Document [original]

Citation: Ziersch, M.; Harms, D.;

Neumair, L.; Kurreck, A.; Johne, R.;

Bock, C.-T.; Kurreck, J. Combining

RNA Interference and RIG-I

Activation to Inhibit Hepatitis E Virus

Replication. Viruses 2024,16, 1378.

https://doi.org/10.3390/v16091378

Academic Editors: Danielle Poulin

Porter and Meghan Vermillion

Received: 15 July 2024

Revised: 19 August 2024

Accepted: 27 August 2024

Published: 29 August 2024

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

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Attribution (CC BY) license (https://

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4.0/).

viruses

Article

Combining RNA Interference and RIG-I Activation to Inhibit

Hepatitis E Virus Replication

Mathias Ziersch 1, Dominik Harms 2, Lena Neumair 1, Anke Kurreck 3,4, Reimar Johne 5, C.-Thomas Bock 2

and Jens Kurreck 1,*

1Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany;

[email protected] (M.Z.); [email protected] (L.N.)

Department of Infectious Diseases, Division of Viral Gastroenteritis and Hepatitis Pathogens and Enterovirus,

Robert Koch Institute, 13353 Berlin, Germany; [email protected] (D.H.); [email protected] (C.-T.B.)

3Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany;

[email protected]

4BioNukleo GmbH, Ackerstrasse 76, 13355 Berlin, Germany

5Department of Biological Safety, German Federal Institute for Risk Assessment, 12277 Berlin, Germany;

reimar.johne@bfr.bund.de

*Correspondence: jens.kurr[email protected]; Tel.: +49-30-314-27581

Abstract: Hepatitis E virus (HEV) poses a significant global health threat, with an estimated 20 million

infections occurring annually. Despite being a self-limiting illness, in most cases, HEV infection can

lead to severe outcomes, particularly in pregnant women and individuals with pre-existing liver

disease. In the absence of specific antiviral treatments, the exploration of RNAi interference (RNAi) as

a targeted strategy provides valuable insights for urgently needed therapeutic interventions against

Hepatitis E. We designed small interfering RNAs (siRNAs) against HEV, which target the helicase

domain and the open reading frame 3 (ORF3). These target regions will reduce the risk of viral

escape through mutations, as they belong to the most conserved regions in the HEV genome. The

siRNAs targeting the ORF3 efficiently inhibited viral replication in A549 cells after HEV infection.

Importantly, the siRNA was also highly effective at inhibiting HEV in the persistently infected

A549 cell line, which provides a suitable model for chronic infection in patients. Furthermore, we

showed that a 5

′

triphosphate modification on the siRNA sense strand activates the RIG-I receptor, a

cytoplasmic pattern recognition receptor that recognizes viral RNA. Upon activation, RIG-I triggers a

signaling cascade, effectively suppressing HEV replication. This dual-action strategy, combining the

activation of the adaptive immune response and the inherent RNAi pathway, inhibits HEV replication

successfully and may lead to the development of new therapies.

Keywords: HEV; siRNA; RNAi therapy; RIG-I; RNA 5′triphosphate

1. Introduction

Hepatitis E virus (HEV) is a significant contributor to acute viral hepatitis cases

globally, affecting approximately 20.1 million individuals annually. It is estimated

that HEV infection causes 70,000 deaths due to liver failure and 3000 miscarriages

each year [

]. Various HEV strains with different regional distributions have been

isolated. The prevalence of genotype 1 (HEV-1) is notably high in Africa and Asia,

while genotype 2 (HEV-2) predominates in specific African regions and Mexico. The

most common mode of transmission is through contaminated drinking water, which

perpetuates outbreaks through the fecal–oral route [

]. HEV-3 and HEV-4 are primarily

observed in developed countries. HEV-4 is geographically restricted to Southeast Asia,

while HEV-3 is distributed worldwide, and both are transmitted zoonotically via food-

borne routes [

]. A recent meta-analysis indicates that approximately 12.5% of the

Viruses 2024,16, 1378. https://doi.org/10.3390/v16091378 https://www.mdpi.com/journal/viruses

Viruses 2024,16, 1378 2 of 19

global population experiences HEV infection during their lifetimes, as discerned through

positive anti-HEV IgG antibody tests [5].

The majority of HEV infections resolve spontaneously. However, 5–30% may progress

to acute icteric hepatitis, usually within six weeks in immunocompetent individuals [

Chronic hepatitis may ensue among certain immunocompromised cohorts, notably solid

and hematopoietic transplant recipients, in human immunodeficiency virus (HIV) co-

infections or during chemotherapy [

–

]. Chronic hepatitis E is defined by the persistence

of HEV RNA in the blood or stool for a period exceeding three months, with the poten-

tial for dire complications, including liver cirrhosis or failure, if viremia persists [

Severe acute HEV-1 infections are particularly prevalent among pregnant women in their

third trimester,

with the potential for significant complications, including liver failure, fetal

loss, and mortality rates of up to 25% [12].

HEV is an icosahedral RNA virus excreted as non-enveloped particles (27–34 nm)

in stool via the bile. Quasi-enveloped particles are found in the blood. Its positive-sense

(+) single-stranded (ss) RNA genome (7.2 kb) contains a 5

′

-7-methylguanosine (5

′

-m7G)-

cap, followed by a 5

′

-untranslated region (UTR), three open reading frames (ORFs), and

polyadenylation (Figure 1A). ORF1 encodes non-structural polyproteins, including func-

tional domains like the methyltransferase (Met), X domain, helicase (Hel), and RNA-

dependent RNA polymerase (RdRP). A cis-acting element between ORF1 and ORF2 con-

trols subgenomic RNA expression [

]. This RNA codes for ORF2 (capsid protein) and

ORF3 (relevant for virus egress) [

–

]. Mutations that impair the activity of ORF3 reduce

the release of virions. In fact, a mutated HEV without ORF3 was unable to cause a pro-

ductive infection in mice [

]. HEV-1 putative ORF4, regulated by an internal ribosome

entry site (IRES), overlaps with X and Hel domains and is activated under endoplasmic

reticulum (ER) stress [19].

There are only limited options for the treatment of HEV infections. Reduction

of immunosuppressive drugs in patients with HEV-induced chronic hepatitis post-

organ transplantation clears the virus in over 30% of cases [

]. Pegylated interferon

(PegIFN

) serves as an alternative, but it is not recommended in kidney transplant

patients due to an increased risk of acute renal failure [

]. Currently, there are no

approved drugs for the treatment of HEV, but ribavirin (RBV), a guanosine nucleoside

analog, is sometimes used off-label for chronic hepatitis E treatment [

]. However,

RBV therapy, typically lasting three to six months, can induce dose-dependent anemia

and other adverse effects [

]. There are also alarming indications that RBV treatment

may favor HEV mutagenesis, potentially reducing treatment efficacy and the emergence

of escape mutants [26–28].

A systemic review by Gorris et al. showed that chronic HEV infections were predom-

inantly of the HEV-3 genotype (97%). Negative serum HEV RNA levels for a minimum

of 3 months post-RBV-treatment cessation were achieved in about 76% of patients after

RBV treatment. A relapse was experienced in 18%, with further treatment needed, and a

non-response was observed in 6% of all cases [

]. Patients failing ribavirin therapy have

no further treatment options.

One effective strategy to hinder viral replication involves employing RNA interference

(RNAi), a well-conserved and highly efficient mechanism of post-transcriptional gene

silencing. RNAi is initiated by short double-stranded RNA molecules, such as small

interfering RNA (siRNA), which prompt the degradation of mRNA in a sequence-specific

manner, ultimately leading to the specific inhibition of gene expression [30,31].

Several studies have explored RNAi strategies against the HEV. Huang et al. targeted

the RdRP sequence in HEV-4 [

], while Kumar et al. aimed at the sequence in HEV-1 [

Huang et al. targeted ORF2 in HEV-4 [

], and Liu et al. focused on ORF3 in HEV-4 [

Zhang et al., utilizing adeno-associated virus (AAV), investigated multiple HEV sequences

(except ORF3) in HEV-3 [36]. A summary can be seen in Table 1.

Viruses 2024,16, 1378 3 of 19

Viruses 2024, 16, x FOR PEER REVIEW 2 of 19

experiences HEV infection during their lifetimes, as discerned through positive anti-HEV

IgG antibody tests [5].

The majority of HEV infections resolve spontaneously. However, 5–30% may

progress to acute icteric hepatitis, usually within six weeks in immunocompetent

individuals [6]. Chronic hepatitis may ensue among certain immunocompromised

cohorts, notably solid and hematopoietic transplant recipients, in human

immunodeficiency virus (HIV) co-infections or during chemotherapy [7–9]. Chronic

hepatitis E is defined by the persistence of HEV RNA in the blood or stool for a period

exceeding three months, with the potential for dire complications, including liver cirrhosis

or failure, if viremia persists [10,11]. Severe acute HEV-1 infections are particularly

prevalent among pregnant women in their third trimester, with the potential for

significant complications, including liver failure, fetal loss, and mortality rates of up to

25% [12].

HEV is an icosahedral RNA virus excreted as non-enveloped particles (27–34 nm) in

stool via the bile. Quasi-enveloped particles are found in the blood. Its positive-sense (+)

single-stranded (ss) RNA genome (7.2 kb) contains a 5′-7-methylguanosine (5′-m7G)-cap,

followed by a 5′-untranslated region (UTR), three open reading frames (ORFs), and

polyadenylation (Figure 1A). ORF1 encodes non-structural polyproteins, including

functional domains like the methyltransferase (Met), X domain, helicase (Hel), and RNA-

dependent RNA polymerase (RdRP). A cis-acting element between ORF1 and ORF2

controls subgenomic RNA expression [13]. This RNA codes for ORF2 (capsid protein) and

ORF3 (relevant for virus egress) [14–16]. Mutations that impair the activity of ORF3 reduce

the release of virions. In fact, a mutated HEV without ORF3 was unable to cause a

productive infection in mice [17,18]. HEV-1 putative ORF4, regulated by an internal

ribosome entry site (IRES), overlaps with X and Hel domains and is activated under

endoplasmic reticulum (ER) stress [19].

Figure 1. Organization of the HEV genome and siRNA target selection. (A) Overview of the HEV

genome. The (+)ssRNA genome is 7.2 kb long. It has a 7-methylguanosine cap at the 5′ end and

Figure 1. Organization of the HEV genome and siRNA target selection. (A) Overview of the

HEV genome. The (+)ssRNA genome is 7.2 kb long. It has a 7-methylguanosine cap at the 5

′

end and polyadenylation at the 3

′

-end. There are three conserved ORFs. ORF1 encodes the non-

structural polyproteins and has several functional domains: methyltransferase (Met), Y domain,

papain-like cysteine protease (PCP), hypervariable region (HVR), X domain, helicase (Hel), and RNA-

dependent RNA polymerase (RdRP). ORF2 encodes the capsid structural protein. ORF3 encodes

a multi-functional phosphoprotein. Expression is mediated by a 7.2 kb subgenomic bicistronic

RNA. In addition to these three ORFs, HEV-1 has an ORF4 that overlaps the X and Hel domains.

(B) Alignment

of all HEV-3 subgenotypes to identify conserved regions within the genotype. Non-

matching sequences are shown in black—conserved regions in gray. (C) Representation of suitable

siRNA target sequences in the helicase and ORF3 regions. Identification was performed using the

online programs Eurofins Genomics (blue), Horizon Discovery (red), OligoWalk Web Server (green),

and siDirect2.0 (purple). Selected siRNA target sequences are shown in yellow.

Table 1. Summary of already published siRNA and shRNA against HEV.

Genotype Organism Target Reference

4 Pig RdRp Huang et al. [32]

1 Human Hel, RdRp, 3′CAE Kumar et al. [33]

4 Human ORF2 Huang et al. [34]

4 Pig ORF3 Liu et al. [35]

3 Human Met, Y, HVR, X, Hel, RdRp, ORF2 Zhang et al. [36]

In our study, we focused on conserved regions in the HEV-3 genome, as the previously

mentioned RBV treatment may lead to mutations that can alter the target sequence of

siRNAs, leading to a significant decrease in RNAi efficacy [37,38].

Viruses 2024,16, 1378 4 of 19

The cytosolic pattern recognition receptor RIG-I is essential for cellular defense against

RNA viruses by initiating the early innate immune response, including the activation of

type-I-interferon. It detects double- and single-stranded RNA with a 5

′

-triphosphate group,

which acts as a ligand for RIG-I [

–

]. Findings demonstrate the negative impact of HEV

proteins on the RIG-I signaling pathway [

–

], suggesting that HEV may circumvent

the host antiviral response via this pathway. Xu et al. used lentiviral transduction to

overexpress RIG-I in HEV-infected Huh7.5, A549, and HepaRG cells and significantly

reduced HEV replication. This is corroborated by the observation that targeted RNAi-

mediated silencing of RIG-I in A549 cells led to a significant increase in HEV RNA levels.

Moreover, they reported that the activation of RIG-I signaling by 5

′

-triphosphate RNA

diminished HEV replication in infected A549 cells [

]. Inhibition of influenza A virus [

]

and hepatitis B virus replication [

] was observed when employing a 5

′

-triphosphate-

modified siRNA, as opposed to a standard siRNA. However, no such strategy has yet been

reported for HEV.

The present study describes the design of an efficient siRNA against a conserved

region of the HEV genome. In an initial experiment, we show that this siRNA prevents

viral spread when transfected into the cells prior to infection. Importantly, it also efficiently

inhibits the virus in a persistently infected cell line. The persistent infection represents the

chronic infection found in patients. To prevent viral escape by simple mutations in the

siRNA target site, we produced a bivalent therapy against HEV by combining an siRNA

with a triphosphate at the 5

′

end of the sense strand. The triphosphate inhibited the virus,

even when attached to an siRNA control sequence. We thus developed a highly efficient

silencing strategy against HEV, which is based on two mechanisms to minimize the risk of

viral escape.

2. Materials and Methods

2.1. Design of siRNAs and Plasmid Construction

All HEV-3 subtype reference genomes (Table S1) were aligned using the Geneious

Prime 2021.2.2 (GraphPad Software LLC, Boston, MA, USA) to identify conserved

regions [

]. The sequence of the helicase in ORF1 and the sequence for the multi-

functional ORF3 protein on the HEV genome were identified as suitable targets for the

siRNAs. The patient-derived 47832c strain (GenBank: KC618403) was used as a reference

sequence to design suitable siRNAs. This HEV-3c subtype is adapted to cell culture, and

an established persistently infected cell line could be used to mimic chronic infection.

This strain was, therefore, selected for all further experiments in the current study. The

siRNA design was done in silico using multiple online software tools (Horizon Discovery,

Eurofins Genomics, OligoWalk [

], siDirect [

]) to take into account both the sequence

and structure of the target RNA [

]. Regions with overlapping siRNAs were chosen as

siRNA targets, and a total of 6 siRNAs were selected for further analysis. The Nucleotide

BLAST program was utilized with default parameters to confirm the absence of seed

sequences in the human transcriptome. The control siRNA, siCon, was verified to

not align with any sequences found in the viral or human genome [

]. All siRNAs

were purchased from Microsynth AG (Balgach, Switzerland), and their corresponding

sequences are shown in Table 2.

The silencing efficacy of the siRNAs was assessed through reporter assays. Two distinct

vectors were generated for each target, both expressing the firefly luciferase reporter.

The HEV-3c-ORF3 and HEV-3c-Hel sequence, both derived from the patient-derived and

cell culture-adapted virus sequence of the 47832c strain, were synthesized by Thermo

Fischer Scientific (Invitrogen, Carlsbad, CA, USA) and sequences were inserted individually

downstream of the Renilla luciferase gene of the psiCHECK™ 2 vector (Promega, Madison,

WI, USA) plasmid using the XhoI/NotI restriction sites in the MCS.

Viruses 2024,16, 1378 5 of 19

Table 2. Sequences of designed siRNAs.

Name Target siRNA-Sequence

siORF3.1 ORF3 5′-GGGCUGUUCUGUUGCUGUUTT-3′

3′-TTCCCGACAAGACAACGACAA-5′

siORF3.2 ORF3 5′-GGGUUGAUUCUCAGCCCUUTT-3′

3′-TTCCCAACUAAGAGUCGGGAA-5′

siORF3.3 1ORF3 5′-CCUAUAUUCAUCCAACCAATT-3′

3′-TTGGAUAUAAGUAGGUUGGUU-5′

siHel.1 Helicase 5′-GGAUGUUGAUGUGGUGGUUTT-3′

3′-TTCCUACAACUACACCACCAA-5′

siHel.2 Helicase 5′-ACCGCAUUUGUUGCUACUATT-3′

3′-TTUGGCGUAAACAACGAUGAU-5′

siHel.3 Helicase 5′-ACUUUCACGGAGACUACAATT-3′

3′-TTUGAAAGUGCCUCUGAUGUU-5′

siCon no target 5′-ACGUGACACGUUCGGAGAATT-3′

3′-TTUGCACUGUGCAAGCCUCUU-5′

1Sequence is identical to ORF3-siRNA2 from Lui et al. [35].

2.2. Cell Culture

The A549 subclone A549/D3, which is highly susceptible to infection with HEV strain

47832c [

] and A549 cells that have been persistently infected with HEV strain 47832c

(A549/pers-HEV) [

] cells were cultured in minimum essential medium (MEM, Biowest,

Nuaillé, France) supplemented with 10% fetal calf serum (FCS, c.c.pro, Oberdorla, Ger-

many) for A549/D3 and with 2% FCS for A549/pers-HEV, 2 mM GlutaMAX (Gibco, Life

Technologies Corporation, Paisley, UK), 1% MEM non-essential amino acids (NEAA, Biow-

est), and 1% penicillin–streptomycin (Biowest). HeLa cells (ACC 57, DSMZ, Braunschweig,

Germany) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Biowest)

supplemented with 10% fetal calf serum, 2 mM GlutaMAX, and 1% NEAA. All cell lines

were cultured in T25 or T75 flasks in a 5% CO2incubator at 37 ◦C.

2.3. Dual Luciferase Reporter Assay

HeLa cells at 10

cells per well were plated in 24-well plates. After 24 h, co-transfection

was carried out by adding 500 ng of the reporter plasmid psiCheck2-HEV-3c-ORF3 or

psiCheck2-HEV-3c-Hel along with 40 nM siRNA, using Lipofectamine™ 2000 (Thermo

Fisher Scientific, Invitrogen) according to the manufacturer’s guidelines. The efficacy

of siRNAs in silencing was evaluated at 48 h post-transfection by measuring relative

luciferase activity using the Dual-Luciferase Reporter Assay System (Promega), according

to the manufacturer’s instructions.

2.4. Synthesis of 5′-Triphosphorylated siRNA

The 5

′

-triphosphorylated sense strands of the siRNAs (3p-siRNAs) were synthesized

through

in vitro

transcription employing the AmpliScribe™ T7-Flash™ Transcription-

skit (LGC Biosearch Technologies, Lucigen Corporation, Middelton, WI, USA). Single-

stranded RNA antisense oligonucleotides were ordered from Microsynth AG. Template

DNA oligonucleotides with a T7 RNA polymerase promoter sequence followed by the

siRNA sense sequence (Table S2) were ordered from Thermo Fisher Scientific (Invitrogen)

and hybridized by incubation at 95

◦

C for two minutes, followed by cooling to 25

◦

C at

−

◦

C/minute. The 3p-sense strand of the siRNA was produced by

in vitro

transcription

at 37

◦

C for 16 h according to the manufacturer’s instructions. The DNA template was

then digested with DNase I, and the resulting 3p-sense strand was purified using the

Monarch RNA Cleanup (New England Biolabs, Ipswich, MA, USA). Its concentration

was measured using the NanoDrop™ 2000 (Thermo Fisher Scientific, Invitrogen). The

3p-sense strand was then paired equimolar with the antisense strand. After denaturation at

75 ◦C

for two minutes, the mixture was cooled to 25

◦

C at

−

◦

C/minute. Non-hybridized

strands were digested with RNase A (Macherey-Nagel, Düren, Germany) to ensure only

Viruses 2024,16, 1378 6 of 19

double-stranded 3p-siRNAs, followed by purification using the Monarch RNA Cleanup

Kit. The final concentration was determined using NanoDrop. To verify that the 3p-siRNAs

were double-stranded, urea polyacrylamide gel electrophoresis (PAGE) was performed. A

15% urea polyacrylamide gel was loaded with 1

g of each sample and separated at 100 V

for approximately two hours. Gels were stained with ethidium bromide for 20 min and

visualized with the Molecular Imager Gel Doc (Bio-Rad Laboratories, Hercules, CA, USA)

using the Image Lab Software 6.1 (Bio-Rad Laboratories).

2.5. Transfection of siRNA and 3p-siRNA

For transfection, A549/D3 cells were seeded at 2.5

cells per well in 12-well

plates and 0.75

cells per well in 24-well plates, reaching 60–70% confluence after

24 h for transfection. A549/pers-HEV cells were seeded at 1.25

cells per well the

day before transfection. The siRNAs were transfected at a concentration of 50 nM using

Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Invitrogen)

in either 24-well or 12-well plates, according to the manufacturer’s protocol. Similarly,

polyinosin-polycytidylacid (Poly(I:C) HMW, InvivoGen, Toulouse, France) was transfected

at concentrations of 0.5 or 0.25 ng/mL using Lipofectamine™ 2000 (Thermo Fisher Scientific,

Invitrogen) as a control for RIG-I activation. Cells were incubated at 37

◦

C for 24 or 48 h,

as indicated.

2.6. HEV-3c Production Using A549/Pers-HEV Cells

HEV particles were harvested as previously described [

] with the modification that

persistent A549/pers-HEV cells were used, and the HEV-3c strain 47832c was isolated.

Briefly, cells were washed with PBS, trypsinized, and resuspended in 500

L fresh

2% MEM

medium. This was followed by three freeze–thaw cycles to lyse the cells and release

intracellular virus particles. The cell components were centrifuged at 16,000

gfor

15 min

and the supernatant containing virus particles was transferred to a fresh reaction tube

and stored at

−

◦

C. Infectious titers were determined by virus titration, followed by

immunofluorescence, as previously described [

]. In contrast, infection with intracellular

viral particles was performed at 37◦, 5% CO2overnight (16 h).

2.7. siRNA Antiviral Replication Assay

For HEV inhibition assays, A549/D3 cells were transfected with each of the siRNAs

as described previously 24 h before the virus challenge. Infection with intracellular virus

particles (HEV-3c strain 47832c) was performed at a multiplicity of infection (MOI) of 1.0

for 16 h. The infection was stopped by changing the medium and washing the cells with

PBS. Supernatants and intracellular viral RNA were harvested 48 h or 96 h post-inoculation.

Viral RNA was extracted from cell culture supernatant with the QIAamp Viral RNA Mini

Kit (Qiagen, Hilden, Germany) and from cells with the RNeasy Mini Kit (Qiagen) according

to the manufacturer’s instructions.

2.8. Reverse Transcription Quantitative PCR of HEV Samples

Viral RNA was quantified via HEV-specific reverse transcription quantitative PCR (RT-

qPCR), specifically amplifying a conserved 70-nucleotide (nt) region within the ORF2/ORF3

genes, as previously reported [

]. An established internal standard was amplified in

parallel to calculate the copy number with the corresponding cycle threshold (CT) value [

RT-qPCR was conducted on a LightCycler

480 Instrument (Roche, Basel, Switzerland) or

on a CFX Opus 96 Real-Time PCR System (Bio-Rad Laboratories) utilizing the LightCycler

Multiplex RNA Virus Master Kit (Roche). The reaction mixture, totaling 20

L, consisted

of 1

qRT-PCR Reaction Mix, 0.2

mol/L probe, 0.4

mol/L of primers, 1

RT Enzyme

Solution, and 5

L of the sample. Cycling conditions were set as follows: 15 min at 50

◦

2 min at 95 ◦C, followed by 45 cycles of [10 s at 95 ◦C, 30 s at 63 ◦C].

Viruses 2024,16, 1378 7 of 19

2.9. Immunoblotting

A549/D3 cells in 12-well plates were washed twice with cold PBS 48 h after trans-

fection and lysed in 50

L RIPA buffer (Thermo Scientific, Pierce, Rockford, IL, USA)

supplemented with Halt™ phosphatase inhibitor cocktail (Thermo Scientific, Pierce™). Pro-

tein concentration was determined using the Pierce™ BCA protein assay (Thermo Scientific,

Pierce™) according to the manufacturer’s instructions. Protein separation was performed

by SDS-PAGE using Mini-PROTEAN TGX Stain-Free gels (Bio-Rad Laboratories). Protein

samples were prepared for running on polyacrylamide gels by adding 4X LDS sample

buffer and heating at 95

◦

C for five minutes. Subsequently, 60

g of the samples and 3

L of

CozyHiTM Prestained Protein Ladder (highQu GmbH, Kraichtal, Germany) were loaded

onto the gel. Proteins were transferred from the SDS gel to a PVDF membrane using the

semi-dry blotting method at 70 mA for 75 min. Subsequently, blocking was performed for

one hour at room temperature in 1% BSA blocking buffer. Incubation with primary anti-

bodies against RIG-I (#3743-1:1000, Cell Signaling Technologies, Danvers, MA, USA) and

actin (#A5441–1:15,000, Sigma-Aldrich, Saint Louis, MO, USA) diluted in blocking buffer

was performed overnight at 4

◦

C. The membranes were then washed three times with TBS

T and incubated with HRP-conjugated secondary antibodies anti-rabbit (

#31460–1:10,000

Thermo Scientific, Pierce™) and anti-mouse (#31430–1:10,000, Thermo Scientific, Pierce™)

for 1 h at room temperature. Chemiluminescence detection was performed using the

Pierce ECL Western Blotting Substrate Kit (Thermo Scientific, Pierce™) according to the

manufacturer’s instructions, and images were captured on the ChemiDoc™ MP Imaging

System (Bio-Rad Laboratories) using Image Lab Software 6.1 (Bio-Rad Laboratories).

2.10. Statistical Analysis

All data are presented as mean and standard deviation (SD) of three independent

experiments. Statistical significance was determined by Brown–Forsythe and Welch’s

one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons using

GraphPad Prism version 8.0.2 for Windows (GraphPad Software).

3. Results

3.1. Design of HEV-3c-ORF3 and HEV-3c-Helicase Sequence Targeting siRNAs

For the development of siRNAs against HEV, the most common genotype in Europe,

HEV-3 was selected. In order to identify suitable regions for siRNA design, a sequence

alignment of existing HEV genotype 3 subtypes was performed (Figure 1B). Only a few

conserved regions within the HEV-3 genome were identified and, therefore, a large part

of the genome is not suitable as a target sequence for siRNA due to the risk of rapidly

emerging escape mutants. The regions of the helicase and ORF3 appear to be the most

highly conserved and are presumably the least susceptible to escape mutations. Once the

HEV target sequences (helicase and ORF3) were selected, several online programs (Eurofins

Genomics, Horizon Discovery, OligoWalk Web Server, siDirect2.0) were used to identify

functional siRNA for the HEV-3c strain 47832c. Each program uses specific bioinformatic

selection criteria. Figure 1C summarizes the results of all online tools. For each target

region, the three siRNA sequences with the highest overlap within the programs were

selected (Table 2). The Nucleotide BLAST program was utilized with default parameters to

confirm the absence of seed sequences in the human transcriptome. The control siRNA,

siCon, was verified to not align with any sequences found in either the viral or human

genome [53].

3.2. RNAi-Mediated Targeting of the ORF3 and Helicase of HEV in Reporter Assays

The activity of the selected siRNAs was evaluated via a dual luciferase assay (DLA).

The target sequence HEV-3c ORF3 or HEV-3c helicase was cloned into the psiCheck2 vector

(Figure 2A) and co-transfected into HeLa cells with the corresponding siRNAs. The relative

Renilla/Firefly activity was quantified 48 h after transfection, normalized against a non-

regulatory control (siCon), and set to 100%. A significant reduction, each of approximately

Viruses 2024,16, 1378 8 of 19

90% in Renilla luciferase expression, was achieved with the three helicase-targeting siRNAs.

Of the siRNAs targeting the ORF3, only two of the three were found to be active. The

reduction in Renilla luciferase expression was ~98.6

0.22% for siORF3.1 and ~96.0

1.4%

for siORF3.3. The two active ORF-specific siRNAs demonstrated a slightly greater efficacy

than the helicase-specific siRNAs. In contrast, no reduction in Renilla luciferase expression

was observed with siORF3.2 siRNA (Figure 2B). The results indicated that both the ORF3

and helicase sequences represent potential target sequences.

Viruses 2024, 16, x FOR PEER REVIEW 8 of 19

relative Renilla/Firefly activity was quantified 48 h after transfection, normalized against

a non-regulatory control (siCon), and set to 100%. A significant reduction, each of approx-

imately 90% in Renilla luciferase expression, was achieved with the three helicase-target-

ing siRNAs. Of the siRNAs targeting the ORF3, only two of the three were found to be

active. The reduction in Renilla luciferase expression was ~98.6 ± 0.22% for siORF3.1 and

~96.0 ± 1.4% for siORF3.3. The two active ORF-specific siRNAs demonstrated a slightly

greater eﬃcacy than the helicase-specific siRNAs. In contrast, no reduction in Renilla lu-

ciferase expression was observed with siORF3.2 siRNA (Figure 2B). The results indicated

that both the ORF3 and helicase sequences represent potential target sequences.

Figure 2. Relative change in luciferase activity by the designed siRNAs. A dual luciferase reporter

assay was used to evaluate the silencing activity of the designed siRNAs. (A) Graphic representation

of the psiCheck2 vectors where GOI is the HEV-3c ORF3 or HEV-3c helicase DNA sequence. (B) Co-

transfection of dual-luciferase vectors (500 ng) and siRNA (50 nM) was performed in HeLa cells.

The relative Renilla/Firefly (Ren/Luc) activity was determined 48 h after transfection. The relative

activity was normalized against a non-regulatory control (siCon) and set to 100%, ns = not signifi-

cant, **** p ≤ 0.0001.

3.3. Inhibition of HEV-3c Replication Using ORF3- and Helicase-Targeting siRNAs in

Persistently Infected A549/Pers-HEV Cells

Having shown that the siRNAs were active in reporter assays, we tested the siRNAs

against infectious HEV. For this, the siRNAs were transfected once into A549/pers-HEV

cells. These cells are persistently infected with the HEV-3c genotype isolated from a pa-

tient and adapted to the A549 cell line [55]. The viral load was determined 48 h after trans-

fection by RT-qPCR. These experiments revealed some results that diﬀered from those

obtained with the dual luciferase assay (Figure 3A). Unexpectedly, no substantial reduc-

tion in viral load was observed for the siRNAs targeting the helicase sequence. In contrast,

the siRNAs siORF3.1 and ORF3.3 directed against the ORF3 sequence showed a pro-

nounced and significant reduction in viral copies down to 40% and 15% relative to the

control, respectively.

Based on these results, the siRNAs ORF3.1 and ORF3.3 were used for further optimi-

zation experiments. The next step was to test whether a longer incubation period and

multiple infections can improve silencing. To this end, A549/pers-HEV cells were trans-

fected with siRNAs, and the viral RNA level was determined 96 h after the initial trans-

fection. In additional experiments, cells were transfected with siRNA 48 h after the first

transfection. The viral RNA was quantified by RT-qPCR. The second transfection 24 h

after the first one resulted in a significantly improved reduction in the number of viral

genomes. As can be seen in Figure 3B, the prolonged cultivation time of 96 h with a single

transfection resulted in a further reduction of the viral load to approximately 9% for

Figure 2. Relative change in luciferase activity by the designed siRNAs. A dual luciferase reporter

assay was used to evaluate the silencing activity of the designed siRNAs. (A) Graphic representation

of the psiCheck2 vectors where GOI is the HEV-3c ORF3 or HEV-3c helicase DNA sequence.

(B) Co

transfection of dual-luciferase vectors (500 ng) and siRNA (50 nM) was performed in HeLa cells.

The relative Renilla/Firefly (Ren/Luc) activity was determined 48 h after transfection. The relative

activity was normalized against a non-regulatory control (siCon) and set to 100%, ns = not significant,

**** p≤0.0001.

3.3. Inhibition of HEV-3c Replication Using ORF3- and Helicase-Targeting siRNAs in Persistently

Infected A549/Pers-HEV Cells

Having shown that the siRNAs were active in reporter assays, we tested the siRNAs

against infectious HEV. For this, the siRNAs were transfected once into A549/pers-HEV

cells. These cells are persistently infected with the HEV-3c genotype isolated from a

patient and adapted to the A549 cell line [

]. The viral load was determined 48 h after

transfection by RT-qPCR. These experiments revealed some results that differed from

those obtained with the dual luciferase assay (Figure 3A). Unexpectedly, no substantial

reduction in viral load was observed for the siRNAs targeting the helicase sequence. In

contrast, the siRNAs siORF3.1 and ORF3.3 directed against the ORF3 sequence showed a

pronounced and significant reduction in viral copies down to 40% and 15% relative to the

control, respectively.

Based on these results, the siRNAs ORF3.1 and ORF3.3 were used for further op-

timization experiments. The next step was to test whether a longer incubation period

and multiple infections can improve silencing. To this end, A549/pers-HEV cells were

transfected with siRNAs, and the viral RNA level was determined 96 h after the initial

transfection. In additional experiments, cells were transfected with siRNA 48 h after the

first transfection. The viral RNA was quantified by RT-qPCR. The second transfection

24 h after the

first one resulted

in a significantly improved reduction in the number of

viral genomes. As can be seen in Figure 3B, the prolonged cultivation time of 96 h with a

single transfection resulted in a further reduction of the viral load to approximately 9% for

siORF3.1 and siORF3.3. A double transfection improved the inhibition to approximately

5% (Figure 3B).

Viruses 2024,16, 1378 9 of 19

Viruses 2024, 16, x FOR PEER REVIEW 9 of 19

siORF3.1 and siORF3.3. A double transfection improved the inhibition to approximately

5% (Figure 3B).

Figure 3. Changes in viral copy number by siRNA treatment of A549/pers-HEV cells. The viral RNA

copy number was determined by qPCR after siRNA transfection of A549/pers-HEV cells. Transfec-

tion of 50 nM siRNA was performed 24 h after cell seeding. (A) At 48 h post-transfection, viral RNA

was isolated, and the viral copies/mL were determined by qPCR. (B) A second transfection (TF) was

then conducted 48 h after seeding. Viral RNA was isolated 96 h after the initial transfection, and the

viral RNA was determined by RT-qPCR. The viral RNA was normalized against a non-regulatory

control (siCon) and set to 100%. Mean ± SD of three independent experiments (n = 3) are shown. ns

= not significant, *** p ≤ 0.001, **** p ≤ 0.0001.

3.4. Inhibition of HEV-3c Replication with Antiorf3-siRNAs Followed by HEV Infection

In addition to the A549/pers-HEV cell line, we also investigated whether the func-

tional siRNAs could inhibit HEV replication in A549/D3 cells. A549/D3 cells were seeded

and transfected with 50 nM siRNA after 24 h. After an additional 24 h, the A549/D3 cells

were infected with HEV isolates from A549/pers-HEV cells at a MOI of 1.0. At 96 h post-

infection, viral RNA was isolated, and viral load was determined by qPCR. Both siRNAs

resulted in a significant inhibition of viral replication by approximately 96%. These exper-

iments confirm the high potency of the designed siRNAs (Figure 4A).

Subsequently, the concentration-dependency of the inhibition of viral replication by

siORF3.1 was performed. Diﬀerent concentrations of the siRNA ranging from 0.01 to 50

nM were transfected. HEV infection was again performed at an MOI of 1 using HEV iso-

lates from A549/pers-HEV cells. Figure 4B shows a clear concentration-dependent reduc-

tion of the HEV RNA level. Virus inhibition was still approximately 65% at a concentration

as low as 0.01 nM, indicating a very high antiviral potency of siORF3.1.

Figure 3. Changes in viral copy number by siRNA treatment of A549/pers-HEV cells. The viral

RNA copy number was determined by qPCR after siRNA transfection of A549/pers-HEV cells.

Transfection of 50 nM siRNA was performed 24 h after cell seeding. (A) At 48 h post-transfection,

viral RNA was isolated, and the viral copies/mL were determined by qPCR. (B) A second transfection

(TF) was then conducted 48 h after seeding. Viral RNA was isolated 96 h after the initial transfection,

and the viral RNA was determined by RT-qPCR. The viral RNA was normalized against a non-

regulatory control (siCon) and set to 100%. Mean

SD of three independent experiments (n= 3) are

shown. ns = not significant, *** p≤0.001, **** p≤0.0001.

3.4. Inhibition of HEV-3c Replication with Antiorf3-siRNAs Followed by HEV Infection

In addition to the A549/pers-HEV cell line, we also investigated whether the functional

siRNAs could inhibit HEV replication in A549/D3 cells. A549/D3 cells were seeded and

transfected with 50 nM siRNA after 24 h. After an additional 24 h, the A549/D3 cells were

infected with HEV isolates from A549/pers-HEV cells at a MOI of 1.0. At 96 h post-infection,

viral RNA was isolated, and viral load was determined by qPCR. Both siRNAs resulted

in a significant inhibition of viral replication by approximately 96%. These experiments

confirm the high potency of the designed siRNAs (Figure 4A).

Subsequently, the concentration-dependency of the inhibition of viral replication by

siORF3.1 was performed. Different concentrations of the siRNA ranging from 0.01 to 50 nM

were transfected. HEV infection was again performed at an MOI of 1 using HEV isolates

from A549/pers-HEV cells. Figure 4B shows a clear concentration-dependent reduction of

the HEV RNA level. Virus inhibition was still approximately 65% at a concentration as low

as 0.01 nM, indicating a very high antiviral potency of siORF3.1.

Viruses 2024,16, 1378 10 of 19

Viruses 2024, 16, x FOR PEER REVIEW 10 of 19

Figure 4. Inhibition of HEV replication by siRNA in infected A549/D3 cells. Cells were transfected

and subsequently infected with viral isolates. At 96 h post-infection, viral RNA was isolated, and

viral copies/mL were determined by qPCR. The viral copy number was normalized against a non-

regulatory control (siCon) and set to 100%. (A) Transfection with 50 nM siRNA was performed 24 h

after seeding. Infection was performed 24 h after transfection with viral isolates at an MOI of 1.0.

(B) The A549/D3 cells were transfected with diﬀerent concentrations (0.01 to 50 nM) of siORF3.1,

and 24 h after transfection, the cells were infected with virus isolates at a MOI of 1.0 for 16 h. Mean

± SD of three independent experiments (n = 3) are shown. ns = not significant, ** p ≤ 0.01, **** p ≤

0.0001.

3.5. RIG-I Activation and Inhibition of HEV Replication with 5′ Triphosphate siRNA

HEV proteins have been shown to negatively aﬀect the RIG-I signaling pathway [42–

44]. A known RIG-I ligand, 5′-triphosphate RNA, was found to decrease HEV replication

in infected A549 cells [45]. To exploit this eﬀect, a strategy was developed in which tri-

phosphorylated siRNA carries this modification only on the sense strand (Figure 5A), as

it is removed from the antisense strand during RISC assembly by the Ago2 protein and is

not required for silencing [60].

As the chemical synthesis of an siRNA strand with a 5′-3p is complex, the sense

strand of the siRNA carrying the 5′-3p was generated by in vitro transcription and an-

nealed to a chemically synthesized antisense strand. Using urea-PAGE, both 3p-siRNAs

(3p-siORF3.1, 3p-siCon) were shown to be double-stranded after hybridization, RNase di-

gestion, and purification (Figure 5B). To test the functionality of the 3p-siRNAs, the 3p-

siRNAs (3p-siORF3.1 and 3p-siCon) and the purchased siRNAs (siORF3.1 and siCon)

were transfected into A549/D3 cells. Poly I:C (0.5 and 0.25 ng/mL) was also transfected as

a positive control, as it is known to activate RIG-I [61]. The cells were lysed 48 h after

transfection and analyzed by PAGE and Western blot. Both 3p-siRNAs (3p-siORF3.1 and

3p-siCon) and poly I:C transfection resulted in strong expression of RIG-I in A549/D3 cells

(Figure 5C). Furthermore, RIG-I activation appears to be concentration dependent, as the

lowest concentration of 10 nM for both 3p-siRNAs did not result in any detectable activa-

tion of RIG-I. Poly I:C at 0.25 ng/mL also failed to induce RIG-I expression. Neither

siORF3.1 (without 5′-triphosphate) nor untreated cells gave a positive band for RIG-I.

A DLA was performed (see Section 3.1) to verify that the newly generated 3p-

siORF3.1 maintained its specific silencing activity against the HEV target site. Relative

Renilla/Firefly activity was quantified 48 h after transfection, normalized against the re-

spective control (siCon, 3p-siCon), and set to 100%. Renilla luciferase expression was sig-

nificantly inhibited by both siORF3.1 and 3p-siORF3.1, with a significant knockdown of

>98% (Figure 5C).

Figure 4. Inhibition of HEV replication by siRNA in infected A549/D3 cells. Cells were transfected

and subsequently infected with viral isolates. At 96 h post-infection, viral RNA was isolated, and

viral copies/mL were determined by qPCR. The viral copy number was normalized against a non-

regulatory control (siCon) and set to 100%. (A) Transfection with 50 nM siRNA was performed

24 h

after seeding. Infection was performed 24 h after transfection with viral isolates at an MOI of 1.0.

(B) The

A549/D3 cells were transfected with different concentrations (0.01 to 50 nM) of siORF3.1, and

24 h after transfection, the cells were infected with virus isolates at a MOI of 1.0 for 16 h. Mean

of three independent experiments (n= 3) are shown. ** p≤0.01, **** p≤0.0001.

3.5. RIG-I Activation and Inhibition of HEV Replication with 5′Triphosphate siRNA

HEV proteins have been shown to negatively affect the RIG-I signaling

pathway [42–44]

A known RIG-I ligand, 5

′

-triphosphate RNA, was found to decrease HEV replication in

infected A549 cells [

]. To exploit this effect, a strategy was developed in which tri-

phosphorylated siRNA carries this modification only on the sense strand (Figure 5A), as it

is removed from the antisense strand during RISC assembly by the Ago2 protein and is not

required for silencing [60].

As the chemical synthesis of an siRNA strand with a 5

′

-3p is complex, the sense

strand of the siRNA carrying the 5

′

-3p was generated by

in vitro

transcription and

annealed to a chemically synthesized antisense strand. Using urea-PAGE, both 3p-

siRNAs (3p-siORF3.1, 3p-siCon) were shown to be double-stranded after hybridization,

RNase digestion, and purification (Figure 5B). To test the functionality of the 3p-siRNAs,

the 3p-siRNAs (3p-siORF3.1 and 3p-siCon) and the purchased siRNAs (siORF3.1 and

siCon) were transfected into A549/D3 cells. Poly I:C (0.5 and 0.25 ng/mL) was also

transfected as a positive control, as it is known to activate RIG-I [

]. The cells were

lysed 48 h after transfection and analyzed by PAGE and Western blot. Both 3p-siRNAs

(3p-siORF3.1 and 3p-siCon) and poly I:C transfection resulted in strong expression

of RIG-I in A549/D3 cells (Figure 5C). Furthermore, RIG-I activation appears to be

concentration dependent, as the lowest concentration of 10 nM for both 3p-siRNAs did

not result in any detectable activation of RIG-I. Poly I:C at 0.25 ng/mL also failed to

induce RIG-I expression. Neither siORF3.1 (without 5

′

-triphosphate) nor untreated cells

gave a positive band for RIG-I.

A DLA was performed (see Section 3.1) to verify that the newly generated 3p-

siORF3.1 maintained its specific silencing activity against the HEV target site. Relative

Renilla/Firefly activity was quantified 48 h after transfection, normalized against the

respective control (siCon, 3p-siCon), and set to 100%. Renilla luciferase expression was

significantly inhibited by both siORF3.1 and 3p-siORF3.1, with a significant knockdown

of >98% (Figure 5C).

Viruses 2024,16, 1378 11 of 19

Viruses 2024, 16, x FOR PEER REVIEW 11 of 19

Figure 5. Design, synthesis, and functional testing of 5′ triphosphate siRNA. (A) Sequence and struc-

ture of 3p-siORF3.1 and 3p-siCon. The siRNA forms a 19 bp double strand with a UU overhang at

the 3′-end and a triphosphate at the 5′-end of the sense strand. (B) Successful hybridization and

purity of 3p-siRNAs (3p-siORF3.1 and 3p-siCon) were verified by a 15% urea polyacrylamide gel (1

= hybridization, 2 = RNase A digestion, 3 = final purification). The chemically synthesized siRNAs

(siORF3.1 and siCon) and the antisense single strands ssORF3.1 and ssCon were used as controls.

with diﬀerent concentrations of 3p-siCon, 3p-siORF3.1 (10 and 50 nM), poly I:C (0.5 and 0.25 ng/mL)

or siORF3.1 (50 nM). Protein extracts from non-transfected A549/D3 cells served as negative control

(NC). Actin was used as an internal control. (D) Dual-luciferase reporter assays were used to evalu-

ate the silencing activity of the designed siRNAs. Co-transfection of dual-luciferase vectors (500 ng)

and siRNA (50 nM) was performed in HeLa cells. The relative Renilla/Firefly (Ren/Luc) activity was

determined 48 h after transfection. The relative activity was normalized against a non-regulatory

control (siCon) and set to 100%. (E) Inhibition of HEV replication by 3p-siRNA: A549/D3 cells were

transfected with the indicated siRNAs (siCon, siORF3.1, 3p-siCon, 3p-siORF3.1) at a concentration

of 50 nM. The cells were infected with HEV at an MOI of 1.0, 24 h after transfection, and incubated

for 16 h. At 96 h after infection, viral RNA was isolated, and viral load was determined by qPCR.

The viral copy number was normalized against a non-regulatory control (siCon) and set to 100%.

Mean ± SD of three independent experiments (n = 3) are shown. ns = not significant, **** p ≤ 0.0001.

These results demonstrate that 3p-siORF3.1 is active against the target sequence in

the dual-luciferase assay and that both 3p-siRNAs are eﬀective stimulators of RIG-I in

A549/D3 cells. To test the therapeutic eﬃcacy of the modified siRNAs (3p-siORF3.1 3p-

siCon) against HEV, A549/D3 cells were transfected with 50 nM of each of the siRNAs and

subsequently infected with HEV as described in Section 3.4. The unmodified siORF3.1 and

siCon, as well as 0.25 ng/mL poly I:C, served as controls. Viral RNA was isolated 96 h post-

Figure 5. Design, synthesis, and functional testing of 5

′

triphosphate siRNA. (A) Sequence and

structure of 3p-siORF3.1 and 3p-siCon. The siRNA forms a 19 bp double strand with a UU overhang

at the 3

′

-end and a triphosphate at the 5

′

-end of the sense strand. (B) Successful hybridization and

purity of 3p-siRNAs (3p-siORF3.1 and 3p-siCon) were verified by a 15% urea polyacrylamide gel

(

1 = hybridization

, 2 = RNase A digestion, 3 = final purification). The chemically synthesized siRNAs

(siORF3.1 and siCon) and the antisense single strands ssORF3.1 and ssCon were used as controls.

with different concentrations of 3p-siCon, 3p-siORF3.1 (10 and 50 nM), poly I:C (0.5 and 0.25 ng/mL)

or siORF3.1 (50 nM). Protein extracts from non-transfected A549/D3 cells served as negative control

(NC). Actin was used as an internal control. (D) Dual-luciferase reporter assays were used to evaluate

the silencing activity of the designed siRNAs. Co-transfection of dual-luciferase vectors (500 ng) and

siRNA (50 nM) was performed in HeLa cells. The relative Renilla/Firefly (Ren/Luc) activity was

determined 48 h after transfection. The relative activity was normalized against a non-regulatory

control (siCon) and set to 100%. (E) Inhibition of HEV replication by 3p-siRNA: A549/D3 cells were

transfected with the indicated siRNAs (siCon, siORF3.1, 3p-siCon, 3p-siORF3.1) at a concentration

of 50 nM. The cells were infected with HEV at an MOI of 1.0, 24 h after transfection, and incubated

for 16 h. At 96 h after infection, viral RNA was isolated, and viral load was determined by qPCR.

The viral copy number was normalized against a non-regulatory control (siCon) and set to 100%.

Mean ±SD

of three independent experiments (n= 3) are shown. ns = not significant, **** p

≤

0.0001.

These results demonstrate that 3p-siORF3.1 is active against the target sequence in

the dual-luciferase assay and that both 3p-siRNAs are effective stimulators of RIG-I in

A549/D3 cells. To test the therapeutic efficacy of the modified siRNAs (3p-siORF3.1 3p-

siCon) against HEV, A549/D3 cells were transfected with 50 nM of each of the siRNAs and

subsequently infected with HEV as described in Section 3.4. The unmodified siORF3.1 and

Viruses 2024,16, 1378 12 of 19

siCon, as well as 0.25 ng/mL poly I:C, served as controls. Viral RNA was isolated 96 h

post-infection and quantified by RT-qPCR. Results were normalized relative to siCon. As

expected, the modified siORF3.1 resulted in a significant inhibition of HEV replication to

3.1% (Figure 5D). Interestingly, compared to the control (siCon), 3p-siCon also resulted

in a downregulation of HEV expression to 3% by stimulation of the RIG-I receptor alone.

The inhibitory activity of 3p-siORF3.1 was comparable to that of siORF3.1 or 3p-siCon.

The control transfection with poly I:C showed a similar inhibition of HEV replication

to about 4.4%. These results demonstrate that HEV can be inhibited by specific RNAi-

silencing or by unspecific induction of an immune response to a similar extent. While

the combination of both mechanisms does not lead to a measurable increase in silencing

activity, the double-edge strategy will still be of value to prevent the potential emergence

of viral escape mutants induced by the siRNA.

4. Discussion

HEV infections have garnered increased attention recently. Although these infections

are typically acute and self-limiting, they can progress to a chronic state with serious

complications, especially in individuals with weakened immune systems. While there is

no specific therapy available, ribavirin has been used off-label with some success [

Nevertheless, treatment with ribavirin frequently results in failure due to mutations in the

viral genome [

–

]. Patients who do not respond to ribavirin therapy have no alternative

treatments available, highlighting the need for new therapeutic options.

The cytosolic genomic RNA of RNA viruses, as well as the translated mRNA of DNA

viruses, are excellent targets for therapeutic intervention using RNAi. Research groups

have shown in various

in vivo

and

in vitro

studies that RNAi can successfully inhibit repli-

cation of virtually every medically relevant virus, including hepatitis B virus (HBV) [

hepatitis C virus [

], coxsackievirus B3 [

], influenza A virus [

], adenovirus [

], human

immunodeficiency virus [

–

], and the severe acute respiratory syndrome coronavirus

type 2 [70,71].

In this study, we designed siRNAs against the ORF3 and the helicase domain of the

HEV-3c strain. A comparison of HEV subtypes showed that the HEV-3 genome has very

few conserved regions. In a comprehensive study, Ju et al. identified conserved regions

across all genotypes of cis-acting elements, with 11 nucleotides in the 5

′

coding region of

ORF1 and six nucleotides in the 3

′

coding region of ORF2 [

]. A very efficient siRNA

targeting the ORF2 cis-element in HEV-1 was already found by Kumar et al. [

]. Since

the siRNA target site requires a conserved region of at least 19 nucleotides, we opted to

select a different region of the genome with a higher degree of conservation for designing

our siRNAs. Only the ORF3 shows longer stretches of conservation (Figure 1). This is also

reflected by the fact that mutations in ORF3 show reduced viral replication or eliminate

HEV infectivity [

]. To ensure good siRNA efficiency, several online tools were used

to obtain the best possible target sequence. A total of three siRNAs against ORF3 and

three siRNAs against the helicase domain were selected. Using a dual luciferase reporter

assay, we were able to show that all but one siRNA (siORF3.2) succeeded in significantly

inhibiting luciferase activity (Figure 2). These experiments also demonstrated that the

siRNAs against ORF3 showed better silencing than those against the helicase domain. All

siRNAs were then tested against the genotype 3c strain 47832c adapted to cell culture. The

A549/pers-HEV cell line persistently infected with the strain [

] was transfected with

the siRNAs. Unexpectedly, the siRNAs against the helicase, found to be highly active

in the reporter assays, did not function to inhibit the infectious virus to a satisfactory

extent. The most likely explanation for this discrepancy is that the full RNA genome

folds into a different structure than the isolated fragment that was used for the reporter

assay, thereby preventing the siRNA targeting the helicase region from binding to their

complementary sequences.

Further experiments with our selected siRNA demonstrate that prolonged cultivation

and double transfection further improved the inhibition of viral replication. Studies by

Viruses 2024,16, 1378 13 of 19

Bartlett et al. have shown that siRNA silencing lasts up to seven days in rapidly dividing

cells and up to three weeks in non-dividing cells and that cell division is an important

factor in RNAi [74]. This effect was further enhanced by Takahashi et al. using 2′-OMe-4′-

thioribonucleoside modification to protect the siRNA from intracellular nucleases [75].

The present study focuses on the RNAi-mediated inhibition of the HEV-3c genotype.

Previous studies have almost exclusively focused on HEV-4 and HEV-1, which are not

particularly relevant in Europe [

–

]. Studies have also shown that subtype 3c is the

most common genotype in Germany and the most relevant genotype for chronic HEV-

infected patients [

–

]. Chronic HEV infections can be treated off-label with ribavirin,

a guanosine nucleoside analog [

]. Despite some side effects, treatment is successful

for many patients. However, ribavirin treatment leads to mutations and this may result

in resistance to the treatment [

–

]. RNAi-based drugs may then serve as a second-line

therapeutic for those who no longer respond to ribavirin treatment.

In our study, we used the A549 cell line for the infection experiments. This cell line

is derived from adenocarcinomic human alveolar basal epithelial cells; it is widely used

in HEV research [

–

]. Other studies have used hepatic cells [

]. However, various

HCC-derived cell lines, including HepG2/C3A or Huh-7, are deficient in RIG-I signaling

and were, therefore, not suitable for our study. We, therefore, decided to carry out the

experiments with the widely used A549 cell line, but we will use HepaRG cell in further

experiments, which have been shown to be infectable with HEV [

] and with which we

even established a bioprinted 3D liver model [88].

For

in vitro

experiments to characterize antiviral siRNAs, it is a common procedure

to transfect siRNAs first and infect the cells with the virus after a certain period to allow

cellular uptake of the siRNAs [

]. While this procedure is highly artificial, as a thera-

peutic drug will be applied after the infection, it is necessary since viruses tend to lyse cells

in vitro

rapidly, preventing any attempt to simulate an ongoing infection in transfected

cells. In the present study, we not only inhibited the virus with the pre-treatment strategy

but also used the persistently HEV-infected cell line A549/pers-HEV [

] and achieved

substantial and statistically significant inhibition of the ongoing infection.

In recent years, RNAi has become an increasingly important therapeutic approach,

with numerous drug approvals every year [

]. The present study used Lipofectamine

RNAiMAX for siRNA transfection into culture cells, an approach that cannot be used in

patients. Efficient delivery of siRNAs to the target organ remains the major challenge in

their therapeutic development. Delivery to the liver, however, is possible using existing

techniques, in contrast to many other internal organs. Most of the siRNAs approved

so far are covalently linked to N-acetylgalactosamine (GalNAc/NAc), which binds to

asialoglycoprotein receptors on hepatocytes, facilitating specific delivery of siRNAs to

the liver [

]. Furthermore, several RNAi therapeutics against chronic HBV infections

are in various stages of development, which also use the GalNAc modification [

The siRNA presented here can thus be used as a GalNAc-linked molecule for future

therapeutic application.

While RNAi is an efficient strategy to suppress viruses, it is well-known that the

selection pressure of the siRNA treatment results in the rapid accumulation of escape

mutations [

]. As a countermeasure, we chose to develop a combination strategy

that uses the RNAi mechanism as a specific silencing pathway to inhibit the virus and,

at the same time, activate the innate immune response by adding a triphosphate to the

′

end of the siRNA. This functionalization is known to activate RIG-I and was shown

to improve the anti-tumoral activity of an siRNA used to treat melanoma [

]. It has

subsequently also been used for antiviral applications. Lin et al. found that a 3p-siRNA

caused a significantly stronger inhibition of influenza A virus replication compared to an

unmodified siRNA in A549 cells [

]. Other studies have shown that 3p-siRNAs against

HBV achieved significantly higher inhibition of viral replication than siRNAs without

′

-triphosphate [

]. Animal studies also confirmed that 3p-siRNAs can inhibit the

replication of influenza A viruses and HBV more effectively [

]. Research findings

Viruses 2024,16, 1378 14 of 19

indicate that HEV proteins have a detrimental effect on the RIG-I signaling pathway [

–

implying that HEV may manipulate the host antiviral response through this pathway.

We tested two different 3p-modified siRNAs and compared their activity to that of the

unmodified counterparts: 3p-siORF3.1, which activates both specific RNAi against the HEV

genome and RIG-I, and 3p-siCon, which induces only the RIG-I response without triggering

RNAi. Initial DLA experiments confirmed that 3p-siORF3.1 retained full silencing activity

when compared to siORF3.1, demonstrating its functionality despite the modification.

RIG-I expression was analyzed by Western blot 48 h after transfection and showed that

both 5′-3p-siRNAs can activate RIG-I.

The next step was to test the approach against the infectious virus. As RIG-I can also

be activated by other synthetic ligands lacking a 5

′

-triphosphate, we used poly I:C, which

is a positive control for RIG-I activation [

]. Treatment with poly I:C induced RIG-I as

confirmed by Western blotting and efficiently inhibited HEV replication, highlighting the

potential of RIG-I stimulation for virus inhibition. We then investigated the inhibitory

activity of HEV-specific and control siRNA with and without 5

′

-triphosphate, respectively.

While the unmodified control siRNA did not exert antiviral activity, the HEV-specific

siRNA, as well as the 5

′

-triphosphate-modified control, efficiently inhibited the virus.

The RIG-I activation by a modified siRNA alone is thus sufficient to suppress HEV. This

finding is in line with an HEV-3a-replicon-based study by Xu et al., which has shown

that 5

′

-triphosphate RNA can inhibit HEV RNA by approximately 67% and which also

reported a significant reduction of HEV replication by overexpression of lentiviral RIG-I

in HEV-infected Huh7.5, A549, and HepaRG cells [

]. Another study by Devhare et al.

showed that transfection of the Huh7.5 cell line, which lacks active RIG-I signaling, with

a RIG-I expression plasmid-reduced HEV-1 replication, while cells transfected with an

empty vector showed higher HEV replication [

]. Our results support the hypothesis

that 5

′

-3p-siRNA can effectively reduce HEV replication by activating the innate immune

response after infection. The experiments of the current study were carried out with the

47832c strain, which is a clinical isolate that is a typical representative of HEV-3c, which

is prevalent in Europe. As shown by Devhare et al. and Xu et al., other HEV strains are

also susceptible to RIG-I signaling [

]. It will, therefore, be of interest to investigate

in further experiments whether our strategy to inhibit HEV by 5

′

-3p-siRNAs will be as

efficient in other strains as it is for HEV-3c.

A last resort therapy for RBV non-responsive patients is PegIFN

, but this therapy is

contra-indicated for some organ transplant recipients, one of the highest risk groups for

developing chronic HEV infections [

]. Our liver-specific targeted 3p-siRNA, which

activates RIG-I and its respective Interferon I response, could fine-tune this therapeutic

approach and provide an alternative to PegIFNαin organ transplant patients.

Interestingly, we did not observe an additive effect, i.e., improved virus inhibition, for

the HEV-specific 5

′

-3p-modified siRNA compared to the unmodified active siRNA or the

modified control. However, the modification was not primarily intended to improve the

silencing activity but to combine two independent antiviral mechanisms. RNA viruses,

with their poor replication fidelity, rapidly generate mutants capable of escaping repression

by targeted siRNAs and thus become resistant to the treatment. Inhibition of viruses,

particularly RNA viruses with a high rate of replicatory errors, will result in the enrichment

of variants with mutations in the siRNA target site, which have become resistant to the

treatment. This can be prevented by attacking the virus with a second, independent

mechanism, the activation of the innate immune response by activating RIG-I signaling.

The siRNA inhibited the virus with high efficiency. While most

in vitro

studies devel-

oping RNAi antiviral approaches pre-treat cells with the siRNA and carry out the infection

step afterwards, we were also able to inhibit HEV in a persistently infected cell line, which

is closer to being a model for a chronic infection found in patients. Finally, we demonstrate

that the addition of a triphosphate to the 5

′

end of the sense strand of the siRNA also exerts

high antiviral activity by activating RIG-I. We therefore suggest the use of a 5

′

-triphosphate

modified siRNA for the treatment of HEV infections. The primary intention of using a

Viruses 2024,16, 1378 15 of 19

double payload is not to increase antiviral activity but rather to prevent viral escape by

combining two different antiviral mechanisms. As the liver is the best-suited organ for

the delivery of siRNAs, this approach can develop into a rescue strategy for patients with

chronic HEV infection that does not respond to the current standard treatments.

Supplementary Materials: The following supporting information can be downloaded at: https:

//www.mdpi.com/article/10.3390/v16091378/s1, Table S1: Reference sequence for HEV-3 Subtype

a-j; Table S2: DNA oligonucleotides for T7 in vitro transcription of 5’- triphosphorylateded siRNA.

Author Contributions: Conceptualization, M.Z., C.-T.B., R.J., A.K. and J.K.; methodology, M.Z., L.N.

and D.H.; validation, M.Z. and J.K.; formal analysis M.Z.; investigation, M.Z. and J.K.; resources,

C.-T.B., R.J. and J.K.; writing—original draft preparation, M.Z.; writing—review and editing, M.Z.,

D.H., L.N., A.K., R.J., C.-T.B. and J.K.; visualization, M.Z.; supervision, J.K.; funding acquisition, A.K.

and J.K. All authors have read and agreed to the published version of the manuscript.

Funding: A.K. and J.K. gratefully acknowledge financial support from the Federal Ministry for

Economic Affairs and Climate Action (through ZIM funding, funding codes 16KN073265 and

16KN073266).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Data are available upon reasonable request.

Acknowledgments: The authors are particularly thankful to Erik Wade for the careful proofreading

of the manuscript and helpful comments.

Conflicts of Interest: Author Anke Kurreck was employed by the BioNukleo GmbH. The other

authors declare no conflicts of interest.

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