bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 In vitro comparison of the internal ribosomal entry site activity

2 from rodent and pegivirus

3

4 Stuart Sims1, Kevin Michaelsen1, Sara Burkhard2, Cornel Fraefel1

5

6 1. Institute of Virology, University of Zurich, Zurich, Switzerland

7 2. Clinic for Internal Medicine, University Hospital of Zurich, Zurich, Switzerland.

8

9 Abstract

10 The 5’ untranslated region (5’ UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contain

11 sequence homology to the HCV type IV internal ribosome entry sites (IRES). Utilizing a

12 monocistronic expression vector with an RNA polymerase I promoter to drive transcription

13 we show cell-specific IRES translation and regions within the IRES required for full

14 functionality. Focusing on RHV we further pseudotyped lentivirus with RHV and showed cell

15 surface expression of the envelope proteins and transduction of murine hepatocytes.

16 Key Words

17 Hepacivirus, IRES, HCV, Pegivirus bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

18 Introduction

19 Hepatitis C (HCV) infects more than 71 million people worldwide(1) leading to liver

20 failure and hepatocellular carcinoma, presenting a major global health burden. While the

21 introduction of new direct-acting antiviral drugs (DAAs) has improved treatment response

22 rates and heralded a new era of HCV treatment(2), the cost and availability of DAAs along

23 with drug resistance, and chronic/non-reversible liver damage due to late onset of

24 symptoms and delayed treatment initiation after HCV infection are an issue(3). A small

25 animal model for HCV would allow for the testing of vaccines which could potentially solve

26 these problems.

27

28 HCV was discovered in humans 20 years ago(4) but for a long-time researchers failed to

29 identify an animal viral homologue, this all changed with the use of high throughput deep

30 sequencing and has shed light on the evolutionary origins of the virus. The first HCV

31 homologue was discovered in 2011 in canines(5) and was quickly followed by the discovery

32 of homologues in rodents(6,7), equine(8,9), primates(10), bovine(11,12) and then, the first

33 nonmammalian species, sharks(13).

34

35 The rodent HCV homologues were termed rodent (RHV) and were first

36 identified in deer mice (Peromyscus maniculatus), a species known to carry hantavirus,

37 desert woodrats (Neotoma lepida) and hispid pocket mice (Chaetodipus hispidus)(7). The

38 same year RHV was described in European bank voles (Myodes glareolus) and South African

39 four-striped mice (Rhabdomys pumilio)(6). This was succeeded by the discovery of an RHV

40 from Norway rats (Rattus norvegicus) in New York City(14).

41 bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

42 The subsequently discovered RHV (RHV-rn1) from Norway rats was used to make a small

43 animal model for hepaciviral infection, utilising a reverse genetics approach. In this system

44 researchers showed its hepatotropic replication in inbred and outbred rat strains(15).

45 Emulating HCV infection they also showed that persistent infection leads to gradual liver

46 damage and that the HCV antiviral drug sofosbuvir suppresses replication of RHV-rn1. This

47 model can be used to study the mechanisms of HCV persistence, immunity, and

48 pathogenesis.

49

50 Though rats are the usual hosts of RHV-rn1, it has also been shown that the virus is capable

51 of establishing a persistent infection in immunocompromised mice lacking type I interferon

52 and adaptive immunity(16). However, immunocompetent mice clear the virus in a few

53 weeks. Because this mouse model only results in an acute infection, a fully

54 immunocompetent mouse model in which a chronic infection and downstream liver

55 damage can be established is still in need. Additionally, the availability of knock out mice

56 would aid the study of the pathogenesis of HCV related liver damage.

57

58 While these studies could lead the way to future vaccines they also present the possibility of

59 zoonotic sources of HCV infection in humans(17). The genomes of RHV encode for a

60 polyprotein, that is predicted to be cleaved into 10 proteins, as with HCV, but shows a 66-

61 77% amino acid divergence from HCV in the structural genes(18),(7), therefore, tropism and

62 pathogenicity may also differ between the . In particular, the 5’ untranslated regions

63 (UTRs) of RHV are highly divergent from the corresponding regions of HCV and there is a

64 large difference between the different rodent clades (RHV, RHV1, RHV2, RHV3, RHV-rn1).

65 bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

66 The high-level expression of mir-122 in liver cells allows HCV, containing two mir-122 target

67 sequences in the 5’UTR, to replicate and is one reason for HCV hepatotrophism(19).

68 Similarly, the RHV (NC_021153) found in deer mice contains one such mir-122 target

69 sequence in its 5’UTR(7) suggesting liver cell specificity and further has a 200nt sequence

70 with no homology to other hepaciviruses 5’UTRs. The RHV-1 (KC411777)) 5’UTR contains

71 structural elements typical of both pegi- and HCV-like IRES and contains one mir-122 target

72 region. While RHV-1 and RHV-2 (KC411784) are identical in structure and only contain a few

73 nucleotide exchanges, with RHV-3 (KC411807) and RHV-rn1 being more similar to typical

74 HCV-like IRES structures(6,15).

75

76 Along with RHV, a rodent pegivirus (RPgV) was also discovered in white-throated wood rats

77 (Neotoma albigula)(7). Pegiviruses are a new genus of the family of the ,

78 encompassing the human GBV-A, GBV-C/HGV/HPgV-1, GBV-D and HPgV-2 viruses. These

79 pegiviruses are considered to be non-pathogenic and in the case of HPgV has even been

80 reported to be beneficial in co-infections with HIV or Ebola(20–22). However, two

81 pegiviruses were discovered in equine, the first, Theiler's disease-associated virus (TDAV) is

82 suspected to be the causative agent for an outbreak of acute hepatic disease occurring on a

83 horse farm(23). The second, equine pegivirus (EPgV), like the human pegiviruses, is

84 considered to be non-pathogenic(24).

85

86 The viral RNA structures play important roles in both translation and replication. Specifically,

87 the 5’UTR containing the IRES promotes the initiation of protein synthesis in a cap-

88 independent manner. IRES’s are diverse in sequence and structure and these differences bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

89 contribute to tropism; the focus of this study is to assess how these differences in the

90 5’UTRs of RHV and RPgV affect translation and to establish a reverse genetics system.

91 Materials and Methods

92 Cells. Hepa1-6 (ATCC® CRL-1830™), MEFs IRF3-/-, NIH 3T3 (ATCC® CRL-1658™), BHK-21

93 (ATCC® CCL-10™), HEK 293T (ATCC® CRL-11268™), Huh-7.5-RFP-MAVS from Dr Charles M

94 Rice, HepG2 (ATCC® HB-8065™) and Vero cells (ATCC® CCL-81™) were cultured in DMEM

95 (Sigma-Aldrich), 10% FBS and 1% penicillin-streptomycin.

96 Plasmid construction. Monocistronic reporter plasmids containing viral 5’UTR were

97 constructed in pUC19. First pUC19 was digested with EcoR1 and HIndIII (NEB), then a

98 minimal RNA polymerase I (RNA Pol I) promoter and terminator (synthesised by Twist

99 Biosciences) was inserted into the digested plasmid by In-Fusion cloning (Takara Bio), this

100 plasmid was named pOLI (Fig S1A).

101 The plasmid pOLI was linearized with PpuMI. Viral 5’ and 3” UTR (synthesised by Twist

102 Biosciences) (HCV IRES taken from pFR_HCV_xb (Addgene)) along mCitrine were cloned into

103 the linearized pOLI by In-Fusion cloning (Takara Bio) (Fig S1B). Deletions and additions to the

104 monocistronic reporter plasmids were created by PCR and In-Fusion cloning (Takara Bio).

105 Plasmids containing viral structural genes were constructed in pUC19 by In-Fusion cloning

106 (Takara Bio) using hepacivirus CE1E2 region (synthesised by Twist Biosciences), along with a

107 CMV promoter and BGH polyA (Fig S1C). Flag tag and c-Myc tag sequences were inserted

108 into this plasmid by PCR and In-Fusion cloning (Takara Bio), this plasmid was named

109 pCMycE1E2Flag. The capsid gene was deleted from pCMycE1E2Flag by PCR and In-Fusion cloning

110 (Takara Bio) resulting in the plasmid pE1E2Flag.

111 To construct plasmids containing full-length RHV1 and RPgV viral genomes, the respective

112 monocistronic vector (pOLI.IRES.Cirtine) were PCR linearized and gene fragments, 1.5-2kb bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

113 (synthesised by Twist Biosciences), covering the full-length viral coding region were then

114 cloned in by In-Fusion cloning (Takara Bio) replacing mCitrine. The plasmids were named

115 pOLI.RHV1 and pOLI.RPGV (Fig S1D).

116 For constructing a reporter plasmid, mScarlet -BSD (synthesised by Twist Biosciences) was

117 cloned into the plasmid containing the full-length virus in-between NS5A and NS5B while

118 also duplicating the cleavage sequence by In-Fusion cloning (Takara Bio). These plasmids

119 were named pOLI.RHV1.SB and pOLI.RPgV.SB (Fig S1E).

120 Transfection. All transfections were carried out using Lipofectamine® LTX with Plus™

121 Reagent (Thermo Fisher Scientific) in Opti-MEM (Gibco) according to the manufacturer's

122 specifications.

123 Western blot. Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.6, 1%

124 Nonidet P40, 0.5% sodium deoxycholate, 5 mM EDTA) supplemented with cOmplete™

125 Protease Inhibitor Cocktail (Roche) for 15 min on ice. The lysate was run on a 12% SDS-Page

126 and transferred to PVDF membrane (Bio-Rad). Membranes were incubated for 1 hour in PBS

127 with 5% non-fat dry milk, then stained with primary and subsequently secondary antibodies

128 for 1 hour in PBS with 1% non-fat dry milk. Immunocomplexes were detected using an

129 Odyssey® Fc Imaging System (LI-COR Biosciences).

130 Immunofluorescence. Intracellular: Cells were grown on glass coverslips, washed with PBS

131 prior to fixation in formaldehyde (3.7% w/v in PBS) for 10 min at room temperature,

132 permeabilized for 5 min with 0.1% Triton X-100 in PBS, and blocked with 3% BSA in PBS for

133 30 min. Primary and secondary antibodies were diluted in PBS containing 1% BSA, cells were

134 stained with primary antibody for 1 hour, washed 3x with PBS then stained with secondary

135 antibody for 1 hour, followed by staining with 0.1 µg/ml DAPI in PBS for 5 min and

136 application of Prolong Gold Antifade reagent (Invitrogen). bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

137 Extracellular: Cells were grown on glass coverslips, incubated with PBS containing 4% FBS

138 (Fetal Bovine Serum) for 30min. Primary and secondary antibodies were diluted in PBS

139 containing 4% FBS, cells were stained with primary antibody for 1 hour, washed 3x with PBS

140 then stained with secondary antibody for 1 hour. Cells were then stained with Wheat Germ

141 Agglutinin, Alexa Fluor™ 594 conjugate as per manufacturers protocol. Cells were fixed in

142 formaldehyde (3.7% w/v in PBS) for 10 min at room temperature, permeabilized for 5 min

143 with 0.1% Triton X-100 in PBS and stained with 0.1 µg/ml DAPI in PBS for 5 min followed by

144 application of Prolong Gold Antifade reagent (Invitrogen).

145 Images were acquired using a confocal microscope (Leica specify type), Z-stacks, and

146 analysed with ImageJ.

147 Antibodies. Rat anti-DYKDDDDK (clone L5, Biolegend), mouse anti-c-myc (clone 9E11,

148 Biolegend), mouse anti-β-actin (clone 2F1-1, Biolegend), mouse J2 anti-dsRNA IgG2a

149 (Sciscons), goat anti-rat IgG (H+L) Alexa Fluor 488 (Invitrogen), IRDye® 800CW goat anti-rat

150 IgG and IRDye® 680RD donkey anti-mouse IgG (LI-COR Biosciences).

151 Flow cytometry. Single cell suspensions were generated and kept in FACS buffer (2% FCS,

152 5mM EDTA in PBS). Cells were analyzed using a Gallios flow cytometer (Beckman Coulter)

153 and FlowJo software, and gated on viable cells using the live/dead fixable near-IR dead cell

154 stain kit (Invitrogen).

155 Lentivirus. Plates were seeded with HEK 293T in DMEM and 3% FCS, then transfected with

156 pLKO-gfp (Addgene), pCMV∆R8.2 (Addgene) and either pMD2.G (Addgene) or pE1E2 at a

157 ratio of 1:1:0.1 respectively using lipofectamine LTK (Thermo Fisher Scientific) and Opti-

158 mem (Gibco). At 6 hours post transfection, media was replaced and 72h post transfection,

159 supernatant was harvested and passed through a 0.45µm filter. bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

160 GeneBank accession numbers. RHV (Hepacivirus E); NC_021153, RHV1 (Hepacivirus J);

161 KC411777, RHV2 (Hepacivirus F); KC411784, RHV3 (Hepacivirus I); KC411807, RHV-rn1

162 (Hepacivirus G); KX905133.1, RPgV; NC_021154. bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

163 Results

164 RHV and RPgV IRES’s are functional in Rodent cells

165 To test viral IRES driven translation, a monocistronic plasmid vector was constructed

166 containing a minimal RNA polymerase I promoter in front of the full-length viral 5’ UTR

167 followed by a fluorescent maker, the viral 3’ UTR, and the RNA polymerase I terminator (Fig

168 1A). The HCV IRES was used as a positive control along with a control plasmid containing a

169 random sequence in place of the viral 5’ UTR. We constructed plasmids containing RHV,

170 RHV1, RHV2, RHV3, RHV-rn1 and RPgV 5’ UTR from previously published sequences.

171

172 These plasmids were transfected into the murine hepatocyte cell line Hepa1-6, with the HCV

173 and RPgV IRES driving the highest level of translation at 72 hours post transfection with a

174 mean fluorescence intensity (MFI) of 30, followed by RHV1 and RHV2 with an MFI of 22 and

175 14, respectively. RHV3 and RHV-rn1 IRES drove translation at a level only slightly above

176 background and RHV was not functional in Hepa1-6 (Fig 1.B).

177

178 To further asses IRES function in murine cells, we tested two murine embryonic fibroblasts

179 cell lines, MEFs and NIH 3T3. Transfection of MEFs with the plasmids revealed that RHV1

180 drove the highest level of translation at an MFI of 9 followed by RHV2 at an MFI of 5 (Fig

181 1.C). HCV, RHV, RHv3, RHV-rn1 and RPgV generated signals only slightly above the negative

182 control. In NIH3T3 the RPgV drove the highest level of translation with an MFI of 17

183 followed by HCV with an MFI of 7, again RH1 and RHV2 were functional but at low levels and

184 RHV, RHV3 and RHV-rn1 were not functional (Fig 1.D).

185 bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

186 The viruses originate from different rodent species therefore the baby hamster kidney cell

187 line was tested to assess if the IRES’s are functional in this cell line. As with the previous cell

188 lines, the RPgV IRES drove high levels of translation, also the RHV1 and RHV2 were capable

189 of driving high levels followed by HCV, again the RHV, RHV3, RHVrn1 were not functional

190 (Fig 1.E).

191

192 RHV and RPgV IRES’s show differing function in human cell lines

193 In the human hepatocyte cell line Huh7.5 which expresses high levels of mir122, the RPgV

194 IRES drives the highest levels of translation followed by HCV and RHV2. The RHV-rn1 drove

195 low levels of translation and, as with murine cells, RHV and RHV3 did not yield any signal

196 (Fig 1.F). Another human hepatocyte cell line, HepG2 that does not express mir122, was also

197 used to test the IRES’s function: in these cells, HCV drove the highest levels followed by

198 RPGV and RHV2, while the expression from RHV, RHV1, RHV3 or RHV-rn1 were at

199 background level (Fig 1.G).

200

201 To investigate if the IRESs are functional in non-hepatocyte cells lines, we used HEK 293T,

202 and Vero cell lines. In HEK 293T the HCV IRES showed the highest level of translation

203 followed by the RPgV IRES and RHV2 while that of RHV, RHV1, RHV3 and RHV-rn1 were at

204 the lowest level (Fig 1.H). In Vero cells The RPgV produced high levels of translation, over

205 twice that of HCV; RHV2 was the only other IRES that was functional in these cells although

206 at a very low level when compared to RPGV (Fig 1.I).

207 bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

208 Deletions abrogate the function of RHV1 and RPgV IRES

209 To further asses the structural requirements of RHV1 IRES for full functionality we made

210 several different constructs. The first contained an additional 20 nucleotides of virus

211 sequences downstream of the start codon. When transfected into Hepa1-6 cells this

212 construct did not lead to a difference in the levels of translation in comparison to the

213 construct containing just the 5’ UTR. Two deletion constructs were made, in RHVΔI the 5’

214 three stem loops (Ia/b/c) were deleted, this deletion decreased the IRES function by 90%.

215 The Va and Vb stem loops were deleted from RHV1ΔII and again led to a decrease in

216 function by 90% in the murine hepatocyte cell line Hepa1-6 (Fig 2.C).

217

218 For the RPgV IRES we also added an additional 20 nucleotides of virus sequences

219 downstream of the start codon. This again had no effect on the levels of translation in

220 comparison to the construct containing just the 5’ UTR. To further asses the sequence

221 required for driving translation, three constructs were made with deletions, RPgVΔI and

222 RPgVΔII have deletions to the 5’ of the IRES with RPgVΔI having the first two stem loops

223 (Ia/b) and RPgVΔII three stem loops deleted (Ia/b and II). RPgVΔIII has two internal stem

224 loops deleted (IIId/e). The 5’ deletions to RPGV had no effect on the levels of translation,

225 however the internal deletions in RPgVΔIII led to a reduction in translation of 53% (Fig 2.D).

226

227 Expression of E1E2

228 Previous studies examining the subcellular localization of HCV E1 and E2 used cells

229 transfected with a plasmid expressing the E1 and E2 proteins(25). These studies concluded

230 that the HCV structural proteins are expressed on the cell surface, based on

231 immunofluorescence detection. In order to examine the localization of RHV structural bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

232 proteins in an expression system we cloned the structural region (capsid-E1-E2) of RHV into

233 a plasmid containing the CMV promoter, we then added the c-Myc tag to the 5’ end of the

234 capsid protein and the Flag tag to the 5’ end of the E2 protein following the E1E2 cleavage

235 sequence (Fig3.A).

236

237 HEK 293T cells were transfected with the vectors pE1E2Flag or pCMycE1E2Flag and after 48

238 hours lysed for SDS-PAGE. The E2 protein was detected in cells transfected with either

239 expression vector using an anti-Flag tag antibody (Fig3.A), and the capsid was detected in

240 cells transfected with the pCMycE1E2Flag expression vector using an anti-c-Myc antibody. The

241 proteins detected were of the predicted size, showing post-translational cleavage was

242 complete.

243

244 In order to determine if the RHV glycoproteins expressed from these vectors also exhibit an

245 intracellular colocalization, we examined transfected cells by immunofluorescence for

246 capsid and E2 expression; both were shown to colocalize (Fig3.B). We also assessed if the

247 envelope protein was expressed on the cell surface. Staining with anti-flag antibody to

248 detect E2 indeed showed punctate staining on the cell membrane and when combined with

249 wheat germ agglutinin to stain the cell membrane it showed colocalization with the flag

250 antibody (Fig3.C). This indicates that a proportion of E2 is surface-expressed.

251

252 To determine if the surface-localized E1E2 could mediate viral entry, we produced a GFP

253 encoding lentivirus vector pseudotyped with the RHV, RHV1, RHV2 and RHV3 E1E2 proteins

254 by transfecting HEK 293T cells with pE1E2 and the lentivirus backbone and packaging

255 plasmid, and 72h later harvesting and filtering the supernatant. We then tested if the E1E2- bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

256 pseudotyped lentivirus vectors were entry-competent. Supernatants from the co-

257 transfected cells were applied to Hepa1-6 cells and GFP reporter expression assayed 72 h

258 later. RHV1 E1E2-pseudotyped lentivirus vectors gave rise to a small number of GFP

259 positive cells, when compared to VSVG pseudotyped lentivirus. Lentivirus vectors

260 pseudotyped with RHV, RHV2, RHV3 or lacking envelope glycoprotein failed to give rise to

261 any GFP positive cells (Fig3.D). This indicates that only RHV1 pseudotyped lentivirus vectors

262 can mediate viral entry in Hepa1-6 cells resulting in reporter gene expression. This data also

263 indicates that surface-expressed RHV1 E1E2 heterodimers are functional.

264

265 Reverse genetics approach did not lead to establishment of an infections clone

266 An important strategy for HCV antagonizing induction of IFN-mediated innate immunity is

267 cleavage of the retinoic acid-inducible gene I pathway adaptor mitochondrial antiviral-

268 signalling protein (MAVS) by the viral NS3-4A protease(26). Here we used a human

269 hepatoma cell line (Huh-7.5) containing a MAVS cleavage reporter. Briefly, the carboxy-

270 terminal region of MAVS encoding the NS3-4A recognition site and a mitochondrial

271 targeting sequence, has been fused to red fluorescent protein (RFP-MAVS), and a nuclear

272 localization sequence (NLS) was included between the RFP and the MAVS segment. Upon

273 HCV NS3-4A cleavage of the reporter, the RFP translocates to the nucleus(27).

274

275 Upon transfection of Huh7-MAVS-RFP cells with a plasmid containing the RHV1 full-length

276 consensus clone, we observed nuclear translocation (Fig. 3E). This confirmed that RHV1

277 translation occurs in human hepatoma cells and that the RHV1 NS3-4A protease is capable

278 of cleaving human MAVS. RHV1 did not replicate in these cells, however, because the bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

279 percentage of cells with nuclear translocation decreased and no dsRNA was detected with

280 an anti-dsRNA antibody.

281

282 To further asses the ability of the RHV1 and RPGV clones to replicate, we transfected Hepa1-

283 6 and BHK-21 cells with full-length consensus clones harbouring a red fluorescent reporter

284 (mScarlet) and the antibiotic resistance gene blasticidin (BSD) inserted in-between

285 NS5A/NS5B and duplicating the cleavage sequence as previously done for HCV

286 reporters(28). After transfection with the plasmids containing the RHV1 or RPgV sequences

287 we were able to detect mScarlet at 48 hours post transfection. We also observed ds-RNA in

288 a few BHK cells transfected with RHV1 using an anti-dsRNA antibody at 72 hours post

289 transfection (Fig3.F). However, we were not able to select for resistant clones using BSD.

290

291 Discussion

292 The five rodent hepacivirus IRESs we tested showed different levels of ability to drive

293 translation using in a monocistronic vector across varying cell lines. While the use of a

294 monocistronic vector, utilizing RNA polymerase I, avoids the potential of readthrough in

295 comparison to bicistronic vectors, its drawback is that we weren’t able to directly compare

296 expression levels between cell types due to their difference in susceptibility to transfection.

297 However, the RHV, RHV3 and RHV-rn1 IRESs were either not functional or drove expression

298 at very low levels. This comes as a surprise as the RHV-rn1 virus has already been shown to

299 replicate in both mice and rats.

300

301 Both RHV1 and RHV2 drive high levels of translation in murine hepatocytes, MEFs and BHK-

302 21 cells. In human hepatocytes the RHV2 IRES out performed RHV1 which is of interest as bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

303 they are similar in sequence and therefore likely to have a similar structure. In the case of

304 RHV1, deleting the predicted initial three stem loops abrogates IRES function, suggesting

305 that the full 5’UTR sequence is required to maintain high levels of expression. Also, unlike

306 HCV, where previous studies have shown that the inclusion of 12-30nt of the core protein

307 coding sequence was essential for an efficient IRES activity(29), additional nucleotides from

308 the core protein of RHV1 did not increase transcription levels.

309

310 The RPgV 5’UTR has no significant similarity with any known pegivirus but drives high levels

311 of expression in all cell types tested. By deleting specific regions, we were able to show that

312 the initial 126nt of the 5’UTR do not contribute to IRES function and that stem loops IIId/e

313 are essential for maintaining high levels of expression. It would be of significance in the

314 future to confirm the predicted structures of RPgV and RHV1 IRES’s potentially using RNA

315 SHAPE.

316

317 RHV1 structural genes (C, E1, E2) expressed from plasmid were shown to be cleaved and

318 yielded proteins of the correct size. Moreover, E1E2 supported transduction of hepatocytes

319 when used to pseudotype lentivirus vectors. Further studies will need to be carried out to

320 find the specific entry receptors, initially blocking CD81 and HCV entry receptors and testing

321 susceptibility of transduction in alternative cell lines, but this initial experiment hints at the

322 hepatotropic potential of RHV1 in mice. This study also confirmed the previous finding that

323 the RHV1 NS3-4A protease is capable of cleaving human MAVS(30).

324

325 Our efforts to make an RHV and RPgV replication competent model in vitro have so far

326 proved unfruitful. This is of no surprise due to the difficulties in attempting to culture HCV. bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

327 Both Hepa1-6 and BHK-21 cells failed to yield a clone when selecting for replication with

328 BSD, even when expressing Sec14L2 and ApoE, both essential for high levels of HCV

329 replication(31,32) (not shown). Further cell lines could be tested along with knocking out

330 the innate immune response.

331

332 In Summary, this study shows that RHV1/2 and RPgV contain IRESs that are capable of

333 driving high levels of protein synthesis. RHV1 structural genes are cleaved by cellular

334 proteases and can be used to pseudotype lentivirus vectors that are capable of transducing

335 murine hepatocytes. bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

336 Fig1. RHV and RPGV IRES activity in different cell types.

337 Schematic of the monocistronic vectors used (A). Hepa1-6 (B), MEFs (C) NIH 3T3 (D), BHK-21

338 (E), Huh7.5 (F), HEK 293T (H) and Vero (I) cells were transfected with the indicated plasmids.

339 Cells were harvested and analysed by flow cytometry at 48 h.p.t, bar graphs show MFI for

340 mCitrine (n≥8, mean±SEM of at least three independent experiments) and representative

341 FACS plots on the right of each graph. bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

342 Fig2. Deletions to RHV1 and RPgV 5’ UTR abrogate IRES translation

343 Predicted RNA secondary structure of RHV1 (A) and RPgV (B). Red circles indicate areas

344 deleted from plasmids.

345 Hepa1-6 cells were transfected with RHV1 (C) and RPgV (D) plasmids. Cells were harvested

346 and analysed by flow cytometry at 48 h.p.t. Bar graphs show MFI for mCitrine (n≥8,

347 mean±SEM of at least three independent experiments) and representative FACS plots on

348 the right of each graph. bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

349 Fig3. Expression of RHC envelope proteins and full-length recombinant virus

350 Schematic of the RHV1 envelope expression vector and Western blot of cell lysate at 48h.p.t

351 from transfected HEK 293T (A).

352 Intracellular immunofluorescence of 293T cells at 48h.p.t with RHV1 envelope expression

353 vector, stained with flag tag in green, c-Myc tag in red and combined with DAPI stain (B).

354 Extracellular immunofluorescence for E2 by Flag tag stain (green) and combined with WGA

355 (Red) and DAPI (Blue) from 293T at 48h.p.t with RHV1 envelope expression vector, images

356 represent one slice from z-stack (C).

357 GFP-lentivirus vector pseudotyped with RHV or RHV1 envelope proteins or VSVG were

358 incubated on Hepa1-6 cells; GFP positive cells indicate transduction (D).

359 Huh7.5-MAVS-RFP cells transfected with full length RHV1 expression plasmid. Translocation

360 of RFP to the nucleus is indicated by arrows (E).

361 BHK-21 cells transfected with full length recombinant RHV1 or RPGV expression plasmid

362 containing mScarlet (Red) as in schematic, combined with dsRNA staining (Green) and DAPI

363 (Blue) (F). bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

364 Fig S1: Illustration of plasmid construction as outlined in methods. bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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A Pol I P 5' UTR mCitrine 3' UTR Pol I T

B HCV RHV1 F HCV RHV1 31±2.99 22±1.56 40 12 7.16±0.52 2.39±0.13

30 9

20 RHV2 RPgV 6 RHV2 RPgV 14±0.9 30±2.59 4.47±0.43 11±0.48

10 3

mCitrine

Huh-7.5 MFI mCitrine 0

Hepa 1-6 mCitrine MFI mCitrine 0 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV IRES IRES HCV RHV1 G HCV RHV1 C 11.00 2.24±0.13 9.26±0.9 22 20±0.99 2.52±0.28

8.25 16.5

5.50 RHV2 RPgV 11 RHV2 RPgV 5.49±0.67 2.37±0.34 10±1.24 14±0.58

2.75 5.5

mCitrine

MEFs

MFI mCitrine

mCitrine

HepG2 0.00 MFI mCitrine 0 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV IRES HCV RHV1 IRES HCV RHV1 7.68±0.21 3.98±0.23 18±0.92 3.7±0.92 D 20 H 20

15 15

10 RHV2 RPgV 10 RHV2 RPgV 5.10±0.41 17±1.73 7.7±0.44 11±0.49

5 5

mCitrine

NIH 3T3 mCitrine

MFI mCitrine

HEK 293T 0 MFI mCitrine 0 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV IRES HCV RHV1 IRES HCV RHV1 E 80 23±1.63 53±2.92 I 80 33±0.92 2.19±0.03

60 60

40 RHV2 RPgV 40 RHV2 RPgV 49.28±3.03 76±2.89 7.94±0.22 77±2.17

20 20

mCitrine

Vero

MFI mCitrine

BHK-21 mCitrine MFI mCitrine 0 0 HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV HCV RHV RHV1 RHV2 RHV3 RHV-rn1 RPgV IRES IRES bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B 200 C C A U IIIb C C A G G A 370 A G C G 150 U U A G A C C C U C G C G 160 A U C U G G U 140 G 180 C C G C G G IVa A G C U G G G U C G U U C 210 A A U U G C G A A A C C U G A G G G U G A U C A A C C G U U G U A G 190 A U C 200 C G G C G C G A U A G II G C U G U C C U C G A 190 C C 130 C G 170 G G C G G G C C U G U G A C A 380 RPgV IRES G C G C 360 U C G G 210 A U C C G C C G U 220 U G G C G C G U C A G RHV1 IRES G A A U G G C 120 C C G IIIc C G A G 180 230 G C G ∆III U A C IIIa A C G U C G G U U G U G A C U A G C C C C G C U A 170 C G G U G 220 350 U G A C 390 C C G A G U G C A U G A G A U A U G C IIIa IIIb A 160 U A C 260 C G G G U G G U G 240 G G U G A G C C G C U U C A 290 U C G U G G U C G G A U IIId G U G C A C G G 110 C G G U U U G A C G 250 G IVb C G G C G ∆II A A U G C G C 400 U 300 A 340 G U C 280 C G G U U C C U 230 G U U C A A U C 310 U C G U G G C C G G G G G U C G A C G G C U U G G U A G C C C C G G A A 270 A C C C A G G C U 150 U G G G A G A A U A 270 C C A 410 C U U G C A U G C C G II G C A G G G G A U U C 420 A G G C C A U 90 C C A C 240 A A A G U A G U C U G G A 320 430 C A C G G A G U G U C C C U U G A C 250 U 330 C G C U C U C U U G 290 U IIIe U A C C G G A 100 U A C G 260 C U G C G A G U 90 U A A G G U A G 100 U U G U C G C 470 A C G A U G C C U C G G G A G C A G A C 440 G C C U U U C G A G C C U A 280 G G C G C U C U G G U G A 140 A C G G G C A A U U C U 460 C A G G 120 G C G G C 80 G A A G A 130 G A G G A C U A Va C C U A C C C C G 480 C A G C G G U 80 A G C A G U G G U 110 G U G G A U A A G C U G U C G A C U 300 U G G C C U A A G G G G C G G G C A C U C G A U C U 450 C G A U G A G G A G 70 U 60 G C C G C U ∆I Ic G A C G 510 ∆II G A C C G U G U G C A G G C G G C G G 60 C G 70 C 490 C G A C C G A A A C A A C G U G A C A A U U U A 50 U U G ∆I G U C C Ib G 520 G U U C C G A G G C U A 50 G G C G C 310 G A U U U Ib U G U G G G G G G G C C A C 40 C U A A G C C A U C Vb A C G G C C C U 500 40 G C 30 C A C G C G C A C C U G IVa U G A C C C G G U G 340 G A C G C G G C G G 30 U C U G C C C 320 C G A C C U U G C A G U G C U U U G A C G A C G U A U C 20 U A A U C 370 U A G C C 20 10 C Ia U G 330 C U G U U G A U U C G G C G A G C A U G U C 530 Ia C U A C U C A U C C A A G G C C G 10 U 360 A C U U A G C A C G C C A U U A U 540 350 U A G A A G U G C C A G C C A G A A U G U G C A G G G A U C C C 570 C U C 560 A C U 550 A A C

C RHV1 RHV1 C D RPgV RPgV ∆I 22±1.56 22±1.68 30±2.59 30±2.19 30 40

23 30

15 RHV1 ∆I RHV1 ∆II 20 RPgV ∆II RPgV ∆III 2.37±0.14 2.24±0.07 33±0.66 14±0.73

8 10

Hepa 1-6 MFI mCitrine 0 Hepa 1-6 MFI mCitrine 0

mCitrine RHV1 RHV1 C RHV1 ∆I RHV1 ∆II mCitrine RPgV RPgV C RPgV ∆I RPgV ∆II RPgV ∆III IRES IRES bioRxiv preprint doi: https://doi.org/10.1101/761379; this version posted September 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B Myc C E1 Flag E2

Flag

Flag E1E2 c-Myc - pE1E2 pC

E2 30kDa (Flag Tag) HEK 293T Intracellular Actin c-Myc Tag-Capsid c-Myc Tag -Capsid Capsid (c-Myc Tag) 18kDa

Actin 40kDa C D Surface HEK 293T Hepa1-6 RHV RHV1 VSVG

WGA - Plasma membrane E F 5'UTR C-NS5A mScarlet-BSD NS5B 3'UTR BHK-21 Huh 7.5-MAVS-RFP Negative control RHV1 RHV1 RHV1 RPgV