1887

Abstract

Equine hepacivirus (EHcV) (now also classified as hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the . Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001316
2019-09-06
2019-09-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/10.1099/jgv.0.001316/jgv001316.html?itemId=/content/journal/jgv/10.1099/jgv.0.001316&mimeType=html&fmt=ahah

References

  1. Jang SK, Kräusslich HG, Nicklin MJ, Duke GM, Palmenberg AC et al. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 1988;62:2636–2643
    [Google Scholar]
  2. Pelletier J, Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 1988;334:320–325 [CrossRef]
    [Google Scholar]
  3. Lozano G, Martínez-Salas E. Structural insights into viral IRES-dependent translation mechanisms. Curr Opin Virol 2015;12:113–120 [CrossRef]
    [Google Scholar]
  4. Borman AM, Kean KM. Intact eukaryotic initiation factor 4G is required for hepatitis A virus internal initiation of translation. Virology 1997;237:129–136 [CrossRef]
    [Google Scholar]
  5. Asnani M, Kumar P, Hellen CUT. Widespread distribution and structural diversity of type IV IRESs in members of Picornaviridae. Virology 2015;478:61–74 [CrossRef]
    [Google Scholar]
  6. Sweeney TR, Dhote V, Yu Y, Hellen CUT. A distinct class of internal ribosomal entry site in members of the Kobuvirus and proposed Salivirus and Paraturdivirus genera of the Picornaviridae. J Virol 2012;86:1468–1486 [CrossRef]
    [Google Scholar]
  7. Sasaki J, Nakashima N. Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro. J Virol 1999;73:1219–1226
    [Google Scholar]
  8. Stewart H, Walter C, Jones D, Lyons S, Simmonds P et al. The non-primate hepacivirus 5' untranslated region possesses internal ribosomal entry site activity. J Gen Virol 2013;94:2657–2663 [CrossRef]
    [Google Scholar]
  9. Ramsay JD, Evanoff R, Wilkinson TE, Divers TJ, Knowles DP et al. Experimental transmission of equine hepacivirus in horses as a model for hepatitis C virus. Hepatology 2015;61:1533–1546 [CrossRef]
    [Google Scholar]
  10. Pfaender S, Walter S, Grabski E, Todt D, Bruening J et al. Immune protection against reinfection with nonprimate hepacivirus. Proc Natl Acad Sci U S A 2017;114:E2430–E2439 [CrossRef]
    [Google Scholar]
  11. Pfaender S, Walter S, Todt D, Behrendt P, Doerrbecker J et al. Assessment of cross-species transmission of hepatitis C virus-related non-primate hepacivirus in a population of humans at high risk of exposure. J Gen Virol 2015;96:2636–2642 [CrossRef]
    [Google Scholar]
  12. Gather T, Walter S, Todt D, Pfaender S, Brown RJP et al. Vertical transmission of hepatitis C virus-like non-primate hepacivirus in horses. J Gen Virol 2016;97:2540–2551 [CrossRef]
    [Google Scholar]
  13. Ray PS, Das S. Inhibition of hepatitis C virus IRES-mediated translation by small RNAs analogous to stem-loop structures of the 5'-untranslated region. Nucleic Acids Res 2004;32:1678–1687 [CrossRef]
    [Google Scholar]
  14. Kalliampakou KI, Psaridi-Linardaki L, Mavromara P. Mutational analysis of the apical region of domain II of the HCV IRES. FEBS Lett 2002;511:79–84 [CrossRef]
    [Google Scholar]
  15. Lukavsky PJ. Structure and function of HCV IRES domains. Virus Res 2009;139:166–171 [CrossRef]
    [Google Scholar]
  16. Berry KE, Waghray S, Doudna JA. The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit. RNA 2010;16:1559–1569 [CrossRef]
    [Google Scholar]
  17. Honda M, Ping LH, Rijnbrand RCA, Amphlett E, Clarke B et al. Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA. Virology 1996;222:31–42 [CrossRef]
    [Google Scholar]
  18. Pestova TV, Shatsky IN, Fletcher SP, Jackson RJ, Hellen CU. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev 1998;12:67–83 [CrossRef]
    [Google Scholar]
  19. Matsuda D, Mauro VP. Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo. Proc Natl Acad Sci USA 2014;111:15385–15389 [CrossRef]
    [Google Scholar]
  20. Malygin AA, Kossinova OA, Shatsky IN, Karpova GG. Hcv IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation. Nucleic Acids Res 2013;41:8706–8714 [CrossRef]
    [Google Scholar]
  21. Kieft JS, Zhou K, Jubin R, Doudna JA. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 2001;7:194–206 [CrossRef]
    [Google Scholar]
  22. Jubin R, Vantuno NE, Kieft JS, Murray MG, Doudna JA et al. Hepatitis C virus internal ribosome entry site (IRES) stem loop IIID contains a phylogenetically conserved GGG triplet essential for translation and IRES folding. J Virol 2000;74:10430–10437 [CrossRef]
    [Google Scholar]
  23. Sun C, Querol-Audí J, Mortimer SA, Arias-Palomo E, Doudna JA et al. Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation. Nucleic Acids Res 2013;41:7512–7521 [CrossRef]
    [Google Scholar]
  24. Sizova DV, Kolupaeva VG, Pestova TV, Shatsky IN, Hellen CU. Specific interaction of eukaryotic translation initiation factor 3 with the 5' nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J Virol 1998;72:4775–4782
    [Google Scholar]
  25. Kapoor A, Simmonds P, Gerold G, Qaisar N, Jain K et al. Characterization of a canine homolog of hepatitis C virus. Proc Natl Acad Sci U S A 2011;108:11608–11613 [CrossRef]
    [Google Scholar]
  26. Burbelo PD, Dubovi EJ, Simmonds P, Medina JL, Henriquez JA et al. Serology-enabled discovery of genetically diverse hepaciviruses in a new host. J Virol 2012;86:6171–6178 [CrossRef]
    [Google Scholar]
  27. Scheel TKH, Kapoor A, Nishiuchi E, Brock KV, Yu Y et al. Characterization of nonprimate hepacivirus and construction of a functional molecular clone. Proc Natl Acad Sci U S A 2015;112:2192–2197 [CrossRef]
    [Google Scholar]
  28. Bradrick SS, Walters RW, Gromeier M. The hepatitis C virus 3'-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase. Nucleic Acids Res 2006;34:1293–1303 [CrossRef]
    [Google Scholar]
  29. Song Y, Friebe P, Tzima E, Junemann C, Bartenschlager R et al. The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site. J Virol 2006;80:11579–11588 [CrossRef]
    [Google Scholar]
  30. Jangra RK, Yi M, Lemon SM. Regulation of hepatitis C virus translation and infectious virus production by the microRNA miR-122. J Virol 2010;84:6615–6625 [CrossRef]
    [Google Scholar]
  31. Yu Y, Scheel TKH, Luna JM, Chung H, Nishiuchi E et al. miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence. PLoS Pathog 2017;13:e1006694 [CrossRef]
    [Google Scholar]
  32. Fletcher SP, Ali IK, Kaminski A, Digard P, Jackson RJ. The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes. RNA 2002;8:1558–1571
    [Google Scholar]
  33. Chard LS, Kaku Y, Jones B, Nayak A, Belsham GJ. Functional analyses of RNA structures shared between the internal ribosome entry sites of hepatitis C virus and the picornavirus porcine teschovirus 1 Talfan. J Virol 2006;80:1271–1279 [CrossRef]
    [Google Scholar]
  34. Chard LS, Bordeleau M-E, Pelletier J, Tanaka J, Belsham GJ. Hepatitis C virus-related internal ribosome entry sites are found in multiple genera of the family Picornaviridae. J Gen Virol 2006;87:927–936 [CrossRef]
    [Google Scholar]
  35. Licursi M, Komatsu Y, Pongnopparat T, Hirasawa K. Promotion of viral internal ribosomal entry site-mediated translation under amino acid starvation. J Gen Virol 2012;93:951–962 [CrossRef]
    [Google Scholar]
  36. Witteveldt J, Martin-Gans M, Simmonds P. Enhancement of the replication of hepatitis C virus replicons of genotypes 1 to 4 by manipulation of CpG and uPA dinucleotide frequencies and use of cell lines expressing SECL14L2 for antiviral resistance testing. Antimicrob Agents Chemother 2016;60:2981–2992 [CrossRef]
    [Google Scholar]
  37. Angulo J, Ulryck N, Deforges J, Chamond N, Lopez-Lastra M et al. Loop IIID of the HCV IRES is essential for the structural rearrangement of the 40S-HCV IRES complex. Nucleic Acids Res 2016;44:1309–1325 [CrossRef]
    [Google Scholar]
  38. Deigan KE, Li TW, Mathews DH, Weeks KM. Accurate SHAPE-directed RNA structure determination. Proc Natl Acad Sci U S A 2009;106:97–102 [CrossRef]
    [Google Scholar]
  39. Willcocks MM, Zaini S, Chamond N, Ulryck N, Allouche D et al. Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites. Nucleic Acids Res 2017;45:13016–13028 [CrossRef]
    [Google Scholar]
  40. Low JT, Weeks KM. Shape-Directed RNA secondary structure prediction. Methods 2010;52:150–158 [CrossRef]
    [Google Scholar]
  41. Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science 2005;309:1577–1581 [CrossRef]
    [Google Scholar]
  42. Otto GA, Puglisi JD. The pathway of HCV IRES-mediated translation initiation. Cell 2004;119:369–380 [CrossRef]
    [Google Scholar]
  43. Locker N, Easton LE, Lukavsky PJ. Hcv and CSFV IRES domain II mediate eIF2 release during 80S ribosome assembly. Embo J 2007;26:795–805 [CrossRef]
    [Google Scholar]
  44. Wang C, Sarnow P, Siddiqui A. A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA. J Virol 1994;68:7301–7307
    [Google Scholar]
  45. Reynolds JE, Kaminski A, Carroll AR, Clarke BE, Rowlands DJ et al. Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon. RNA 1996;2:867–878
    [Google Scholar]
  46. Kamoshita N, Tsukiyama-Kohara K, Kohara M, Nomoto A. Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA: implication for involvement of the highly ordered structure and cell type-specific transacting factors. Virology 1997;233:9–18 [CrossRef]
    [Google Scholar]
  47. Kolupaeva VG, Pestova TV, Hellen CU. An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J Virol 2000;74:6242–6250 [CrossRef]
    [Google Scholar]
  48. Tanaka T, Otoguro T, Yamashita A, Kasai H, Fukuhara T et al. Roles of the 5′ untranslated region of nonprimate Hepacivirus in translation initiation and viral replication. J Virol 2018;92: [CrossRef]
    [Google Scholar]
  49. Friebe P, Lohmann V, Krieger N, Bartenschlager R. Sequences in the 5' nontranslated region of hepatitis C virus required for RNA replication. J Virol 2001;75:12047–12057 [CrossRef]
    [Google Scholar]
  50. Merino EJ, Wilkinson KA, Coughlan JL, Weeks KM. RNA structure analysis at single nucleotide resolution by selective 2'-hydroxyl acylation and primer extension (shape). J Am Chem Soc 2005;127:4223–4231 [CrossRef]
    [Google Scholar]
  51. Mortimer SA, Weeks KM. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by shape chemistry. J Am Chem Soc 2007;129:4144–4145 [CrossRef]
    [Google Scholar]
  52. Odreman-Macchioli FE, Tisminetzky SG, Zotti M, Baralle FE, Buratti E. Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES. Nucleic Acids Res 2000;28:875–885 [CrossRef]
    [Google Scholar]
  53. Hashem Y, des Georges A, Dhote V, Langlois R, Liao HY et al. Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature 2013;503:539–543 [CrossRef]
    [Google Scholar]
  54. Thibault PA, Huys A, Amador-Cañizares Y, Gailius JE, Pinel DE et al. Regulation of hepatitis C virus genome replication by XRN1 and microRNA-122 binding to individual sites in the 5' untranslated region. J Virol 2015;89:6294–6311 [CrossRef]
    [Google Scholar]
  55. Baron AL, Schoeniger A, Becher P, Baechlein C. Mutational analysis of the bovine Hepacivirus internal ribosome entry site. J Virol 2018;92: [CrossRef]
    [Google Scholar]
  56. Schult P, Roth H, Adams RL, Mas C, Imbert L et al. microRNA-122 amplifies hepatitis C virus translation by shaping the structure of the internal ribosomal entry site. Nat Commun 2018;9:2613 [CrossRef]
    [Google Scholar]
  57. Amador-Cañizares Y, Panigrahi M, Huys A, Kunden RD, Adams HM et al. miR-122, small RNA annealing and sequence mutations alter the predicted structure of the hepatitis C virus 5' UTR RNA to stabilize and promote viral RNA accumulation. Nucleic Acids Res 2018;46:9776–9792 [CrossRef]
    [Google Scholar]
  58. Chahal J, Gebert LFR, Gan HH, Camacho E, Gunsalus KC et al. miR-122 and ago interactions with the HCV genome alter the structure of the viral 5' terminus. Nucleic Acids Res 2019;47:5307–5324 [CrossRef]
    [Google Scholar]
  59. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 1982;42:3858–3863
    [Google Scholar]
  60. Oguma K, Ishida M, Maeda K, Sentsui H. Efficient propagation of equine viruses in a newly established equine cell line, FHK-13.1 cells. J Vet Med Sci 2013;75:1223–1225 [CrossRef]
    [Google Scholar]
  61. Qiao C, Yuan Z, Li J, He B, Zheng H et al. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver. Gene Ther 2011;18:403–410 [CrossRef]
    [Google Scholar]
  62. Pisarev AV, Unbehaun A, Hellen CUT, Pestova TV. Assembly and analysis of eukaryotic translation initiation complexes. Methods Enzymol 2007;430:147–177 [CrossRef]
    [Google Scholar]
  63. Karabiber F. A peak alignment algorithm with novel improvements in application to electropherogram analysis. J Bioinform Comput Biol 2013;11:1350011 [CrossRef]
    [Google Scholar]
  64. Reuter JS, Mathews DH. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 2010;11:129 [CrossRef]
    [Google Scholar]
  65. Darty K, Denise A, Ponty Y. Varna: interactive drawing and editing of the RNA secondary structure. Bioinformatics 2009;25:1974–1975 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001316
Loading
/content/journal/jgv/10.1099/jgv.0.001316
Loading

Data & Media loading...

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error