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In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles

In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and... Hindawi Advances in Virology Volume 2021, Article ID 5569844, 10 pages https://doi.org/10.1155/2021/5569844 Research Article In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles 1 1 2 1 Stuart Sims , Kevin Michaelsen, Sara Burkhard, and Cornel Fraefel Institute of Virology, University of Zurich, Zurich, Switzerland Department of Infectious Diseases, University Hospital of Zurich, Zurich, Switzerland Correspondence should be addressed to Stuart Sims; stuart.sims@hotmail.com Received 4 February 2021; Accepted 20 July 2021; Published 30 July 2021 Academic Editor: Jay C. Brown Copyright © 2021 Stuart Sims et al. &is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. &e 5′ untranslated region (5′ UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contains sequence homology to the HCV type III internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription, we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV, we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes and we then constructed full-length RHV and RPgV replicons with reporter genes. Using the replicon system, we show that the RHV NS3-4A protease cleaves a mitochondrial antiviral signaling protein reporter. However, liver- derived cells did not readily support the complete viral life cycle. primates [10], bovine [11, 12], and then, the first non- 1. Introduction mammalian species, sharks [13]. &e hepatitis C virus (HCV) infects more than 71 million &e rodent HCV homologues were termed rodent people worldwide [1], which can lead to liver failure and hepaciviruses (RHV) and were first identified in deer mice hepatocellular carcinoma and presents a major global health (Peromyscus maniculatus), a species known to carry han- burden. While the introduction of new direct-acting anti- tavirus, desert woodrats (Neotomalepida), and hispid pocket viral drugs (DAAs) has improved treatment response rates mice (Chaetodipus hispidus) [7]. &e same year RHV was and heralded a new era of HCV treatment [2], the cost and described in European bank voles (Myodes glareolus) and availability of DAAs along with drug resistance and chronic/ South African four-striped mice (Rhabdomys pumilio) [6]. nonreversible liver damage due to late onset of symptoms &is was succeeded by the discovery of an RHV from Norway rats (Rattus norvegicus) in New York City [14]. and delayed treatment initiation after HCV infection remain an issue [3]. A small animal model for HCV would allow for &e subsequently discovered RHV (RHV-rn1) from Norway rats was used to make a small animal model for the testing of vaccines which could potentially solve these problems. hepaciviral infection, utilising a reverse genetics approach. HCV was discovered in humans 20 years ago [4] but for a In this system, the researchers showed its hepatotropic long time, researchers failed to identify an animal viral replication in inbred and outbred rat strains [15]. Emulating homologue. &is all changed with the use of high throughput HCV infection, they also showed that persistent infection deep sequencing, which has shed light on the evolutionary leads to gradual liver damage and that the HCV antiviral origins of the virus. &e first HCV homologue was dis- drug sofosbuvir suppresses replication of RHV-rn1. &is covered in 2011 in canines [5] and was quickly followed by model can be used to study the mechanisms of HCV per- the discovery of homologues in rodents [6, 7], equine [8, 9], sistence, immunity, and pathogenesis. 2 Advances in Virology terminator in a DNA transfection system [25, 26] to allow &ough rats are the usual hosts of RHV-rn1, it has also been shown that the virus is capable of establishing a per- for quick and easy mutation and screening. sistent infection in immunocompromised mice lacking type I interferon and adaptive immunity [16]. However, im- munocompetent mice clear the virus in a few weeks. Because 2. Materials and Methods this mouse model only results in an acute infection, a fully -/- Cells. Hepa1-6 (ATCC CRL-1830 ), MEFs IRF3 , immunocompetent mouse model in which a chronic in- NIH 3T3 (ATCC CRL-1658 ), BHK-21 (ATCC ® ® fection and downstream liver damage can be established is CCL-10 ), HEK 293T (ATCC CRL-11268 ), Huh- ™ ™ still in need. Additionally, the availability of knockout mice 7.5-RFP-MAVS from Dr Charles M Rice, HepG2 would aid the study of the pathogenesis of HCV related liver (ATCC HB-8065 ) and Vero cells (ATCC CCL- ® ® damage. 81 ) were cultured in DMEM (Sigma-Aldrich), 10% While these studies could lead the way to future vaccines, FBS and 1% penicillin-streptomycin. they also present the possibility of zoonotic sources of HCV Plasmid Construction. Monocistronic reporter plas- infection in humans [17]. &e genomes of RHV encode for a mids containing viral 5′UTR were constructed in polyprotein that is predicted to be cleaved into 10 proteins, pUC19. First, pUC19 was digested with EcoR1 and as with HCV, but shows a 66–77% amino acid divergence HIndIII (NEB), and then a minimal RNA polymerase I from HCV in the structural genes [7, 18]; therefore, tropism (RNA Pol I) promoter and terminator (synthesised by and pathogenicity may also differ between the viruses. In Twist Biosciences) was inserted into the digested particular, the 5′ untranslated regions (UTRs) of RHV are plasmid by In-Fusion cloning (Takara Bio); this plas- highly divergent from the corresponding regions of HCV, mid was named pOLI (Figure S1A). and there is a large difference between the different rodent clades (RHV, RHV1, RHV2, RHV3, and RHV-rn1). &e plasmid pOLI was linearized with PpuMI. Viral 5′ and 3″ UTR (synthesised by Twist Biosciences) (HCV &e high-level expression of mir-122 in liver cells allows HCV, containing two mir-122 target sequences in the IRES taken from pFR_HCV_xb (Addgene)) along mCitrine were cloned into the linearized pOLI by In- 5′UTR, to replicate and is one reason for HCV hepato- tropism [19]. Similarly, the RHV (NC_021153) found in deer Fusion cloning (Takara Bio) (Figure S1B). Deletions mice contains one such mir-122 target sequence in its 5′UTR and additions to the monocistronic reporter plasmids were created by PCR and In-Fusion cloning (Takara [7] suggesting liver cell specificity and further has a 200 nt sequence with no homology to other hepaciviruses 5′UTRs. Bio). &e RHV-1 (KC411777)) 5′UTR contains structural ele- Plasmids containing viral structural genes were con- ments typical of both pegi- and HCV-like IRES and contains structed in pUC19 by In-Fusion cloning (Takara Bio) one mir-122 target region, while RHV-1 and RHV-2 using hepacivirus CE1E2 region (synthesised by Twist (KC411784) are identical in structure and only contain a few Biosciences), along with a CMV promoter and BGH nucleotide exchanges, with RHV-3 (KC411807) and RHV- polyA (Figure S1C). Flag tag and c-Myc tag sequences rn1 being more similar to typical HCV-like IRES structures were inserted into this plasmid by PCR and In-Fusion My- [6, 15] (Figure 1). cloning (Takara Bio); this plasmid was named pC c Flag My- Along with RHV, a rodent pegivirus (RPgV) was also E1E2 . &e capsid gene was deleted from pC c Flag discovered in white-throated wood rats (Neotoma albigula) E1E2 by PCR and In-Fusion cloning (Takara Bio) Flag [7]. Pegiviruses are a new genus of the family of the Fla- resulting in the plasmid pE1E2 . viviridae, encompassing the human GBV-A, GBV-C/HGV/ To construct plasmids containing full-length RHV1 HPgV-1, GBV-D and HPgV-2 viruses. &ese pegiviruses and RPgV viral genomes, the respective monocistronic are considered to be nonpathogenic and in the case of vector (pOLI.IRES.Cirtine) was PCR linearized, and HPgV has even been reported to be beneficial in coin- gene fragments, 1.5–2 kb (synthesised by Twist Bio- fections with HIV or Ebola [20–22]. However, two pegi- sciences), covering the full-length viral coding region viruses were discovered in equine. &e first, &eiler’s were then cloned in by In-Fusion cloning (Takara Bio) disease-associated virus (TDAV), is suspected to be the replacing mCitrine. &e plasmids were named causative agent for an outbreak of acute hepatic disease pOLI.RHV1 and pOLI.RPGV (Figure S1D). occurring on a horse farm [23]. &e second, equine For constructing a reporter plasmid, mScarlet-BSD pegivirus (EPgV), like the human pegiviruses, is considered (synthesised by Twist Biosciences) was cloned into the to be nonpathogenic [24]. plasmid containing the full-length virus in-between &e viral RNA structures play important roles in both NS5A and NS5B, while also duplicating the cleavage translation and replication. Specifically, the 5′UTR sequence by In-Fusion cloning (Takara Bio). &ese containing the IRES promotes the initiation of protein plasmids were named pOLI.RHV1.SB and synthesis in a cap-independent manner. IRES’s are di- pOLI.RPgV.SB (Figure S1E). verse in sequence and structure and these differences contribute to tropism; the focus of this study is to assess Transfection. All transfections were carried out using how these differences in the 5′UTRs of RHV and RPgV Lipofectamine LTX with Plus Reagent (&ermo affect translation and to establish a reverse genetics Fisher Scientific) in Opti-MEM (Gibco) according to system utilizing the RNA polymerase I promoter and the manufacturer’s specifications. Advances in Virology 3 HCV RHV RHV1 IIIb II IIIb IIIc IIIa IIIa IIIa IIIb IIIc II IVb Va IIId Ic IIId IIIe IIIf Ic IIIf IIIe Ib Ib IV I Vb Ia Ia IV RHV2 RHV3 RHV-rn1 IIIb IIIb II IIIa IIIc II IIIa IVa IIId IIIb Ic IIIe Ib Ic IVb IIIf Ia IIb IIIa IIb Va Ib Ia Vb IV IIIf IIIc Ia IIIe IIa VI Ib IIId RPgV IIIb IIIc IIIa IIId IIIe Ib IV Ia Figure 1: Predicted RNA secondary structures within HCV, RHV, RHV1, RHV2, RHV3, RHV-rn1, and RPgV 5′ UTR. &e sequence corresponding to the mir-122 binding sites within the 5′ UTR is highlighted in orange; the first binding sites are represented by filled circles and the second by the outline. HCV contains two mir-122 binding sites and RHV, RHV1, and RHV-rn1 contain one predicted binding site. RHV2/3 and RPgV do not contain a mir-122 binding site. &e nucleotides corresponding to the AUG start codon are highlighted by filled green circles. 4 Advances in Virology Western Blot. Cells were lysed in RIPA buffer (150 mm pMD2.G (Addgene) or pE1E2 at a ratio of 1 :1:0.1, NaCl, 50 mm Tris/HCl pH 7.6, 1% Nonidet P40, 0.5% respectively, using lipofectamine LTK (&ermo Fisher sodium deoxycholate, and 5 mm EDTA) supplemented Scientific) and Opti-mem (Gibco). At 6 hours after with cOmplete Protease Inhibitor Cocktail (Roche) transfection, media were replaced and 72 h after for 15 min on ice. &e lysate was run on a 12% SDS- transfection, supernatant was harvested and passed Page and transferred to PVDF membrane (Bio-Rad). through a 0.45µm filter. Membranes were incubated for 1 hour in PBS with 5% nonfat dry milk and then stained with primary and 2.1. RNA Structure Prediction. &e RNA sequences sec- subsequently secondary antibodies for 1 hour in PBS ondary structure was predicted with RNA fold and visual- with 1% nonfat dry milk. Immunocomplexes were ization was performed using force directed graph layout detected using an Odyssey Fc Imaging System (LI- (Forna); both are hosted on ViennaRNA Web services COR Biosciences). (http://rna.tbi.univie.ac.at/forna/). Intracellular Immunofluorescence. Cells were grown on glass coverslips, washed with PBS prior to fixation in 2.2. RNA Extraction and cDNA Generation. Cells from 24- formaldehyde (3.7% w/v in PBS) for 10 min at room well plate were washed in PBS and resuspended in Trizol temperature, permeabilized for 5 min with 0.1% Triton (Sigma-Aldrich, USA), and RNA was isolated by iso- X-100 in PBS, and blocked with 3% BSA in PBS for propanol precipitation, washed with 70% ethanol, and 30 min. Primary and secondary antibodies were diluted resuspended in DEPC-water. RNA was DNAse treated in PBS containing 1% BSA, and cells were stained with (Invitrogen, Paisley, UK) and subjected to RT-PCR using primary antibody for 1 hour, washed 3x with PBS, and 100 ng purified RNA. For RT-PCR, the high-capacity cDNA then stained with secondary antibody for 1 hour, fol- archive Kit from Applied Biosystem (ABI PRISM, War- lowed by staining with 0.1µg/ml DAPI in PBS for 5 min rington, United Kingdom) was used according to the and application of Prolong Gold Antifade reagent specifications of the manufacturer. (Invitrogen). Extracellular. Cells were grown on glass coverslips, incubated with PBS containing 4% FBS (Fetal Bovine 2.3. Quantitative Real-Time PCR for Selected Genes. Serum) for 30 min. Primary and secondary antibodies Quantitative real-time PCR was performed using a Light were diluted in PBS containing 4% FBS, cells were cycler 480 Real-Time PCR System (Roche Diagnostics) and stained with primary antibody for 1 hour, washed 3x the LightCycler 480 probes master reaction mix (Roche with PBS, and then stained with secondary antibody for Diagnostics) following the manufacturer’s protocol. Data 1 hour. Cells were then stained with Wheat Germ analysis was performed with LightCycler 480 Software Agglutinin, Alexa Fluor 594 conjugate as per man- (Roche Diagnostics). Oligonucleotides sequences used as ufacturers protocol. Cells were fixed in formaldehyde primers for quantitative real-time PCR and corresponding (3.7% w/v in PBS) for 10 min at room temperature, probes were designed according to the guidance of the permeabilized for 5 min with 0.1% Triton X-100 in PBS, Universal Probe library from Roche applied Science. and stained with 0.1µg/ml DAPI in PBS for 5 min &ermal cycling started with HotStarTaq activation during followed by application of Prolong Gold Antifade re- ° 10 min at 95 C. &ereafter, 45 cycles of amplification were ° ° ° agent (Invitrogen). run consisting of 10 s at 95 C, 30 s 60 C, and 20 s of 72 C. A negative control containing reagents only and serial dilu- Images were acquired using a confocal microscope (Leica specify type) and Z-stacks and analysed with tions of cDNA was included in each run. Each sample was measured as a triplicate and the average concentration was ImageJ. used. For LightCycler analysis, expression of hypoxanthine Antibodies. Rat anti-DYKDDDDK (clone L5, Biol- phosphoribosyltransferase gene (HPRT) was used for nor- egend), mouse anti-c-myc (clone 9E11, Biolegend), malization. Relative expression of samples was calculated by mouse anti-β-actin (clone 2F1-1, Biolegend), mouse J2 the comparative cycling threshold method (ΔΔCT) and then anti-dsRNA IgG2a (Sciscons), goat anti-rat IgG (H + L) setting the samples transfected with HCV IRES as the Alexa Fluor 488 (Invitrogen), IRDye 800CW goat benchmark. anti-rat IgG, and IRDye 680RD donkey anti-mouse IgG (LI-COR Biosciences). GeneBank Accession Numbers. RHV (Hepacivirus E), Flow Cytometry. Single cell suspensions were generated NC_021153; RHV1 (Hepacivirus J), KC411777; RHV2 (Hep- and kept in FACS buffer (2% FCS, 5 mm EDTA in PBS). acivirus F), KC411784; RHV3 (Hepacivirus I), KC411807; Cells were analysed using a Gallios flow cytometer RHV-rn1 (Hepacivirus G), KX905133.1; RPgV, NC_021154. (Beckman Coulter) and FlowJo software and gated on viable cells using the live/dead fixable near-IR dead cell 3. Results stain kit (Invitrogen). Lentivirus. Plates were seeded with HEK 293T in 3.1. RHV and RPgV IRESs Are Functional in Rodent Cells. DMEM and 3% FCS and then transfected with pLKO- To test viral IRES driven translation, a monocistronic gfp (Addgene), pCMV∆R8.2 (Addgene), and either plasmid vector was constructed containing a minimal RNA Advances in Virology 5 that of RHV, RHV1, RHV3, and RHV-rn1 was at the lowest polymerase I promoter in front of the full-length viral 5′ UTR followed by a fluorescent maker, the viral 3′ UTR, and level (Figure 2(h)). In Vero cells, the RPgV produced high levels of translation, over twice that of HCV; RHV2 was the the RNA polymerase I terminator (Figure 2(a)). &e HCV IRES was used as a positive control along with a control only other IRES that was functional in these cells although at plasmid containing a scrambled sequence in place of the a very low level when compared to RPgV (Figure 2(i)). viral 5′ UTR. We constructed plasmids containing RHV, RHV1, RHV2, RHV3, RHV-rn1, and RPgV 5′ UTR from 3.3.DeletionsAbrogatetheFunctionofRHV1andRPgVIRES. previously published sequences. To further assess the structural requirements of RHV1 IRES &ese plasmids were transfected into the murine hepa- for full functionality, we made several different constructs. tocyte cell line Hepa1-6, with the HCV and RPgV IRES driving &e first contained an additional 20 nucleotides of virus the highest level of translation at 72 hours after transfection sequences downstream of the start codon. When transfected with a mean fluorescence intensity (MFI) of 30, followed by into Hepa1-6 cells, this construct did not lead to a difference RHV1 and RHV2 with an MFI of 22 and 14, respectively. in the levels of translation in comparison with the construct RHV3 and RHV-rn1 IRES drove translation at a level only containing just the 5′ UTR. Two deletion constructs were slightly above background and RHV was not functional in made; in RHVΔI, the 5′ three stem loops (Ia/b/c) were deleted Hepa1-6 (Figure 2(b)). To further assess the level of RNA (Figure 3(a)). &is deletion decreased the IRES function by transcripts of mCitrine within the cells, we preformed qRT- 90%. &e Va and Vb stem loops were deleted from RHV1ΔII PCR on transfected cells. &ere was no statistical difference in and again led to a decrease in function by 90% in the murine the level of transcripts between HCV, RHV, RHV1, RHV2, hepatocyte cell line Hepa1-6 (Figure 3(c)). RHV3, RHV-rn1, RPgV, and the control plasmid containing a For the RPgV IRES, we also added an additional 20 scrambled sequence in place of the viral 5′UTR. &e other nucleotides of virus sequences downstream of the start control plasmid contains RHV1 5′UTR but no Pol I promoter codon. &is again had no effect on the levels of translation in did yield a readily detectable level of RNA (Figure S2). comparison to the construct containing just the 5′ UTR. To To further assess IRES function in murine cells, we tested further assess the sequence required for driving translation, two murine embryonic fibroblasts cell lines, MEFs and NIH three constructs were made with deletions, RPgVΔI and 3T3. Transfection of MEFs with the plasmids revealed that RPgVΔII have deletions to the 5′ of the IRES with RPgVΔI RHV1 drove the highest level of translation at an MFI of 9 having the first two stem loops (Ia/b) and RPgVΔII three followed by RHV2 at an MFI of 5 (Figure 3(c)). HCV, RHV, stem loops deleted (Ia/b and II) (Figure 3(b)). RPgVΔIII has RHv3, RHV-rn1, and RPgV generated signals only slightly two internal stem loops deleted (IIId/e). &e 5′ deletions to above the negative control. In NIH3T3, the RPgV drove the RPGV had no effect on the levels of translation. However, highest level of translation with an MFI of 17 followed by the internal deletions in RPgVΔIII led to a reduction in HCV with an MFI of 7; again, RH1 and RHV2 were translation of 53% (Figure 3(d)). functional but at low levels, and RHV, RHV3, and RHV-rn1 were not functional (Figure 3(d)). &e viruses originate from different rodent species; 3.4. Expression of E1E2. Previous studies examining the therefore, the baby hamster kidney cell line was tested to subcellular localization of HCV E1 and E2 used cells assess if the IRESs are functional in this cell line. As with the transfected with a plasmid expressing the E1 and E2 proteins previous cell lines, the RPgV IRES drove high levels of [27]. &ese studies concluded that the HCV structural translation; also the RHV1 and RHV2 were capable of proteins are expressed on the cell surface, based on im- driving high levels followed by HCV; again, the RHV, munofluorescence detection. In order to examine the lo- RHV3, and RHV-rn1 were not functional (Figure 2(e)). calization of RHV structural proteins in an expression system, we cloned the structural region (capsid-E1-E2) of RHV into a plasmid containing the CMV promoter; we then 3.2.RHVandRPgVIRESsShowDifferingFunctionsinHuman added the c-Myc tag to the 5′ end of the capsid protein and CellLines. In the human hepatocyte cell line, Huh7.5 which the Flag tag to the 5′ end of the E2 protein following the expresses high levels of mir122, and the RPgV IRES drives E1E2 cleavage sequence (Figure 4(a)). the highest levels of translation followed by HCV and RHV2. HEK 293T cells were transfected with the vectors Flag Myc Flag &e RHV-rn1 drove low levels of translation and, as with pE1E2 or pC E1E2 and after 48 hours lysed for murine cells, RHV and RHV3 did not yield any signal SDS-PAGE. &e E2 protein was detected in cells transfected (Figure 2(f)). Another human hepatocyte cell line, HepG2 with either expression vector using an anti-Flag tag antibody that does not express mir122, was also used to test the IRES’s (Figure 4(a)), and the capsid was detected in cells transfected Myc Flag function: in these cells, HCV drove the highest levels fol- with the pC E1E2 expression vector using an anti-c- lowed by RPgV and RHV2, while the expressions from RHV, Myc antibody. &e proteins detected were of the predicted RHV1, RHV3, or RHV-rn1 were at background level size, showing that posttranslational cleavage was complete. (Figure 2(g)). In order to determine if the RHV glycoproteins To investigate if the IRESs are functional in non- expressed from these vectors also exhibit an intracellular hepatocyte cells lines, we used HEK 293Tand Vero cell lines. colocalization, we examined transfected cells by immuno- In HEK 293T, the HCV IRES showed the highest level of fluorescence for capsid and E2 expression; both were shown translation followed by the RPgV IRES and RHV2, while to colocalize (Figure 4(b)). We also assessed if the envelope IRES IRES IRES IRES IRES IRES IRES IRES 6 Advances in Virology HCV RHV1 PolIP 5′UTR mCitrine 3′UTR PolIT 31 ± 2.99 22 ± 1.56 RHV2 RPgV 14 ± 0.9 30 ± 2.59 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (a) (b) HCV RHV1 HCV RHV1 2.24 ± 0.13 9.26 ± 0.9 7.68 ± 0.21 3.98 ± 0.23 11.00 20 8.25 15 RPgV RHV2 5.50 RHV2 10 RPgV 2.37 ± 0.34 5.10 ± 0.41 5.49 ± 0.67 17 ± 1.73 2.75 5 0.00 0 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (c) (d) HCV RHV1 HCV RHV1 80 12 23 ± 1.63 53 ± 2.92 7.16 ± 0.52 2.39 ± 0.13 60 9 6 RHV2 RPgV 40 RHV2 RPgV 49.28 ± 3.03 76 ± 2.89 4.47 ± 0.43 11 ± 0.48 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (e) (f) HCV RHV1 HCV RHV1 22 20 ± 0.99 2.52 ± 0.28 18 ± 0.92 3.7 ± 0.92 16.5 11 RHV2 RPgV RHV2 RPgV 10 ± 1.24 14 ± 0.58 7.7 ± 0.44 11 ± 0.49 5.5 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (g) (h) HCV RHV1 33 ± 0.92 2.19 ± 0.03 40 RHV2 RPgV 7.94 ± 0.22 77 ± 2.17 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (i) Figure 2: RHV and RPGV IRES activity in different cell types. Schematic of the monocistronic vectors used (a). Hepa1-6 (b), MEFs (c), NIH 3T3 (d), BHK-21 (e), Huh7.5 (f), HEK 293T (h), and Vero (i) cells were transfected with the indicated plasmids. Cells were harvested and analysed by flow cytometry at 48 h.p.t.; bar graphs show MFI for mCitrine (n≥ 8, mean± SEM of at least three independent experiments), and representative FACS plots on the right of each graph numbers represent the MFI (mean± SEM). &e dashed line represents the fluorescent output of the control plasmid containing a scrambled sequence in place of the viral 5′ UTR. MEFs MFI mCitrine BHK-21 MFI mCitrine HepG2 MFI mCitrine Vero MFI mCitrine mCitrine mCitrine mCitrine Hepa1-6 MFI mCitrine NIH3T3 MFI mCitrine HEK 293T MFI mCitrine Huh-7.5 MFI mCitrine mCitrine mCitrine mCitrine mCitrine mCitrine IRES IRES Advances in Virology 7 C C 200 G A 370 G C C 150 U G A A C A U IIIb C C U A U G G G 160 A 140 C 180 C C IVa G C G G G A C U G G G U U C G A A C G U G U U C A U G A C C U G A G C C G G U G A U C U A A C C G G U G U A G C A G G 200 U G C A G G II C C 210 G A C U U G A C G U C A U C U U 130 C G 190 170 C 190 A G G C G G C C U U G A G C G A 380 360 C U G C G G 210 A C C G C G U G G RPgVIRES U G G C C U C U G RHV1IRES G A C G G G C C C 220 120 G C G A G A U A G A U C U U U C G IIIc A G C U A G 230 ΔIII G 220 350 IIIa C A C G C 390 C G U G G U A U G C IIIa IIIb A U C U C A A G G G C C C G 170 U C U C G U G G A G U G G C C C A G G A G A A G U U G C A U G A 290 160 U U G G G U C 260 G C U G A U G G 110 C G G G 240 U G A C G U C G C G IVb C C G G U G C A U G C C 400 G III 280 C 300 A 340 G G G U C A C 230 G U U G A U U C 310 U C G C U U U C A C G G G G G U C G 250 C C G G ΔII C G G G G U U A G C C C C G G A A A C C C A U C G G C G G A G A U U U C C U A 270 G 410 A C C C A G G G C A U G C C G A G G G U C 420 A U A U 270 A G 240 C A C A U 150 A C U C A A A U G U G G G A 320 430 A G U G U G 250 U C C G II U U G U U A C C U U 330 U G C U C C G A G 100 U A C A G C 260 C U G C G 90 C G 90 G U A G U C U A U U G C A G G C G C C 470 C C C A C A C G C U IIIe G 440 G C C G 290 U A G G C G C G A C U U C A G A U G A C G G G G A G 100 U G C A C G U U C U 460 C A G U C G A C G G C G A G C C G U A Va U U U 280 G A C C C G 480 C A C U A G 80 A G G A 140 U U G A U A G G A 80 G 120 U A G A 130 A G G G C C C U A G C C G G C A A G C C G G U U C U 450 C G G U A G G G A G 110 U ΔI Ic C G C ΔII G G G A U G C A C G 510 C G A C C U U 300 G U G U U G G G U G G C G C C C A A G G C G G C U G C G A 60 C 70 A U G A G G G 70 C C A 490 U 60 C C A G G A U G A C A C C G U G A A A G C U U A C U G A U C A Ib G 520 U G C U A U G C G U G 50 G C G ΔI 50 A G G U G A G G U U G C G U G G G C C A C G 40 C U A A Vb C C C A C C U C C U 500 U 30 C 310 G U Ib U G G C A G C 40 G G C U C A C C C G C A G C C C U G IVa 20 C A G C C G G C C G C U G G 340 C A G 30 G U C U G C C C 320 Ia U U G A C C G U U G C A G U U U C G G A G A G A U G A C G C C U U A A C A G G U 530 370 U A U G C C C U 20 10 A C G C 330 U C C A U G C A U U C G G G C A U 10 Ia C U A U C C G A C 360 A C A C U 540 U A U A A A G G G C G C C C A A C U U G U A G A A A C G G 350 570 U C C U C G C U 560 A A C G A C G 550 A C (a) (b) RHV1 RHV1C RPgV RPgV∆I 22 ± 1.56 22 ± 1.68 30 30 ± 2.59 30 ± 2.19 RHV1∆I RHV1∆II RPgV∆II RPgV∆III 2.37 ± 0.14 2.24 ± 0.07 33 ± 0.66 14 ± 0.73 RHV1 RHV1C RHV1∆I RHV1∆II RPgV RPgVC RPgV∆I RPgV∆II RPgV∆III (c) (d) Figure 3: Deletions to RHV1 and RPgV 5′ UTR abrogate IRES translation. Predicted RNA secondary structure of RHV1 (a) and RPgV (b). Red circles indicate areas deleted from plasmids. Hepa1-6 cells were transfected with RHV1 (c) and RPgV (d) plasmids. Cells were harvested and analysed by flow cytometry at 48 h.p.t. Bar graphs show MFI for mCitrine (n≥ 8, mean± SEM of at least three independent experiments) and representative FACS plots on the right of each graph; numbers represent the MFI (mean± SEM). &e dashed line represents the fluorescent output of the control plasmid containing a scrambled sequence in place of the viral 5′ UTR. protein was expressed on the cell surface. Staining with anti- 4. Discussion flag antibody to detect E2 indeed showed punctate staining &e five rodent hepacivirus IRESs we tested showed different on the cell membrane and when combined with wheat germ levels of ability to drive translation using in a monocistronic agglutinin to stain the cell membrane, it showed colocali- vector across varying cell lines. While the use of a mono- zation with the flag antibody (Figure 4(c)). &is indicates cistronic vector, utilizing RNA polymerase I, avoids the that a proportion of E2 is surface-expressed. potential of readthrough in comparison to bicistronic vec- To determine if the surface-localized E1E2 could mediate tors; its drawback is that we were not able to directly viral entry, we produced a GFP encoding lentivirus vector compare expression levels between cell types due to their pseudotyped with the RHV, RHV1, RHV2, and RHV3 E1E2 difference in susceptibility to transfection. However, the proteins by transfecting HEK 293T cells with pE1E2 and the RHV, RHV3, and RHV-rn1 IRESs were either not functional lentivirus backbone and packaging plasmid, and 72 h later or drove expression at very low levels. &is comes as a harvesting and filtering the supernatant. We then tested if the surprise as the RHV-rn1 virus has already been shown to E1E2-pseudotyped lentivirus vectors were entry-competent. replicate in both mice and rats. Supernatants from the cotransfected cells were applied to Both RHV1 and RHV2 drive high levels of translation in Hepa1-6 cells and GFP reporter expression assayed 72 h later. murine hepatocytes, MEFs and BHK-21 cells. In human RHV1 E1E2-pseudotyped lentivirus vectors gave rise to a hepatocytes, the RHV2 IRES outperformed RHV1 which is of small number of GFP positive cells, when compared to VSVG interest as they are similar in sequence and therefore are likely pseudotyped lentivirus. Lentivirus vectors pseudotyped with to have a similar structure. In the case of RHV1, deleting the RHV, RHV2, and RHV3 or lacking envelope glycoprotein predicted initial three stem loops abrogates IRES function, failed to give rise to any GFP positive cells (Figure 4(d)). &is suggesting that the full 5′UTR sequence is required to indicates that only RHV1 pseudotyped lentivirus vectors can maintain high levels of expression. Also, unlike HCV, where mediate viral entry in Hepa1-6 cells resulting in reporter gene previous studies have shown that the inclusion of 12–30 nt of expression. &is data also indicates that surface-expressed the core protein coding sequence was essential for an efficient RHV1 E1E2 heterodimers are functional. Hepa1-6 MFI mCitrine mCitrine Hepa1-6 MFImCitrine mCitrine 8 Advances in Virology Myc C E1 Flag E2 E2 E2 E2 30 kDa (Flag tag) Capsid 18 kDa (c-Myc tag) Flag tag-E2 c-Myc tag-capsid Flag tag-E2 c-Myc Tag-capsid Actin 40 kDa DAPI-nucleus (a) (b) E1 Flag E2 RHV RHV1 VSVG Flag tag-E2 Flag tag-E2 DAPI-nucleus DAPI-nucleus WGA-plasma membrane (c) (d) Figure 4: Expression of RHV envelope proteins. Schematic of the RHV1 envelope expression vector and western blot of cell lysate at 48 h.p.t. from transfected HEK 293T (a) Intracellular immunofluorescence of 293T cells at 48 h.p.t. with RHV1 envelope expression vector, stained with flag tag in green, c-Myc tag in red, and combined with DAPI stain (b) Extracellular immunofluorescence for E2 by flag tag stain (green) and combined with WGA (red) and DAPI (blue) from 293Tat 48 h.p.t. with RHV1 envelope expression vector, and images represent one slice from z-stack (c) GFP-lentivirus vector pseudotyped with RHV or RHV1 envelope proteins or VSVG were incubated on Hepa1-6 cells; GFP positive cells indicate transduction (d). IRES activity [28], additional nucleotides from the core study cell tropism in a murine model and the antigenicity of protein of RHV1 did not increase transcription levels. the functional E1 and E2 glycoproteins. &e RPgV 5′UTR has little significant similarity with Our efforts to make an RHV and RPgV replication any known pegivirus but drives high levels of expression competent model in vitro have so far proved unfruitful. in all cell types tested. By deleting specific regions, we were Using full-length viral constructs, we tested the human hepatoma cell line (Huh-7.5) containing a MAVS able to show that the initial 126 nt of the 5′UTR does not contribute to IRES function and that stem loops IIId/e are cleavage reporter where upon HCV NS3-4A cleavage of essential for maintaining high levels of expression. It the reporter, the RFP translocates to the nucleus [29]. would be of significance in the future to confirm the Translocation of RFP was observed with full-length predicted structures of RPgV and RHV1 IRES’s poten- RHV1, confirming the previous finding that the RHV1 tially using RNA SHAPE. NS3-4A protease is capable of cleaving human MAVS RHV1 structural genes (C, E1, and E2) expressed from [30]; however, the number of cells with RFP translocation plasmid were shown to be cleaved and yielded proteins of the did not increase over time (Figure S3.A). correct size. Moreover, RHV1 E1E2 supported transduction We further tested full-length viral constructs containing of hepatocytes when used to pseudotype lentivirus vectors. m-Scarlet and BSD inserted between NS4A-B in both Further studies will need to be carried out to find the specific Hepa1-6 and BHK-21, mScarlet was expressed in cells but the number of cells expressing mScarlet did not increase entry receptors, initially blocking CD81 and HCV entry receptors and testing susceptibility of transduction in al- overtime and failed to yield a clone when selecting for ternative cell lines, but this initial experiment hints at the replication with BSD, even when expressing Sec14L2 and hepatotropic potential of RHV1 in mice. ApoE, both essential for high levels of HCV replication [31, 32] (Figure S3.B). Further cell lines could be tested along &e generation of viral pseudotypes is one of the most with knocking out the innate immune response in future widely used methods for assaying functional receptors, experiments. allowing attachment, penetration, and uncoating to be In Summary, this study shows that RHV1/2 and RPgV studied. &is study lays the groundwork for using RHV1 contain IRESs that are capable of driving high levels of pseudotype particles to be used to asses these important protein synthesis. RHV1 structural genes are cleaved by parts for the viral replication cycle and could also be used to HEK 293T surface Flag pE1E2 c-Myc Flag pC E1E2 HEK 293T Hepa1-6 intracellular Advances in Virology 9 [10] M. Lauck, S. D. Sibley, J. Lara et al., “A novel hepacivirus with cellular proteases and can be used to pseudotype lentivirus an unusually long and intrinsically disordered NS5A protein vectors that are capable of transducing murine hepatocytes. in a wild old world primate,” Journal of Virology, vol. 87, pp. 8971–8981, 2013. Data Availability [11] C. Baechlein, N. Fischer, A. Grundhoff et al., “Identification of a novel hepacivirus in domestic cattle from Germany,” &e data used to support the findings of this study are Journal of Virology, vol. 89, pp. 7007–7015, 2015. available from the corresponding author upon request. [12] V. M. Corman, A. Grundhoff, C. Baechlein et al., “Highly divergent hepaciviruses from African cattle,” Journal of Vi- rology, vol. 89, pp. 5876–5882, 2015. Disclosure [13] M. Shi, X.-D. Lin, N. Vasilakis et al., “Divergent viruses discovered in arthropods and vertebrates revise the evolu- &is paper was previously made available in preprint on tionary history of the flaviviridae and related viruses,” Journal bioRxiv (doi: https://doi.org/10.1101/761379). of Virology, vol. 90, pp. 659–669, 2016. [14] C. Firth, M. Bhat, M. A. Firth et al., “Detection of zoonotic Conflicts of Interest pathogens and characterization of novel viruses carried by commensal Rattus norvegicus in New York city,” MBio, vol. 5, &e authors declare that they have no conflicts of interest. pp. e01933–e02014, 2014. [15] S. Trivedi, S. Murthy, H. Sharma et al., “Viral persistence, liver Authors’ Contributions disease, and host response in a hepatitis C-like virus rat model,” Hepatology, vol. 68, pp. 435–448, 2018. SS designed and planned all the experiments. SS, SB, and KM [16] E. Billerbeck, R. Wolfisberg, U. Fahnøe et al., “Mouse models performed the experiments and analysed results. SS and SB of acute and chronic hepacivirus infection,” Science, vol. 357, wrote the manuscript. CF edited the manuscript and ac- pp. 204–208, 2017. quired funding. [17] O. G. Pybus and J. &ez ´ e, ´ “Hepacivirus cross-species trans- mission and the origins of the hepatitis C virus,” Current Opinion in Virology, vol. 16, pp. 1–7, 2016. Supplementary Materials [18] S. Pfaender, R. J. P. Brown, T. Pietschmann, and E. Steinmann, “Natural reservoirs for homologs of hepatitis C (1) Illustration of plasmid construction as outlined in virus,” Emerging Microbes and Infections, vol. 3, no. 3, methods. (2) Supplementary analyses of level of RNA e21 pages, 2014. transcripts produced by viral 5′ UTRs. (3) MAVS cleavage [19] C. L. Jopling, M. Yi, A. M. Lancaster, S. M. Lemon, and and dsRNA analysis of full-length replicons. (Supplementary P. Sarnow, “Modulation of hepatitis C virus RNA abundance Materials) by a liver-specific MicroRNA,” Science, vol. 309, pp. 1577– 1581, 2005. References [20] E. L. 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In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles

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Copyright © 2021 Stuart Sims et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Hindawi Advances in Virology Volume 2021, Article ID 5569844, 10 pages https://doi.org/10.1155/2021/5569844 Research Article In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles 1 1 2 1 Stuart Sims , Kevin Michaelsen, Sara Burkhard, and Cornel Fraefel Institute of Virology, University of Zurich, Zurich, Switzerland Department of Infectious Diseases, University Hospital of Zurich, Zurich, Switzerland Correspondence should be addressed to Stuart Sims; stuart.sims@hotmail.com Received 4 February 2021; Accepted 20 July 2021; Published 30 July 2021 Academic Editor: Jay C. Brown Copyright © 2021 Stuart Sims et al. &is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. &e 5′ untranslated region (5′ UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contains sequence homology to the HCV type III internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription, we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV, we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes and we then constructed full-length RHV and RPgV replicons with reporter genes. Using the replicon system, we show that the RHV NS3-4A protease cleaves a mitochondrial antiviral signaling protein reporter. However, liver- derived cells did not readily support the complete viral life cycle. primates [10], bovine [11, 12], and then, the first non- 1. Introduction mammalian species, sharks [13]. &e hepatitis C virus (HCV) infects more than 71 million &e rodent HCV homologues were termed rodent people worldwide [1], which can lead to liver failure and hepaciviruses (RHV) and were first identified in deer mice hepatocellular carcinoma and presents a major global health (Peromyscus maniculatus), a species known to carry han- burden. While the introduction of new direct-acting anti- tavirus, desert woodrats (Neotomalepida), and hispid pocket viral drugs (DAAs) has improved treatment response rates mice (Chaetodipus hispidus) [7]. &e same year RHV was and heralded a new era of HCV treatment [2], the cost and described in European bank voles (Myodes glareolus) and availability of DAAs along with drug resistance and chronic/ South African four-striped mice (Rhabdomys pumilio) [6]. nonreversible liver damage due to late onset of symptoms &is was succeeded by the discovery of an RHV from Norway rats (Rattus norvegicus) in New York City [14]. and delayed treatment initiation after HCV infection remain an issue [3]. A small animal model for HCV would allow for &e subsequently discovered RHV (RHV-rn1) from Norway rats was used to make a small animal model for the testing of vaccines which could potentially solve these problems. hepaciviral infection, utilising a reverse genetics approach. HCV was discovered in humans 20 years ago [4] but for a In this system, the researchers showed its hepatotropic long time, researchers failed to identify an animal viral replication in inbred and outbred rat strains [15]. Emulating homologue. &is all changed with the use of high throughput HCV infection, they also showed that persistent infection deep sequencing, which has shed light on the evolutionary leads to gradual liver damage and that the HCV antiviral origins of the virus. &e first HCV homologue was dis- drug sofosbuvir suppresses replication of RHV-rn1. &is covered in 2011 in canines [5] and was quickly followed by model can be used to study the mechanisms of HCV per- the discovery of homologues in rodents [6, 7], equine [8, 9], sistence, immunity, and pathogenesis. 2 Advances in Virology terminator in a DNA transfection system [25, 26] to allow &ough rats are the usual hosts of RHV-rn1, it has also been shown that the virus is capable of establishing a per- for quick and easy mutation and screening. sistent infection in immunocompromised mice lacking type I interferon and adaptive immunity [16]. However, im- munocompetent mice clear the virus in a few weeks. Because 2. Materials and Methods this mouse model only results in an acute infection, a fully -/- Cells. Hepa1-6 (ATCC CRL-1830 ), MEFs IRF3 , immunocompetent mouse model in which a chronic in- NIH 3T3 (ATCC CRL-1658 ), BHK-21 (ATCC ® ® fection and downstream liver damage can be established is CCL-10 ), HEK 293T (ATCC CRL-11268 ), Huh- ™ ™ still in need. Additionally, the availability of knockout mice 7.5-RFP-MAVS from Dr Charles M Rice, HepG2 would aid the study of the pathogenesis of HCV related liver (ATCC HB-8065 ) and Vero cells (ATCC CCL- ® ® damage. 81 ) were cultured in DMEM (Sigma-Aldrich), 10% While these studies could lead the way to future vaccines, FBS and 1% penicillin-streptomycin. they also present the possibility of zoonotic sources of HCV Plasmid Construction. Monocistronic reporter plas- infection in humans [17]. &e genomes of RHV encode for a mids containing viral 5′UTR were constructed in polyprotein that is predicted to be cleaved into 10 proteins, pUC19. First, pUC19 was digested with EcoR1 and as with HCV, but shows a 66–77% amino acid divergence HIndIII (NEB), and then a minimal RNA polymerase I from HCV in the structural genes [7, 18]; therefore, tropism (RNA Pol I) promoter and terminator (synthesised by and pathogenicity may also differ between the viruses. In Twist Biosciences) was inserted into the digested particular, the 5′ untranslated regions (UTRs) of RHV are plasmid by In-Fusion cloning (Takara Bio); this plas- highly divergent from the corresponding regions of HCV, mid was named pOLI (Figure S1A). and there is a large difference between the different rodent clades (RHV, RHV1, RHV2, RHV3, and RHV-rn1). &e plasmid pOLI was linearized with PpuMI. Viral 5′ and 3″ UTR (synthesised by Twist Biosciences) (HCV &e high-level expression of mir-122 in liver cells allows HCV, containing two mir-122 target sequences in the IRES taken from pFR_HCV_xb (Addgene)) along mCitrine were cloned into the linearized pOLI by In- 5′UTR, to replicate and is one reason for HCV hepato- tropism [19]. Similarly, the RHV (NC_021153) found in deer Fusion cloning (Takara Bio) (Figure S1B). Deletions mice contains one such mir-122 target sequence in its 5′UTR and additions to the monocistronic reporter plasmids were created by PCR and In-Fusion cloning (Takara [7] suggesting liver cell specificity and further has a 200 nt sequence with no homology to other hepaciviruses 5′UTRs. Bio). &e RHV-1 (KC411777)) 5′UTR contains structural ele- Plasmids containing viral structural genes were con- ments typical of both pegi- and HCV-like IRES and contains structed in pUC19 by In-Fusion cloning (Takara Bio) one mir-122 target region, while RHV-1 and RHV-2 using hepacivirus CE1E2 region (synthesised by Twist (KC411784) are identical in structure and only contain a few Biosciences), along with a CMV promoter and BGH nucleotide exchanges, with RHV-3 (KC411807) and RHV- polyA (Figure S1C). Flag tag and c-Myc tag sequences rn1 being more similar to typical HCV-like IRES structures were inserted into this plasmid by PCR and In-Fusion My- [6, 15] (Figure 1). cloning (Takara Bio); this plasmid was named pC c Flag My- Along with RHV, a rodent pegivirus (RPgV) was also E1E2 . &e capsid gene was deleted from pC c Flag discovered in white-throated wood rats (Neotoma albigula) E1E2 by PCR and In-Fusion cloning (Takara Bio) Flag [7]. Pegiviruses are a new genus of the family of the Fla- resulting in the plasmid pE1E2 . viviridae, encompassing the human GBV-A, GBV-C/HGV/ To construct plasmids containing full-length RHV1 HPgV-1, GBV-D and HPgV-2 viruses. &ese pegiviruses and RPgV viral genomes, the respective monocistronic are considered to be nonpathogenic and in the case of vector (pOLI.IRES.Cirtine) was PCR linearized, and HPgV has even been reported to be beneficial in coin- gene fragments, 1.5–2 kb (synthesised by Twist Bio- fections with HIV or Ebola [20–22]. However, two pegi- sciences), covering the full-length viral coding region viruses were discovered in equine. &e first, &eiler’s were then cloned in by In-Fusion cloning (Takara Bio) disease-associated virus (TDAV), is suspected to be the replacing mCitrine. &e plasmids were named causative agent for an outbreak of acute hepatic disease pOLI.RHV1 and pOLI.RPGV (Figure S1D). occurring on a horse farm [23]. &e second, equine For constructing a reporter plasmid, mScarlet-BSD pegivirus (EPgV), like the human pegiviruses, is considered (synthesised by Twist Biosciences) was cloned into the to be nonpathogenic [24]. plasmid containing the full-length virus in-between &e viral RNA structures play important roles in both NS5A and NS5B, while also duplicating the cleavage translation and replication. Specifically, the 5′UTR sequence by In-Fusion cloning (Takara Bio). &ese containing the IRES promotes the initiation of protein plasmids were named pOLI.RHV1.SB and synthesis in a cap-independent manner. IRES’s are di- pOLI.RPgV.SB (Figure S1E). verse in sequence and structure and these differences contribute to tropism; the focus of this study is to assess Transfection. All transfections were carried out using how these differences in the 5′UTRs of RHV and RPgV Lipofectamine LTX with Plus Reagent (&ermo affect translation and to establish a reverse genetics Fisher Scientific) in Opti-MEM (Gibco) according to system utilizing the RNA polymerase I promoter and the manufacturer’s specifications. Advances in Virology 3 HCV RHV RHV1 IIIb II IIIb IIIc IIIa IIIa IIIa IIIb IIIc II IVb Va IIId Ic IIId IIIe IIIf Ic IIIf IIIe Ib Ib IV I Vb Ia Ia IV RHV2 RHV3 RHV-rn1 IIIb IIIb II IIIa IIIc II IIIa IVa IIId IIIb Ic IIIe Ib Ic IVb IIIf Ia IIb IIIa IIb Va Ib Ia Vb IV IIIf IIIc Ia IIIe IIa VI Ib IIId RPgV IIIb IIIc IIIa IIId IIIe Ib IV Ia Figure 1: Predicted RNA secondary structures within HCV, RHV, RHV1, RHV2, RHV3, RHV-rn1, and RPgV 5′ UTR. &e sequence corresponding to the mir-122 binding sites within the 5′ UTR is highlighted in orange; the first binding sites are represented by filled circles and the second by the outline. HCV contains two mir-122 binding sites and RHV, RHV1, and RHV-rn1 contain one predicted binding site. RHV2/3 and RPgV do not contain a mir-122 binding site. &e nucleotides corresponding to the AUG start codon are highlighted by filled green circles. 4 Advances in Virology Western Blot. Cells were lysed in RIPA buffer (150 mm pMD2.G (Addgene) or pE1E2 at a ratio of 1 :1:0.1, NaCl, 50 mm Tris/HCl pH 7.6, 1% Nonidet P40, 0.5% respectively, using lipofectamine LTK (&ermo Fisher sodium deoxycholate, and 5 mm EDTA) supplemented Scientific) and Opti-mem (Gibco). At 6 hours after with cOmplete Protease Inhibitor Cocktail (Roche) transfection, media were replaced and 72 h after for 15 min on ice. &e lysate was run on a 12% SDS- transfection, supernatant was harvested and passed Page and transferred to PVDF membrane (Bio-Rad). through a 0.45µm filter. Membranes were incubated for 1 hour in PBS with 5% nonfat dry milk and then stained with primary and 2.1. RNA Structure Prediction. &e RNA sequences sec- subsequently secondary antibodies for 1 hour in PBS ondary structure was predicted with RNA fold and visual- with 1% nonfat dry milk. Immunocomplexes were ization was performed using force directed graph layout detected using an Odyssey Fc Imaging System (LI- (Forna); both are hosted on ViennaRNA Web services COR Biosciences). (http://rna.tbi.univie.ac.at/forna/). Intracellular Immunofluorescence. Cells were grown on glass coverslips, washed with PBS prior to fixation in 2.2. RNA Extraction and cDNA Generation. Cells from 24- formaldehyde (3.7% w/v in PBS) for 10 min at room well plate were washed in PBS and resuspended in Trizol temperature, permeabilized for 5 min with 0.1% Triton (Sigma-Aldrich, USA), and RNA was isolated by iso- X-100 in PBS, and blocked with 3% BSA in PBS for propanol precipitation, washed with 70% ethanol, and 30 min. Primary and secondary antibodies were diluted resuspended in DEPC-water. RNA was DNAse treated in PBS containing 1% BSA, and cells were stained with (Invitrogen, Paisley, UK) and subjected to RT-PCR using primary antibody for 1 hour, washed 3x with PBS, and 100 ng purified RNA. For RT-PCR, the high-capacity cDNA then stained with secondary antibody for 1 hour, fol- archive Kit from Applied Biosystem (ABI PRISM, War- lowed by staining with 0.1µg/ml DAPI in PBS for 5 min rington, United Kingdom) was used according to the and application of Prolong Gold Antifade reagent specifications of the manufacturer. (Invitrogen). Extracellular. Cells were grown on glass coverslips, incubated with PBS containing 4% FBS (Fetal Bovine 2.3. Quantitative Real-Time PCR for Selected Genes. Serum) for 30 min. Primary and secondary antibodies Quantitative real-time PCR was performed using a Light were diluted in PBS containing 4% FBS, cells were cycler 480 Real-Time PCR System (Roche Diagnostics) and stained with primary antibody for 1 hour, washed 3x the LightCycler 480 probes master reaction mix (Roche with PBS, and then stained with secondary antibody for Diagnostics) following the manufacturer’s protocol. Data 1 hour. Cells were then stained with Wheat Germ analysis was performed with LightCycler 480 Software Agglutinin, Alexa Fluor 594 conjugate as per man- (Roche Diagnostics). Oligonucleotides sequences used as ufacturers protocol. Cells were fixed in formaldehyde primers for quantitative real-time PCR and corresponding (3.7% w/v in PBS) for 10 min at room temperature, probes were designed according to the guidance of the permeabilized for 5 min with 0.1% Triton X-100 in PBS, Universal Probe library from Roche applied Science. and stained with 0.1µg/ml DAPI in PBS for 5 min &ermal cycling started with HotStarTaq activation during followed by application of Prolong Gold Antifade re- ° 10 min at 95 C. &ereafter, 45 cycles of amplification were ° ° ° agent (Invitrogen). run consisting of 10 s at 95 C, 30 s 60 C, and 20 s of 72 C. A negative control containing reagents only and serial dilu- Images were acquired using a confocal microscope (Leica specify type) and Z-stacks and analysed with tions of cDNA was included in each run. Each sample was measured as a triplicate and the average concentration was ImageJ. used. For LightCycler analysis, expression of hypoxanthine Antibodies. Rat anti-DYKDDDDK (clone L5, Biol- phosphoribosyltransferase gene (HPRT) was used for nor- egend), mouse anti-c-myc (clone 9E11, Biolegend), malization. Relative expression of samples was calculated by mouse anti-β-actin (clone 2F1-1, Biolegend), mouse J2 the comparative cycling threshold method (ΔΔCT) and then anti-dsRNA IgG2a (Sciscons), goat anti-rat IgG (H + L) setting the samples transfected with HCV IRES as the Alexa Fluor 488 (Invitrogen), IRDye 800CW goat benchmark. anti-rat IgG, and IRDye 680RD donkey anti-mouse IgG (LI-COR Biosciences). GeneBank Accession Numbers. RHV (Hepacivirus E), Flow Cytometry. Single cell suspensions were generated NC_021153; RHV1 (Hepacivirus J), KC411777; RHV2 (Hep- and kept in FACS buffer (2% FCS, 5 mm EDTA in PBS). acivirus F), KC411784; RHV3 (Hepacivirus I), KC411807; Cells were analysed using a Gallios flow cytometer RHV-rn1 (Hepacivirus G), KX905133.1; RPgV, NC_021154. (Beckman Coulter) and FlowJo software and gated on viable cells using the live/dead fixable near-IR dead cell 3. Results stain kit (Invitrogen). Lentivirus. Plates were seeded with HEK 293T in 3.1. RHV and RPgV IRESs Are Functional in Rodent Cells. DMEM and 3% FCS and then transfected with pLKO- To test viral IRES driven translation, a monocistronic gfp (Addgene), pCMV∆R8.2 (Addgene), and either plasmid vector was constructed containing a minimal RNA Advances in Virology 5 that of RHV, RHV1, RHV3, and RHV-rn1 was at the lowest polymerase I promoter in front of the full-length viral 5′ UTR followed by a fluorescent maker, the viral 3′ UTR, and level (Figure 2(h)). In Vero cells, the RPgV produced high levels of translation, over twice that of HCV; RHV2 was the the RNA polymerase I terminator (Figure 2(a)). &e HCV IRES was used as a positive control along with a control only other IRES that was functional in these cells although at plasmid containing a scrambled sequence in place of the a very low level when compared to RPgV (Figure 2(i)). viral 5′ UTR. We constructed plasmids containing RHV, RHV1, RHV2, RHV3, RHV-rn1, and RPgV 5′ UTR from 3.3.DeletionsAbrogatetheFunctionofRHV1andRPgVIRES. previously published sequences. To further assess the structural requirements of RHV1 IRES &ese plasmids were transfected into the murine hepa- for full functionality, we made several different constructs. tocyte cell line Hepa1-6, with the HCV and RPgV IRES driving &e first contained an additional 20 nucleotides of virus the highest level of translation at 72 hours after transfection sequences downstream of the start codon. When transfected with a mean fluorescence intensity (MFI) of 30, followed by into Hepa1-6 cells, this construct did not lead to a difference RHV1 and RHV2 with an MFI of 22 and 14, respectively. in the levels of translation in comparison with the construct RHV3 and RHV-rn1 IRES drove translation at a level only containing just the 5′ UTR. Two deletion constructs were slightly above background and RHV was not functional in made; in RHVΔI, the 5′ three stem loops (Ia/b/c) were deleted Hepa1-6 (Figure 2(b)). To further assess the level of RNA (Figure 3(a)). &is deletion decreased the IRES function by transcripts of mCitrine within the cells, we preformed qRT- 90%. &e Va and Vb stem loops were deleted from RHV1ΔII PCR on transfected cells. &ere was no statistical difference in and again led to a decrease in function by 90% in the murine the level of transcripts between HCV, RHV, RHV1, RHV2, hepatocyte cell line Hepa1-6 (Figure 3(c)). RHV3, RHV-rn1, RPgV, and the control plasmid containing a For the RPgV IRES, we also added an additional 20 scrambled sequence in place of the viral 5′UTR. &e other nucleotides of virus sequences downstream of the start control plasmid contains RHV1 5′UTR but no Pol I promoter codon. &is again had no effect on the levels of translation in did yield a readily detectable level of RNA (Figure S2). comparison to the construct containing just the 5′ UTR. To To further assess IRES function in murine cells, we tested further assess the sequence required for driving translation, two murine embryonic fibroblasts cell lines, MEFs and NIH three constructs were made with deletions, RPgVΔI and 3T3. Transfection of MEFs with the plasmids revealed that RPgVΔII have deletions to the 5′ of the IRES with RPgVΔI RHV1 drove the highest level of translation at an MFI of 9 having the first two stem loops (Ia/b) and RPgVΔII three followed by RHV2 at an MFI of 5 (Figure 3(c)). HCV, RHV, stem loops deleted (Ia/b and II) (Figure 3(b)). RPgVΔIII has RHv3, RHV-rn1, and RPgV generated signals only slightly two internal stem loops deleted (IIId/e). &e 5′ deletions to above the negative control. In NIH3T3, the RPgV drove the RPGV had no effect on the levels of translation. However, highest level of translation with an MFI of 17 followed by the internal deletions in RPgVΔIII led to a reduction in HCV with an MFI of 7; again, RH1 and RHV2 were translation of 53% (Figure 3(d)). functional but at low levels, and RHV, RHV3, and RHV-rn1 were not functional (Figure 3(d)). &e viruses originate from different rodent species; 3.4. Expression of E1E2. Previous studies examining the therefore, the baby hamster kidney cell line was tested to subcellular localization of HCV E1 and E2 used cells assess if the IRESs are functional in this cell line. As with the transfected with a plasmid expressing the E1 and E2 proteins previous cell lines, the RPgV IRES drove high levels of [27]. &ese studies concluded that the HCV structural translation; also the RHV1 and RHV2 were capable of proteins are expressed on the cell surface, based on im- driving high levels followed by HCV; again, the RHV, munofluorescence detection. In order to examine the lo- RHV3, and RHV-rn1 were not functional (Figure 2(e)). calization of RHV structural proteins in an expression system, we cloned the structural region (capsid-E1-E2) of RHV into a plasmid containing the CMV promoter; we then 3.2.RHVandRPgVIRESsShowDifferingFunctionsinHuman added the c-Myc tag to the 5′ end of the capsid protein and CellLines. In the human hepatocyte cell line, Huh7.5 which the Flag tag to the 5′ end of the E2 protein following the expresses high levels of mir122, and the RPgV IRES drives E1E2 cleavage sequence (Figure 4(a)). the highest levels of translation followed by HCV and RHV2. HEK 293T cells were transfected with the vectors Flag Myc Flag &e RHV-rn1 drove low levels of translation and, as with pE1E2 or pC E1E2 and after 48 hours lysed for murine cells, RHV and RHV3 did not yield any signal SDS-PAGE. &e E2 protein was detected in cells transfected (Figure 2(f)). Another human hepatocyte cell line, HepG2 with either expression vector using an anti-Flag tag antibody that does not express mir122, was also used to test the IRES’s (Figure 4(a)), and the capsid was detected in cells transfected Myc Flag function: in these cells, HCV drove the highest levels fol- with the pC E1E2 expression vector using an anti-c- lowed by RPgV and RHV2, while the expressions from RHV, Myc antibody. &e proteins detected were of the predicted RHV1, RHV3, or RHV-rn1 were at background level size, showing that posttranslational cleavage was complete. (Figure 2(g)). In order to determine if the RHV glycoproteins To investigate if the IRESs are functional in non- expressed from these vectors also exhibit an intracellular hepatocyte cells lines, we used HEK 293Tand Vero cell lines. colocalization, we examined transfected cells by immuno- In HEK 293T, the HCV IRES showed the highest level of fluorescence for capsid and E2 expression; both were shown translation followed by the RPgV IRES and RHV2, while to colocalize (Figure 4(b)). We also assessed if the envelope IRES IRES IRES IRES IRES IRES IRES IRES 6 Advances in Virology HCV RHV1 PolIP 5′UTR mCitrine 3′UTR PolIT 31 ± 2.99 22 ± 1.56 RHV2 RPgV 14 ± 0.9 30 ± 2.59 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (a) (b) HCV RHV1 HCV RHV1 2.24 ± 0.13 9.26 ± 0.9 7.68 ± 0.21 3.98 ± 0.23 11.00 20 8.25 15 RPgV RHV2 5.50 RHV2 10 RPgV 2.37 ± 0.34 5.10 ± 0.41 5.49 ± 0.67 17 ± 1.73 2.75 5 0.00 0 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (c) (d) HCV RHV1 HCV RHV1 80 12 23 ± 1.63 53 ± 2.92 7.16 ± 0.52 2.39 ± 0.13 60 9 6 RHV2 RPgV 40 RHV2 RPgV 49.28 ± 3.03 76 ± 2.89 4.47 ± 0.43 11 ± 0.48 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (e) (f) HCV RHV1 HCV RHV1 22 20 ± 0.99 2.52 ± 0.28 18 ± 0.92 3.7 ± 0.92 16.5 11 RHV2 RPgV RHV2 RPgV 10 ± 1.24 14 ± 0.58 7.7 ± 0.44 11 ± 0.49 5.5 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (g) (h) HCV RHV1 33 ± 0.92 2.19 ± 0.03 40 RHV2 RPgV 7.94 ± 0.22 77 ± 2.17 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV (i) Figure 2: RHV and RPGV IRES activity in different cell types. Schematic of the monocistronic vectors used (a). Hepa1-6 (b), MEFs (c), NIH 3T3 (d), BHK-21 (e), Huh7.5 (f), HEK 293T (h), and Vero (i) cells were transfected with the indicated plasmids. Cells were harvested and analysed by flow cytometry at 48 h.p.t.; bar graphs show MFI for mCitrine (n≥ 8, mean± SEM of at least three independent experiments), and representative FACS plots on the right of each graph numbers represent the MFI (mean± SEM). &e dashed line represents the fluorescent output of the control plasmid containing a scrambled sequence in place of the viral 5′ UTR. MEFs MFI mCitrine BHK-21 MFI mCitrine HepG2 MFI mCitrine Vero MFI mCitrine mCitrine mCitrine mCitrine Hepa1-6 MFI mCitrine NIH3T3 MFI mCitrine HEK 293T MFI mCitrine Huh-7.5 MFI mCitrine mCitrine mCitrine mCitrine mCitrine mCitrine IRES IRES Advances in Virology 7 C C 200 G A 370 G C C 150 U G A A C A U IIIb C C U A U G G G 160 A 140 C 180 C C IVa G C G G G A C U G G G U U C G A A C G U G U U C A U G A C C U G A G C C G G U G A U C U A A C C G G U G U A G C A G G 200 U G C A G G II C C 210 G A C U U G A C G U C A U C U U 130 C G 190 170 C 190 A G G C G G C C U U G A G C G A 380 360 C U G C G G 210 A C C G C G U G G RPgVIRES U G G C C U C U G RHV1IRES G A C G G G C C C 220 120 G C G A G A U A G A U C U U U C G IIIc A G C U A G 230 ΔIII G 220 350 IIIa C A C G C 390 C G U G G U A U G C IIIa IIIb A U C U C A A G G G C C C G 170 U C U C G U G G A G U G G C C C A G G A G A A G U U G C A U G A 290 160 U U G G G U C 260 G C U G A U G G 110 C G G G 240 U G A C G U C G C G IVb C C G G U G C A U G C C 400 G III 280 C 300 A 340 G G G U C A C 230 G U U G A U U C 310 U C G C U U U C A C G G G G G U C G 250 C C G G ΔII C G G G G U U A G C C C C G G A A A C C C A U C G G C G G A G A U U U C C U A 270 G 410 A C C C A G G G C A U G C C G A G G G U C 420 A U A U 270 A G 240 C A C A U 150 A C U C A A A U G U G G G A 320 430 A G U G U G 250 U C C G II U U G U U A C C U U 330 U G C U C C G A G 100 U A C A G C 260 C U G C G 90 C G 90 G U A G U C U A U U G C A G G C G C C 470 C C C A C A C G C U IIIe G 440 G C C G 290 U A G G C G C G A C U U C A G A U G A C G G G G A G 100 U G C A C G U U C U 460 C A G U C G A C G G C G A G C C G U A Va U U U 280 G A C C C G 480 C A C U A G 80 A G G A 140 U U G A U A G G A 80 G 120 U A G A 130 A G G G C C C U A G C C G G C A A G C C G G U U C U 450 C G G U A G G G A G 110 U ΔI Ic C G C ΔII G G G A U G C A C G 510 C G A C C U U 300 G U G U U G G G U G G C G C C C A A G G C G G C U G C G A 60 C 70 A U G A G G G 70 C C A 490 U 60 C C A G G A U G A C A C C G U G A A A G C U U A C U G A U C A Ib G 520 U G C U A U G C G U G 50 G C G ΔI 50 A G G U G A G G U U G C G U G G G C C A C G 40 C U A A Vb C C C A C C U C C U 500 U 30 C 310 G U Ib U G G C A G C 40 G G C U C A C C C G C A G C C C U G IVa 20 C A G C C G G C C G C U G G 340 C A G 30 G U C U G C C C 320 Ia U U G A C C G U U G C A G U U U C G G A G A G A U G A C G C C U U A A C A G G U 530 370 U A U G C C C U 20 10 A C G C 330 U C C A U G C A U U C G G G C A U 10 Ia C U A U C C G A C 360 A C A C U 540 U A U A A A G G G C G C C C A A C U U G U A G A A A C G G 350 570 U C C U C G C U 560 A A C G A C G 550 A C (a) (b) RHV1 RHV1C RPgV RPgV∆I 22 ± 1.56 22 ± 1.68 30 30 ± 2.59 30 ± 2.19 RHV1∆I RHV1∆II RPgV∆II RPgV∆III 2.37 ± 0.14 2.24 ± 0.07 33 ± 0.66 14 ± 0.73 RHV1 RHV1C RHV1∆I RHV1∆II RPgV RPgVC RPgV∆I RPgV∆II RPgV∆III (c) (d) Figure 3: Deletions to RHV1 and RPgV 5′ UTR abrogate IRES translation. Predicted RNA secondary structure of RHV1 (a) and RPgV (b). Red circles indicate areas deleted from plasmids. Hepa1-6 cells were transfected with RHV1 (c) and RPgV (d) plasmids. Cells were harvested and analysed by flow cytometry at 48 h.p.t. Bar graphs show MFI for mCitrine (n≥ 8, mean± SEM of at least three independent experiments) and representative FACS plots on the right of each graph; numbers represent the MFI (mean± SEM). &e dashed line represents the fluorescent output of the control plasmid containing a scrambled sequence in place of the viral 5′ UTR. protein was expressed on the cell surface. Staining with anti- 4. Discussion flag antibody to detect E2 indeed showed punctate staining &e five rodent hepacivirus IRESs we tested showed different on the cell membrane and when combined with wheat germ levels of ability to drive translation using in a monocistronic agglutinin to stain the cell membrane, it showed colocali- vector across varying cell lines. While the use of a mono- zation with the flag antibody (Figure 4(c)). &is indicates cistronic vector, utilizing RNA polymerase I, avoids the that a proportion of E2 is surface-expressed. potential of readthrough in comparison to bicistronic vec- To determine if the surface-localized E1E2 could mediate tors; its drawback is that we were not able to directly viral entry, we produced a GFP encoding lentivirus vector compare expression levels between cell types due to their pseudotyped with the RHV, RHV1, RHV2, and RHV3 E1E2 difference in susceptibility to transfection. However, the proteins by transfecting HEK 293T cells with pE1E2 and the RHV, RHV3, and RHV-rn1 IRESs were either not functional lentivirus backbone and packaging plasmid, and 72 h later or drove expression at very low levels. &is comes as a harvesting and filtering the supernatant. We then tested if the surprise as the RHV-rn1 virus has already been shown to E1E2-pseudotyped lentivirus vectors were entry-competent. replicate in both mice and rats. Supernatants from the cotransfected cells were applied to Both RHV1 and RHV2 drive high levels of translation in Hepa1-6 cells and GFP reporter expression assayed 72 h later. murine hepatocytes, MEFs and BHK-21 cells. In human RHV1 E1E2-pseudotyped lentivirus vectors gave rise to a hepatocytes, the RHV2 IRES outperformed RHV1 which is of small number of GFP positive cells, when compared to VSVG interest as they are similar in sequence and therefore are likely pseudotyped lentivirus. Lentivirus vectors pseudotyped with to have a similar structure. In the case of RHV1, deleting the RHV, RHV2, and RHV3 or lacking envelope glycoprotein predicted initial three stem loops abrogates IRES function, failed to give rise to any GFP positive cells (Figure 4(d)). &is suggesting that the full 5′UTR sequence is required to indicates that only RHV1 pseudotyped lentivirus vectors can maintain high levels of expression. Also, unlike HCV, where mediate viral entry in Hepa1-6 cells resulting in reporter gene previous studies have shown that the inclusion of 12–30 nt of expression. &is data also indicates that surface-expressed the core protein coding sequence was essential for an efficient RHV1 E1E2 heterodimers are functional. Hepa1-6 MFI mCitrine mCitrine Hepa1-6 MFImCitrine mCitrine 8 Advances in Virology Myc C E1 Flag E2 E2 E2 E2 30 kDa (Flag tag) Capsid 18 kDa (c-Myc tag) Flag tag-E2 c-Myc tag-capsid Flag tag-E2 c-Myc Tag-capsid Actin 40 kDa DAPI-nucleus (a) (b) E1 Flag E2 RHV RHV1 VSVG Flag tag-E2 Flag tag-E2 DAPI-nucleus DAPI-nucleus WGA-plasma membrane (c) (d) Figure 4: Expression of RHV envelope proteins. Schematic of the RHV1 envelope expression vector and western blot of cell lysate at 48 h.p.t. from transfected HEK 293T (a) Intracellular immunofluorescence of 293T cells at 48 h.p.t. with RHV1 envelope expression vector, stained with flag tag in green, c-Myc tag in red, and combined with DAPI stain (b) Extracellular immunofluorescence for E2 by flag tag stain (green) and combined with WGA (red) and DAPI (blue) from 293Tat 48 h.p.t. with RHV1 envelope expression vector, and images represent one slice from z-stack (c) GFP-lentivirus vector pseudotyped with RHV or RHV1 envelope proteins or VSVG were incubated on Hepa1-6 cells; GFP positive cells indicate transduction (d). IRES activity [28], additional nucleotides from the core study cell tropism in a murine model and the antigenicity of protein of RHV1 did not increase transcription levels. the functional E1 and E2 glycoproteins. &e RPgV 5′UTR has little significant similarity with Our efforts to make an RHV and RPgV replication any known pegivirus but drives high levels of expression competent model in vitro have so far proved unfruitful. in all cell types tested. By deleting specific regions, we were Using full-length viral constructs, we tested the human hepatoma cell line (Huh-7.5) containing a MAVS able to show that the initial 126 nt of the 5′UTR does not contribute to IRES function and that stem loops IIId/e are cleavage reporter where upon HCV NS3-4A cleavage of essential for maintaining high levels of expression. It the reporter, the RFP translocates to the nucleus [29]. would be of significance in the future to confirm the Translocation of RFP was observed with full-length predicted structures of RPgV and RHV1 IRES’s poten- RHV1, confirming the previous finding that the RHV1 tially using RNA SHAPE. NS3-4A protease is capable of cleaving human MAVS RHV1 structural genes (C, E1, and E2) expressed from [30]; however, the number of cells with RFP translocation plasmid were shown to be cleaved and yielded proteins of the did not increase over time (Figure S3.A). correct size. Moreover, RHV1 E1E2 supported transduction We further tested full-length viral constructs containing of hepatocytes when used to pseudotype lentivirus vectors. m-Scarlet and BSD inserted between NS4A-B in both Further studies will need to be carried out to find the specific Hepa1-6 and BHK-21, mScarlet was expressed in cells but the number of cells expressing mScarlet did not increase entry receptors, initially blocking CD81 and HCV entry receptors and testing susceptibility of transduction in al- overtime and failed to yield a clone when selecting for ternative cell lines, but this initial experiment hints at the replication with BSD, even when expressing Sec14L2 and hepatotropic potential of RHV1 in mice. ApoE, both essential for high levels of HCV replication [31, 32] (Figure S3.B). Further cell lines could be tested along &e generation of viral pseudotypes is one of the most with knocking out the innate immune response in future widely used methods for assaying functional receptors, experiments. allowing attachment, penetration, and uncoating to be In Summary, this study shows that RHV1/2 and RPgV studied. &is study lays the groundwork for using RHV1 contain IRESs that are capable of driving high levels of pseudotype particles to be used to asses these important protein synthesis. RHV1 structural genes are cleaved by parts for the viral replication cycle and could also be used to HEK 293T surface Flag pE1E2 c-Myc Flag pC E1E2 HEK 293T Hepa1-6 intracellular Advances in Virology 9 [10] M. Lauck, S. D. Sibley, J. 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Vasilakis et al., “Divergent viruses discovered in arthropods and vertebrates revise the evolu- &is paper was previously made available in preprint on tionary history of the flaviviridae and related viruses,” Journal bioRxiv (doi: https://doi.org/10.1101/761379). of Virology, vol. 90, pp. 659–669, 2016. [14] C. Firth, M. Bhat, M. A. Firth et al., “Detection of zoonotic Conflicts of Interest pathogens and characterization of novel viruses carried by commensal Rattus norvegicus in New York city,” MBio, vol. 5, &e authors declare that they have no conflicts of interest. pp. e01933–e02014, 2014. [15] S. Trivedi, S. Murthy, H. Sharma et al., “Viral persistence, liver Authors’ Contributions disease, and host response in a hepatitis C-like virus rat model,” Hepatology, vol. 68, pp. 435–448, 2018. SS designed and planned all the experiments. SS, SB, and KM [16] E. Billerbeck, R. Wolfisberg, U. Fahnøe et al., “Mouse models performed the experiments and analysed results. 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Advances in VirologyHindawi Publishing Corporation

Published: Jul 30, 2021

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