Technologies). The recombinant pDEST8 was purified and used to transform DH10Baccompetent cells (InvitrogenTM Life Technologies) were transformed with pDEST8 producing recombinant bacmids containing the VP1 gene. Expression in insect cells. Sf9 cells (Riken Cell Bank, Japan) were transfected with recombinant bacmids containing the VP1 gene; the resultant recombinant baculoviruses were collected as previously described (Hansman, Natori et al. 2005). Confluent Tn5 cells (InvitrogenTM Life Technologies) were infected at a multiplicity of infection (MOI) of 5-10 with the recombindant baculoviruses in 1.5 ml of Ex-Cell 405 medium (JRH Biosciences) followed by incubation at 26°C. 5 to 6 days post infection, culture medium was harvested and centrifuged for 10 min at 3,000 g, and further centrifuged for 30 min at 10,000 g. VLPs were concentrated by ultracentrifugation for 2h at 45,000 rpm at 4°C (Beckman TLA-55 rotor). Electron Microscopy. VLPs were prepared for cryo-electron microscopy at a concentration of 1.0mg/ml, loaded onto a glow-discharged R1.2/1.3 Mo 200 mesh holey carbon grid (Quantifoil). Samples were blotted for 7/8 seconds and quickly punged into liquid ethane using an FEI MarkIV Vitrobot. Specimens were observed using a JEOL JEM2200FFC microscope equipped with a field emis sion gun. Images of the samples were collected at a magnification of 80,000 using a charge-coupled-device camera, with a resulting sample size of 1.6Å/pixel. Three-Dimensional Image Reconstruction. Two reconstructions of the VLPs were generated using different methods. To generate the first reconstruciton, David Taylor used the following methods: EMAN2 software was used to select and extract particles from micrographs; individual particles were normalized and phase flipped. A high-pass filter was applied prior to analysis; ~8,000 particle images were aligned and classified through multireference alignment and multivariate statistical analysis in IMAGIC (van Heel, Harauz et al. 1996); the initial model was generated in EMAN2, considering the best match Ultrastructural features of calicivirus infection | 2013 53 | P a g e between reprojections, and further refinement was accomplished by sequential rounds of matching projections, cultimating in a 10Å resolution final reconstruction. Complementarily, we generated the second reconstruction using the micrographs recorded by David Taylor.. Images of VLPs particles were extracted and the contrast transfer function (CTF) was corrected using the BSOFT program BSHOW (Heymann 2001). Images were centered, oriented and reconstructed using PFT2/EM3DR2 (Baker and Cheng 1996). The generated FCV reconstruction was visualized using UCSF Chimera software (Pettersen, Goddard et al. 2004). 3. Results Recombinant Sapporo virus-like particles exhibit common features of caliciviruses. A three-dimensional reconstruction of SV-like particles (SVLPs) was generated from 2943 particles selected from a total of 222 micrographs (Fig.2.1A) recorded by David Taylor and provided by Grant Hansman. The final reconstruction has a calculated resolution of 9 Å. Cryo-electron microscopy of SVLPs showed that SV-VP1 expressed in insect cells assembles into virion-like particles approximately 40nm in diameter (Fig.2.1B). SVLPs presented mainly with two different morphologies: (1) a well-defined organization, with a circular shell, clear protruding spikes arranged radially and extending from the shell, and a less dense interior (black arrows in Fig.2.1B); (2) a more disordered structure, with eccentric shells filled with a denser material inside (white arrows in Fig.2.1B). As predicted, three-dimensional reconstruction of SVLPs showed 90 arch-like dimers of capsomeres (Fig.2.1D) arranged in a pattern consistent with T=3 icosahedral symmetry, similar to the other members of the Caliciviridae family. CC dimers, arranged at the 2-fold axes, and AB dimers, arranged around the 5-fold axes, present P2 domains pointing outwards from the centre of the particle (Fig.2.1C). A clipped view of the threedimensional reconstruction cut to show the VLP interior, showed that SVLPs have no material inside the capsid (Fig.2.1E). The inner surface presents a series of organized features: the imprint of the 3 fold axis and a major indentation located at the 5fold axis (Fig.2.1F). Ultrastructural features of calicivirus infection | 2013 60 | P a g e organization of P2 domains between sapovirus and MNV pinpoints the latter as a better surrogate model for sapovirus studies than to FCV. Although studies show MNV replication is enteric and leads to infection only in immunocompromised mice, with broader symptomatology than human noroand sapoviruses, MNV does not produce clinical symptoms in wild-type mice. Major significant structural differences in SVLPs and MNV are focused on the orientation of the P domains of dimers relative to the S domain. We hypothesized that this shift in the orientation of the P domains between species of different genera may be an important factor for receptor binding, as rotation of the P domain has been shown to occur upon receptor binding (Bhella and Goodfellow 2011). Since all the receptors identified so far for caliciviruses are protein members of the histo-blood compatibility group, it is fair to admit that the organization of these molecules in cell surface between different hosts and different cell types would a limiting factor for virus propagation in cell culture. Therefore, the study of structural organization of caliciviruses is essential to understand the limitations of in vitro cell systems and develop new ways to approach and overcome this problem. Studies on virus structure depend deeply on electron microscopy and image reconstruction software. It is critical then to perfect and validate the software and approaches used to generate 3D reconstruction. Moreover, it is important to judge the quality of reconstruction both qualitatively and quantitatively, since it does not absolutely define the resolution achieved in a study if overfitting is not adequately controlled (Cohen 2013, Mao, Wang et al. 2013). In this study we compared two different software packages by generating SVLP reconstructions: EMAN and EM3DR2. As described previously, the structure of SVLP generated by EMAN showed an amplification of noise in the micrographs, generating random and disorder structures above and inside the capsid. Such structures are not represented in EM3DR2’s reconstruction. Such differences can be due to a possible defective filtering in EMAN or low contrast threshold. P2 domains, initially, appeared to be better resolved in the reconstruction using EMAN, this was found to be a consequence of the B-factor compensation that was applied. When a similar correction was applied to our map, similar high-resolution features emerged, however our map had AB and CC dimers that were more self-consistent. Ultrastructural features of calicivirus infection | 2013 61 | P a g e CHAPTER 3: A study of Feline calicivirus conformational changes in low-pH environment A study of feline calicivirus conformational changes in low-pH environment Ultrastructural features of calicivirus infection | 2013 62 | P a g e Ultrastructural features of calicivirus infection | 2013 63 | P a g e 1. Introduction FCV structure has been determined by cryoEM and X-ray crystallography studies, showing that its capsid is comprised of 180 copies of VP1, the major capsid protein, forming 90 arch-like dimers arranged in a T=3 icosahedral lattice. VP1 can thus assume three different conformations, known as A, B, and C, generating two classes of dimers: AB dimers, arranged around the 5fold symmetry axes, and the CC located at the 2fold symmetry axes (Prasad, Matson et al. 1994, Prasad, Hardy et al. 1999). The cellular receptor for FCV has been determined: fJAM-A binds to FCV, soluble forms neutralize the virus, and transfection of non-permissive cells with the fJAM-A gene renders them susceptible to infection by virus (Makino, Shimojima et al. 2006, Pesavento, Chang et al. 2008). Studies of soluble fJAM-A bound to FCV have shown that the capsid undergoes major conformational changes leading to the loss of the icosahedral symmetry. It has been hypothesized that theses conformational changes may represent the initial steps of the uncoating and penetration process (Bhella, Gatherer et al. 2008, Bhella and Goodfellow 2011). After receptor recognition, FCV enters the cell by clathrin-mediated endocytosis (Stuart & Brown, 2006). The virion is endocytosed into a vesicle coated with clathrin; subsequently clathrin dissociates and the vesicle fuses with an early endosome. At later stages in the pathway, the particle is transported to the late endosome. At some point the virus particle penetrates the membrane of the endosome, escaping or exporting its genome to the cytosol (Smith et al. 2004). Studies in endosomal acidification inhibition with chloroquine showed that a low-pH step is required for a successful FCV infection (Kreutz et al. 1995, Stuart et al. 2004). Kreutz et al. identified this critical step up to 2h after virus adsorption. In this study, we attempted to determine the structure of FCV bound to fJAM-A in a low-pH environment and analyze the conformational changes associated to this step. 2. Materials and Methods Viruses culture and purification. Feline calicivirus strain F9 was propagated in FEA and CRFK cells (kindly provided by Dr. Brian Willett, Faculty of Veterinary Medicine, University of Glasgow, Scotland, United Kingdom) in Dubecco’s Modified Eagle’s medium (DMEM - Gibco® Life Technologies) supplemented with 10% Fetal Bovine Serum (Gibco® Invitrogen) and 1% Penicillin Streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin, Gibco® Life Technologies), using a multiplicity of infection (MOI) of 0.1 or 0.01. Cells were Ultrastructural features of calicivirus infection | 2013 64 | P a g e lysed by a single freeze-thaw, and the supernatant was clarified by centrifugation (4000rpm, 30min at 4°C, Thermo Scientific® Heraeus Megafuge40R centrifuge) and then filtered through a 0.45μm filter (Minisart® Sartorius). Virus particles were precipitated by the addition of solid polyethylene glycol (PEG) with a molecular weight of 3,350 (Sigma®) to a concentration of 10%(w/v) and sodium chloride (0.2 M, AnalaR Normapur®). After 14, 17, and 20 hours incubation at 4°C, precipitated virus was recovered by centrifugation (11500rpm/17948 g, 30min at 4°C, Thermo Scientific® Sorvall Centrifuge Rotor SureSpin 630) and resuspended in 0.2 M boric acid buffer (Normapur®, pH 7.4, in Dulbecco’s Phosphate Buffered Saline 1x Sigma®) containing 0.5 M sodium chloride (AnalaR Normapur®), for different periods of time (1, 2 and 3 hours). Insoluble material was removed by centrifugation (11500rpm/17948 g, 30min at 4°C, Thermo Scientific® Sorvall Centrifuge Rotor SureSpin 630), and virus particles were partially purified by centrifugation through a 30%(w/v) sucrose cushion (Sigma®) at 24600rpm/112398 g for 15h at 4°C. The virus pellet was resuspended in phosphate-buffered saline and banded in an isopycnic cesium chloride gradient (1.31 g/ml, Melford®) by centrifugation at 40200rpm for 20 h at 4°C (Thermo Scientific® Sorvall Centrifuge Rotor AH-650). Virus was removed from the gradient, dialyzed against phosphate-buffered saline, and subsequently concentrated using a 100-kDa-molecular-mass-cutoffcentrifugal concentrator (Vivaspin 4 Sartorius). Other FCV preparation held in stock was also used and was prepared as described previously (Bhella, Gatherer et al. 2008). Virus Titration. To access the cell culture infectious dose 50% (CCID50) of FCV in FEA and CRFK cells, virus titrations were carried out in 96 well plates. Initially, 10μl of the sample was diluted into 5ml of DMEM, followed by a sequential dilution of 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, 1:2048000, 1:8192000, and 1:3276800, in quadruplicates. FEA or CRFK cells were seeded in each well to a final concentration of 2x105 cells/ml. Plates were then incubated at 37°C and examined post infection. Electron Microscopy. Purified FCV was incubated in the presence of soluble fJAM-A (kindly provided by Dr. Ian Goodfellow, Department of Virology, Faculty of Medicine, Imperial College London) for 1h at 4°C. The pH of virus containing solution was modified by the addition of a 100m citrate phosphate buffer (Citric Acid, Sodium Phosphate AnalaR®, pH=5;5.5;6) for 5 or 30min at 4 or 37°C. FCV particles were prepared for cryo-electron microscopy: glow-discharged (Emitech-K100X) C-flatTM holey Ultrastructural features of calicivirus infection | 2013 65 | P a g e carbon grids (CF-22-4C, Protochips®) were covered with 4μl of FCV particles in PBS. Grids were blotted for a few seconds and then plunged into liquid ethane kept cold by liquid nitrogen. Frozen FCV-covered grids were observed at low temperature in a JEOL 2200 FS Cryomicroscope equipped with a Gatan 626 cryo-stage. Samples were imaged at a magnification of 100,000 . Negative-staining electron microscopy was also used for rapid evaluation identification of FCV virions at certain stages during the purification procedure. Prior to the staining, square mesh grids (AGG2400C Square 400 Mesh Copper 3.05mm, Agar Scientific, Ltd.) were coated with a thin Formvar film (Agar Scientific, Ltd.). A uniform carbon layer was deposited onto the Formvar film using a Quorum Q150T Carbon Coater with a ramping current of 40A. Formvar was then dissolved using a chloroform solution (VWR Prolabo 0,6% ethanol). 4μL aliquots of FCV samples were loaded onto freshly glowdischarged carbon-coated grids. The grids were washed three times in deionized water and finally stained in an ammonium molybdate solution (2% pH=7, Agar Scientific, Ltd.) and air dried. Negative stained samples were imaged in a JEOL 1200 EXII electron transmission microscope. Three-dimensional image reconstruction. To calculate reconstructions, 223 micrographs of FCV strain F9 virions were processed. Micrographs were binned by a factor of two, giving a sample frequency of 2.06 Å/pixel. Images of FCV particles (1944 in total) were extracted and the CTF was corrected using the BSOFT program BSHOW (Heymann 2001). Images were centered, oriented and reconstructed using the EM3DR2 program (Baker and Cheng 1996). The generated FCV reconstruction was calculated from 1745 particles and visualized using UCSF Chimera software (Pettersen, Goddard et al. 2004). 3. Results FCV-fJAM-A microscopic structure is significantly affected by low pH environment Purified FCV virions were allowed to bind to soluble fJAM-A as described previously, generating FCV virions decorated with fJAM-A. The pH environment was changed after initial decoration with the receptor and particles were observed by cryoelectron microscopy (Fig.3.1) Ultrastructural features of calicivirus infection | 2013 66 | P a g e Micrographs of FCV-fJAM-A complexes in different pH environments showed that particles undergo a substantial structural change. At neutral pH (pH=7.0), fJAM-decorated FCV particles display a concentric shell with distinctive VP1-fJAM protrusion, measuring about 50nm in diameter (Fig.3.1A). At pH 5.5 and 5 (Fig.3.1B and 3.1C, respectively) particles show a smaller diameter, 30nm approximately, with a disordered exterior. The shell line is no longer recognized, suggesting that the particles collapsed into the centre of the virion but maintained a morphology that still resembles virus particles. To investigate this structural change, data were collected with the intention of processing particle images to calculate a three-dimensional image reconstruction. This aim was hampered by a persistent aggregation of virus particles that was found in all FCV samples (Fig.3.1D). In cryo-electron microscopy, aggregation and overlapping of virus particles usually obscures features of the particles and they can not be used for a reliable three-dimensional image reconstruction. Consequently we decided to work towards identifying an FCV preparation suitable for microscopy studies. Ultrastructural features of calicivirus infection | 2013 67 | P a g e Figure 3.1 - FCV-fJAM-A cryoEM micrographs 100,000X at low pH. (A) FCV particles bound to soluble fJAM-A in neutral pH showing a rounder structure, (B) FCV-fJAM-A complexes in pH 5.5, and (C) in pH 5. Black arrows in images A, B, and C point to FCV virions labeled with fJAM-A. (D) FCV-fJAM-A complexes aggregated in pH 5. Preliminary cryo-electron microscopy and three-dimensional reconstruction studies were performed to assess the viability of another FCV preparation held in stock in the laboratory. Cryo-electron microscopy studies of FCV virions showed that particles were well separated from each other, presenting the distinctive features of calicivirus morphology (Fig.3.2A). A three-dimensional reconstruction of FCV was generated from 1745 particles selected from a total of 223 micrographs. The final reconstruction has a calculated Ultrastructural features of calicivirus infection | 2013 68 | P a g e resolution of 13Å (Fig.3.2B). This structure showed similar features of those previously described but high resolution features were not well resolved, suggesting that the particles were not well preserved. Such a preparation would not allow the study of conformational changes upon fJAM binding and low-pH environment. To overcome this problem, we decided to propagate FCV in cell culture and attempt to optimize the purification process. FCV propagation in FEA cells FCV was propagated in FEA cells using an MOI of 0.1 and 0.01 and virus titrations were performed for a period of 72 hours post-infection to optimize the conditions of virus replication and purification (Fig.3.3). It was observed that the most efficient MOI was 0.1 at 36/48h postinfection. This was the time used for further FCV purification. The cytopathic effect caused by FCV in FEA cells was also observed during the titration (Fig.3.4). A confluent layer of FEA cells (Fig.3.4A) was cultivated as described in the Materials and Methods sections. FEA cells showed the first signs of FCV infection at 6/9h post-infection, at which time some cells had started to detach from the surface and become rounded. At 48 hours after infection, 50-60% of the cells were detached and were seen to be aggregated in small clusters that floated above the monolayer (Fig.3.4C). It was also observed that cellular debris was present in the aggregates. At this point, the culture medium Figure 3.2 – Undecorated FCV cryoEM micrographs 100,000X (A). Three-dimensional representation of FCV reconstruction (B). Detail of representation of FCV reconstruction (C) Ultrastructural features of calicivirus infection | 2013 69 | P a g e turned yellow indicating that pH had fallen below 6.8. Finally, at 72 hours post-infection, almost all the cells lost adhesion to the surface and cellular debris was observed throughout the plate (Fig.3.4D). Figure 3.3 - FCV titration using FEA cells. Virus growth curves were used to evaluate the infection profile of FCV using different MOIs. 0 1 2 3 4 5 6 7 8 9 012 24 36 48 60 72 84 Virus Titration (logCCID50) Time post-infection (h) MOI=0.1 MOI=0.01 Ultrastructural features of calicivirus infection | 2013 76 | P a g e Ultrastructural features of calicivirus infection | 2013 77 | P a g e 1. Introduction Virus cell entry is usually mediated by receptor-binding and, in order to replicate, viruses must hijack cell machinery to synthesize their genome, capsid and other essential proteins. Some other positive-sense RNA viruses replicate their genomes in the cytoplasm, leading to the formation of small vesicles where viral RNA synthesis occurs (Egger and Bienz 2002, den Boon and Ahlquist 2010). FCV infection has shown the formation of small membrane-bound vesicles occurs and these vesicles are enzymatically active, containing all the units required for RNA replication (Green, Mory et al. 2002). However, little is known about the formation of these vesicles, how they relate to each other and from which organelle they originate. In this study, we observed resin embedded FCV infected cells using TEM and three dimensional image reconstruction software to determine the relationship between FCV induced vesicles and the organelles in the cell. 2. Materials and Methods Viruses culture. FEA cells were maintained in DMEM (Gibco® Life Technologies) supplemented with 10% Fetal Bovine Serum (Gibco® Invitrogen) and 1% Penicillin Streptomycin. FCV at an MOI of 10 was adsorbed to an 80% confluent FEA monolayer. A non-infected (mock-infected) FEA control was prepared in which the same procedure was followed in the absence of virus. Growth medium was removed 6, 9, and 12h after infection and cells were prepared for microscopy. Resin Embedding. Cells were washed with PBS and fixed with cold glutaraldehyde fix (2.5% in PBS, TAAB Laboratories Equipment, Ltd.) for 1h at 4°C. After fixation, glutaraldehyde was removed and cells were washed with PBS. A 1% osmium tetroxide solution was added to the cells, incubating at room temperature for 1h. Osmium tetroxide was removed and cells were again washed in water. Samples were stained with a 2% uranyl acetate in water (AnalaR) at room temperature for 45min. Cells were scraped into water and pelleted at low-speed centrifugation. A 2% LGT agarose solution was incorporated into the cells, pelleted, and solidified on ice for 45min. The cell pellet was cut away from the rest of the agar and dehydrated with a series of ethanol solutions (Fisher Scientific UK, Ltd). Samples were infiltrated with a TAAB 812 Resin (TAAB Laboratories Equipment, Ltd.) and left at 65°C for two days. Ultrastructural features of calicivirus infection | 2013 78 | P a g e Electron Microscopy. Resin embedded samples were prepared for electron microscopy. Sections of the embedded cells were cut using a Leica EM UC6 microtome, equipped with a 45° diamond knife (DiATOME). Sections were directly absorbed into G200Hex-C3 Ø3.05mm Copper Grids (Gilder Grids) and photographed on a JEOL 1200 EXII transmission electron microscope. Tomographic series of the samples were collected using a tilt range of 120° ( ) in a JEOL 2200 FS Cryomicroscope equipped with Gatan 626 specimen rod. Three-dimensional image reconstruction. Tomograms were calculated by using etomo in the IMOD software package. Subtomograms were analyzed by using Amira (Visage Imaging GmbH, Berlin, Germany). A bilateral filter was applied to tomograms and contrast invert using SPIDER technology (System for Processing Image Data from Electron Microscopy and Related fields, Wadsworth Centre, Albany, New York, USA) The reconstructions were then segmented into interior and membrane regions. 3. Results FCV infected cells show several characteristic features of positive-sense RNA virus infection. FCV-infected and mock-infected FEA cells were embedded in resin, sectioned and observed in TEM. FCV-infected cells showed typical signs of pathology at 9 and 12 hours post-infection. Mock-infected cells are generally presented in an elongated shape, displaying a bold and regular nuclear outline and homogeneous chromatin density, with a highly organized cytoplasm (Fig4.1.A). On the other hand, FCV-infected cells presented with a rounder shape, with an uneven nuclear outline and irregular morphology – in some cases the nuclear envelope appears broken or fragmented, with protrusions of nuclear material into the cytoplasm, and fragmented. The cytoplasm is often more disordered with swollen organelles (Fig.4.1B). Virus particles were only observed in FCV-infected cells. FCV virions were mainly observed in three different arrangements: small non-crystaline clusters, paracrystaline arrays (arrow in Fig.4.1C), and in association with membranes (arrow in Fig.4.1D). Virus particles often appeared to be associated with the vesicles, and their number per cell increased relative to the durantion infection. In later stages of infection, virus appeared to be packed in unusual cisternae and again in close association to the membrane (Fig.4.1E and F). Ultrastructural features of calicivirus infection | 2013 79 | P a g e Figure 4.1 – FCV cytopathic effect in FEA cells in EM using epoxy resin embedding preparation. (A) non infected FEA cells, (B) FCV-infected FEA cells 9h post-infection. (C), paracrystaline arrays of FCV particles (arrow). (D) membrane-associated FCV particles (arrow). (E and F) unusual shaped cisternae containing FCV particles. (G and H) FCV-induced membrane-bound vesicles. Ultrastructural features of calicivirus infection | 2013 80 | P a g e Vesicles were frequently located near to a virus cluster, presenting a broad range of sizes with a spherical or elliptical shape (Fig.4.1G). These vesicles appeared to have a single membrane bilayer – in contrast to the mitochondria membranes – and to have irregularly shaped dark material generally located at the centre of the vesicle (Fig.4.1H). All these features are characteristic of positive-sense RNA viruses as reviewed in the chapter 1. Golgi apparatus disappearance may be related to the formation of FCVinduced vesicles. Cytoplasm disorganization was shown to increase over the time of infection. Organelle swallowing was accompanied by an increase in the numbers of vesicles and virus particles per cell. In particular, two organelles showed significant changes throughout the infection: the Golgi apparatus and the ER. In mock-infected cells, the Golgi apparatus was easily observed (Fig.4.2A). However, in FCV-infected cells, the Golgi apparatus became more difficult to identify. Still, some cellular structures in early stages of infection resembled the Golgi apparatus. Such an example is the expanded tubular complex associated with smaller vesicles (shown in Fig.4.2B). In mock-infected cells, ER was commonly associated with the nucleus but could also be observed far from it. ER in mockinfected cells was present as thin tubular complexes, frequently packed in two or three folds. Although ER was still observed in FCV-infected cells, its morphology was expanded and sometimes it was associated with vesicles that resembled FCV-induced vesicles. It is plausible to admit that since FCV intereferes with the ER, the secretory pathway is also altered, which may lead to the disappearance of the Golgi apparatus. Ultrastructural features of calicivirus infection | 2013 81 | P a g e Figure 4.2 - Golgi Apparatus (white arrow) in non-infected cells (A). Unusual tubular shaped compartments (black arrow) in FCV-infected cells 9h post-infection (B). Tomography and 3D image reconstruction of FCV-infected cells shows that ER is associated with vesicle membranes. In order to recover three-dimensional information on the spatial organization of FCV-induced vesicles within the cell, a TEM tilt series was performed to generate a tomogram (Suplementary Material 1). ER expansion and disorganization is closely detailed in Fig.4.3A, which shows a frame from the tomogram. Vesicles are found near to compartments that resemble the ER (Ve in Fig.4.3A and B) and in close proximity to arrays of virus particles (black arrows in Fig.4.3). A closer look at the ER compartment in relation to the vesicles showed that some of the vesicles appear to be membrane-associated (Fig.4.3C and D). To further investigate the relationship between ER and vesicles, a three-dimensional image reconstruction of this region was generated as described in Materials and Methods. The tomographic reconstruction of the ER-vesicles suggests that four vesicles appear to be in contact with the ER compartment (Fig. 4.3E). Membranes of the ER and FCV-induced vesicles are represented as a continuous membrane (same density) suggesting that they might be directly associated. These vesicles contain a darker material that was reconstructed as disordered matter (yellow regions in Fig.4.3E and F). Material of similar density is also found in the lumen of ER with a more random distribution. The ER membrane appears to be thicker than the vesicle membranes, with a rougher surface in contact with the cytoplasm. Some of the vesicle and ER membranes depicted here appear Ultrastructural features of calicivirus infection | 2013 82 | P a g e to have gaps, suggesting a variation of membrane density or even absence in certain regions. Figure 4.3 – Tomography and 3D image reconstruction of FCV-infected cells shows that ER is associated with vesicle membranes. Slices through a tomogram of a 400nm section of epoxy resin embedded FEA cells infected with FCV (A, B, and C). Ve: FCVinduced membrane-bound vesicles; ER: endoplasmic reticulum; black arrows: FCV Ultrastructural features of calicivirus infection | 2013 83 | P a g e particle arrays. (D) Enlargement of the boxed region in C. (E and F) Three-dimensional reconstruction of FCV induced vesicles in association with ER. FCV-induced vesicles are organized in a three-dimensional complex with connections between them. Within the tomogram, a discrete area containing FCV-induced vesicles was isolated and three-dimensional information was retrieved in order to generate an image reconstruction, where vesicles membranes are represented in blue and internal densities in yellow (Fig.4.4C). FCV-induced vesicles were distributed across the entire depth of the 400nm section. However, the reconstructed vesicles are depicted as sectioned spheres due to the loss of information at the poles caused by the missing wedge effect (Fig.4.4D). Vesicle size varied greatly from 800Å to 2000Å in diameter. No relation was found between the time of infection and the size of vesicles. As previously shown for ER-vesicles reconstructions, vesicles appear to have variations in membrane density and in some cases “holes” are observed in the membrane. The darker material inside the vesicle is not evenly distributed. However, in most cases it is located in the centre with an extended part associated with the membrane (Fig.4.4C). In several cases, vesicles appear to be associated with each other and joined by a small connecting region (black arrows in Fig.4.4B), the extent of which is better explored in the three-dimensional image reconstruction (Fig.4.4E). Overlapping of vesicles appears to occur very often throughout the network; however the contact region is not shown in great detail. A distortion of the membrane was commonly observed at the position of the connection regions between vesicles, resembling a membrane fusion structure (Fig.4.4F). Ultrastructural features of calicivirus infection | 2013 84 | P a g e Figure 4.4 – FCV-induced vesicles are organized in a three-dimensional complex with connections between them. Slices through a tomogram of a 400nm section of epoxy resin embedded FEA cells infect with FCV (A), (B) Enlargement of the boxed region in A. (C to E) Three-dimensional reconstruction of FCV induced vesicles in association with ER Ultrastructural features of calicivirus infection | 2013 85 | P a g e 4. Discussion In this study, we analyzed the early stages of FCV infection in FEA cells using electron transmission microscopy. We identified several features characteristic of FCV infection. In all FCV-infected cells observed in this study, vesicles were present in the cytoplasm. As described previously (Love and Sabine 1975, Green, Mory et al. 2002), this is a common feature of caliciviruses and other positive-sense RNA viruses. The number of these vesicles in cells is time-dependent and they often associate with virus particles, suggesting that they have a role in the development of cytopathogenesis. As shown previously by Green et al. (2002), these membranous structures have enzymatic activity and are able to synthesize FCV RNA in vitro. A study accomplished by Bailey et al. (2010) showed that three non-structural proteins – predicted as transmembrane proteins – are localized to the ER and when co-expressed with a KDEL motif lead to similar membrane rearrangements. Our data suggest that ER and vesicles are directly associated while pronounced rearrangements of ER were observed during infection. Furthermore, the disappearance of the Golgi apparatus in the early stages of infection suggests that FCV may interfere with the vesicular traffic between the ER and the Golgi apparatus. Some of the FCV-induced vesicles were found to be connected by channels or partially fused. However, the vast majority of vesicles appear to be isolated in the cytoplasm. Also, only vesicles with a considerable diameter (>1300nm) have interconnectivity with other vesicles. Some studies on poliovirus reported similar structures that resemble this connecting regions (Schlegel, Giddings et al. 1996, Egger and Bienz 2002). The apparent connecting regions could be a consequence of the low-resolution of reconstruction of epoxy resin embedding sections. These findings suggest that FCVinduced vesicles may derive primarily from ER, although further in the infection process vesicles can fuse, generating larger compartments for viral RNA replication. To determine the structural relationship between ER and FCV-induced vesicles, microscopy studies producing greater image detail should be carried out. In alternative to epoxy resin sections, cryo-electron tomography sectioning can be use; in such techniques, sections of the infected cells are performed from the frozen block that maintain the biological structures in its native conformation, providing high-resolution images (Diebolder, Koster et al. 2012). Ultrastructural features of calicivirus infection | 2013 88 | P a g e Khan, K. N., G. J. Kociba, M. L. Wellman and J. A. Reiter (1992). 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