Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3’ end- to 5’ end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3’ untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3’ UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3’ UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3′ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3’ UTR acts as a cis-acting element for encapsidation.
References
[1]
Khromykh AA, Varnavski AN, Sedlak PL, Westaway EG (2001) Coupling between replication and packaging of flavivirus RNA: Evidence derived from the use of DNA-based full-length cDNA clones of kunjin virus. J Virol 75: 4633–4640. pmid:11312333 doi: 10.1128/jvi.75.10.4633-4640.2001
[2]
Annamalai P, Rao ALN (2006) Packaging of brome mosaic virus subgenomic RNA is functionally coupled to replication-dependent transcription and translation of coat protein. J Virol 80: 10096–10108. pmid:17005687 doi: 10.1128/jvi.01186-06
[3]
Nugent CI, Johnson KL, Sarnow P, Kirkegaard K (1999) Functional coupling between replication and packaging of poliovirus replicon RNA. J Virol 73: 427–435. pmid:9847348
[4]
Moradpour D, Penin F, Rice CM (2007) Replication of hepatitis C virus. Nat Rev Microbiol 5: 453–463. pmid:17487147 doi: 10.1038/nrmicro1645
[5]
Bai Y, Zhou KH, Doudna JA (2013) Hepatitis C virus 3 ' UTR regulates viral translation through direct interactions with the host translation machinery. Nucleic Acids Res 41: 7861–7874. doi: 10.1093/nar/gkt543. pmid:23783572
[6]
Yi M, Lemon SM (2003) 3' nontranslated RNA signals required for replication of hepatitis C virus RNA. J Virol 77: 3557–3568. pmid:12610131 doi: 10.1128/jvi.77.6.3557-3568.2003
[7]
Friebe P, Bartenschlager R (2002) Genetic analysis of sequences in the 3 ' nontranslated region of hepatitis C virus that are important for RNA replication. J Virol 76: 5326–5338. pmid:11991961 doi: 10.1128/jvi.76.11.5326-5338.2002
[8]
You S, Rice CM (2008) 3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U). J Virol 82: 184–195. pmid:17942554 doi: 10.1128/jvi.01796-07
[9]
Shimizu YK, Hijikata M, Oshima M, Shimizu K, Yoshikura H (2006) Detection of a 5' end subgenome of hepatitis C virus terminating at nucleotide 384 in patients' plasma and liver tissues. J Viral Hepat 13: 746–755. pmid:17052274 doi: 10.1111/j.1365-2893.2006.00745.x
[10]
Quadri R, Negro F (2001) Are there any subgenomic forms of hepatitis C virus RNA in the liver? Digestive and Liver Disease 33: 480–486. pmid:11572575 doi: 10.1016/s1590-8658(01)80026-8
[11]
Herod MR, Schregel V, Hinds C, Liu M, McLauchlan J, et al. (2014) Genetic complementation of hepatitis C virus nonstructural protein functions associated with replication exhibits requirements that differ from those for virion assembly. J Virol 88: 2748–2762. doi: 10.1128/JVI.03588-13. pmid:24352463
[12]
Pietschmann T, Zayas M, Meuleman P, Long G, Appel N, et al. (2009) Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations. PLoS Pathog 5: e1000475 doi: 10.1371/journal.ppat.1000475. pmid:19521536
[13]
Masaki T, Suzuki R, Saeed M, Mori K, Matsuda M, et al. (2010) Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription. J Virol 84: 5824–5835. doi: 10.1128/JVI.02397-09. pmid:20237083
[14]
Akazawa D, Date T, Morikawa K, Murayama A, Miyamoto M, et al. (2007) CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection. J Virol 81: 5036–5045. pmid:17329343 doi: 10.1128/jvi.01573-06
[15]
Fatemi RP, Salah-Uddin S, Modarresi F, Khoury N, Wahlestedt C, et al. (2015) Screening for Small-Molecule Modulators of Long Noncoding RNA-Protein Interactions Using AlphaScreen. J Biomol Screen 20: 1132–1141. doi: 10.1177/1087057115594187. pmid:26173710
[16]
Manders EM, Stap J, Brakenhoff GJ, van Driel R, Aten JA (1992) Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci 103 (Pt 3): 857–862. pmid:1478975
[17]
Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, et al. (2004) A syntaxin 1, G alpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: Analysis by quantitative immunocolocalization. J Neurosci 24: 4070–4081. pmid:15102922 doi: 10.1523/jneurosci.0346-04.2004
[18]
Friebe P, Boudet J, Simorre JP, Bartenschlager R (2005) Kissing-loop interaction in the 3' end of the hepatitis C virus genome essential for RNA replication. J Virol 79: 380–392. pmid:15596831 doi: 10.1128/jvi.79.1.380-392.2005
[19]
Romero-López C, Barroso-Deljesus A, García-Sacristán A, Briones C, Berzal-Herranz A (2014) End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 3'X-tail region. Nucleic Acids Res 42: 567–582. doi: 10.1093/nar/gkt841. pmid:24049069
[20]
Shetty S, Stefanovic S, Mihailescu MR (2013) Hepatitis C virus RNA: molecular switches mediated by long-range RNA-RNA interactions? Nucleic Acids Res 41: 2526–2540. doi: 10.1093/nar/gks1318. pmid:23275555
[21]
Tuplin A, Struthers M, Simmonds P, Evans DJ (2012) A twist in the tail: SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication. Nucleic Acids Res 40: 6908–6921. doi: 10.1093/nar/gks370. pmid:22561372
[22]
Friebe P, Bartenschlager R (2009) Role of RNA structures in genome terminal sequences of the hepatitis C virus for replication and assembly. J Virol 83: 11989–11995. doi: 10.1128/JVI.01508-09. pmid:19740989
[23]
Kim DY, Firth AE, Atasheva S, Frolova EI, Frolov I (2011) Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution. J Virol 85: 8022–8036. doi: 10.1128/JVI.00644-11. pmid:21680508
[24]
Junker-Niepmann M, Bartenschlager R, Schaller H (1990) A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. EMBO J 9: 3389–3396. pmid:2209549
[25]
Bartenschlager R, Penin F, Lohmann V, Andre P (2011) Assembly of infectious hepatitis C virus particles. Trends Microbiol 19: 95–103. doi: 10.1016/j.tim.2010.11.005. pmid:21146993
[26]
Jones CT, Murray CL, Eastman DK, Tassello J, Rice CM (2007) Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 81: 8374–8383. pmid:17537845 doi: 10.1128/jvi.00690-07
[27]
Steinmann E, Penin F, Kallis S, Patel AH, Bartenschlager R, et al. (2007) Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions. PLoS Pathog 3: 962–971. doi: 10.1371/journal.ppat.0030103
[28]
Ma Y, Anantpadma M, Timpe J, Shanmugam S, Singh S, et al. (2011) Hepatitis C virus NS2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins. J Virol 85: 86–97. doi: 10.1128/JVI.01070-10. pmid:20962101
[29]
Popescu C-I, Callens N, Trinel D, Roingeard P, Moradpour D, et al. (2011) NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly. PLoS Pathog 7: e1001278. doi: 10.1371/journal.ppat.1001278. pmid:21347350
[30]
Suzuki R, Matsuda M, Watashi K, Aizaki H, Matsuura Y, et al. (2013) Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2. PLoS Pathog 9: e1003589. doi: 10.1371/journal.ppat.1003589. pmid:24009510
[31]
Jones DM, Patel AH, Targett-Adams P, McLauchlan J (2009) The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus. J Virol 83: 2163–2177. doi: 10.1128/JVI.01885-08. pmid:19073716
[32]
Steinmann E, Brohm C, Kallis S, Bartenschlager R, Pietschmann T (2008) Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles. J Virol 82: 7034–7046. doi: 10.1128/JVI.00118-08. pmid:18480457
[33]
Appel N, Zayas M, Miller S, Krijnse-Locker J, Schaller T, et al. (2008) Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 4: e1000035. doi: 10.1371/journal.ppat.1000035. pmid:18369481
[34]
Ma YH, Yates J, Liang YQ, Lemon SM, Yi MK (2008) NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly. J Virol 82: 7624–7639. doi: 10.1128/JVI.00724-08. pmid:18508894
[35]
Masaki T, Suzuki R, Murakami K, Aizaki H, Ishii K, et al. (2008) Interaction of hepatitis c virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles. J Virol 82: 7964–7976. doi: 10.1128/JVI.00826-08. pmid:18524832
[36]
Ross-Thriepland D, Amako Y, Harris M (2013) The C terminus of NS5A domain II is a key determinant of hepatitis C virus genome replication, but is not required for virion assembly and release. J Gen Virol 94: 1009–1018. doi: 10.1099/vir.0.050633-0. pmid:23324467
[37]
Appel N, Herian U, Bartenschlager R (2005) Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A. J Virol 79: 896–909. pmid:15613318 doi: 10.1128/jvi.79.2.896-909.2005
[38]
Li Q, Pène V, Krishnamurthy S, Cha H, Liang TJ (2013) Hepatitis C virus infection activates an innate pathway involving IKK-α in lipogenesis and viral assembly. Nat Med 19: 722–729. doi: 10.1038/nm.3190. pmid:23708292
[39]
Paul D, Madan V, Bartenschlager R (2014) Hepatitis C virus RNA replication and assembly: living on the fat of the land. Cell Host & Microbe 16: 569–579. doi: 10.1016/j.chom.2014.10.008
[40]
Salloum S, Wang H, Ferguson C, Parton RG, Tai AW (2013) Rab18 binds to hepatitis C virus NS5A and promotes interaction between sites of viral replication and lipid droplets. PLoS Pathog 9: e1003513. doi: 10.1371/journal.ppat.1003513. pmid:23935497
[41]
Masaki T, Matsunaga S, Takahashi H, Nakashima K, Kimura Y, et al. (2014) Involvement of Hepatitis C Virus NS5A Hyperphosphorylation Mediated by Casein Kinase I-α in Infectious Virus Production. J Virol 88: 7541–7555. doi: 10.1128/JVI.03170-13. pmid:24760886
[42]
Takeuchi T, Katsume A, Tanaka T, Abe A, Inoue K, et al. (1999) Real-time detection system for quantification of hepatitis C virus genome. Gastroenterology 116: 636–642. pmid:10029622 doi: 10.1016/s0016-5085(99)70185-x
