1887

Abstract

The hepatitis C virus genotype 2a isolate, JFH-1, exhibits much more efficient genome replication than other isolates. Although basic replication mechanisms must be conserved, this raises the question of whether the regulation of replication might exhibit isolate- and/or genotype-specific characteristics. Exemplifying this, the phenotype of NS5A hyperphosphorylation is genotype-dependent; in genotype 1b a loss of hyperphosphorylation correlates with an enhancement of replication. In contrast, the replication of JFH-1 is not regulated by hyperphosphorylation. We previously identified a novel phosphorylation site in JFH-1 NS5A: S146. A phosphomimetic substitution (S146D) had no effect on replication but correlated with a loss of hyperphosphorylation. In genotype 1b, residue 146 is alanine and we therefore investigated whether the substitution of A146 with a phosphorylatable (S), or phosphomimetic, residue would recapitulate the JFH-1 phenotype, decoupling hyperphosphorylation from replication. This was not the case, as A146D exhibited both a loss of hyperphosphorylation and a reduction in replication, accompanied by a perinuclear restriction of replication complexes, reductions in lipid droplet and PI4P lipid accumulation, and a disruption of NS5A dimerization. In contrast, the S232I culture-adaptive mutation in the low-complexity sequence I (LCSI) also exhibited a loss of hyperphosphorylation, but was associated with an increase in replication. Taken together, these data imply that hyperphosphorylation does not directly regulate replication. In contrast, the loss of hyperphosphorylation is a consequence of perturbing genome replication and NS5A function. Furthermore, we show that mutations in either domain I or LCSI of NS5A can disrupt hyperphosphorylation, demonstrating that multiple parameters influence the phosphorylation status of NS5A.

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2019-12-10
2020-01-27
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References

  1. Webster DP, Klenerman P, Dusheiko GM. Hepatitis C. Lancet 2015;385:1124–1135 [CrossRef]
    [Google Scholar]
  2. Simmonds P, Becher P, Bukh J, Gould EA, Meyers G et al. ICTV virus taxonomy profile: Flaviviridae. J Gen Virol 2017;98:2–3 [CrossRef]
    [Google Scholar]
  3. Ross-Thriepland D, Harris M. Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!. J Gen Virol 2015;96:727–738 [CrossRef]
    [Google Scholar]
  4. Tellinghuisen TL, Marcotrigiano J, Gorbalenya AE, Rice CM. The NS5A protein of hepatitis C virus is a zinc metalloprotein. J Biol Chem 2004;279:48576–48587 [CrossRef]
    [Google Scholar]
  5. Tellinghuisen TL, Marcotrigiano J, Rice CM. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 2005;435:374–379 [CrossRef]
    [Google Scholar]
  6. Love RA, Brodsky O, Hickey MJ, Wells PA, Cronin CN. Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus. J Virol 2009;83:4395–4403 [CrossRef]
    [Google Scholar]
  7. Lambert SM, Langley DR, Garnett JA, Angell R, Hedgethorne K et al. The crystal structure of NS5A domain 1 from genotype 1A reveals new clues to the mechanism of action for dimeric HCV inhibitors. Protein Sci 2014;23:723–734 [CrossRef]
    [Google Scholar]
  8. Ross-Thriepland D, Harris M. Insights into the complexity and functionality of hepatitis C virus NS5A phosphorylation. J Virol 2014;88:1421–1432 [CrossRef]
    [Google Scholar]
  9. Chen YC, Su WC, Huang JY, Chao TC, Jeng KS et al. Polo-Like kinase 1 is involved in hepatitis C virus replication by hyperphosphorylating NS5A. J Virol 2010;84:7983–7993 [CrossRef]
    [Google Scholar]
  10. Masaki T, Matsunaga S, Takahashi H, Nakashima K, Kimura Y et al. Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production. J Virol 2014;88:7541–7555 [CrossRef]
    [Google Scholar]
  11. Chong WM, Hsu SC, Kao WT, Lo CW, Lee KY et al. Phosphoproteomics identified an NS5A phosphorylation site involved in hepatitis C virus replication. J Biol Chem 2016;291:3918–3931 [CrossRef]
    [Google Scholar]
  12. Reiss S, Harak C, Romero-Brey I, Radujkovic D, Klein R et al. The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A. PLoS Pathog 2013;9:e1003359 [CrossRef]
    [Google Scholar]
  13. Quintavalle M, Sambucini S, Summa V, Orsatti L, Talamo F et al. Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors. J Biol Chem 2007;282:5536–5544 [CrossRef]
    [Google Scholar]
  14. Reed KE, Xu J, Rice CM. Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase. J Virol 1997;71:7187–7197
    [Google Scholar]
  15. Tanji Y, Kaneko T, Satoh S, Shimotohno K. Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J Virol 1995;69:3980–3986
    [Google Scholar]
  16. Lemay KL, Treadaway J, Angulo I, Tellinghuisen TL. A hepatitis C virus NS5A phosphorylation site that regulates RNA replication. J Virol 2013;87:1255–1260 [CrossRef]
    [Google Scholar]
  17. Eyre NS, Hampton-Smith RJ, Aloia AL, Eddes JS, Simpson KJ et al. Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation. Virology 2016;491:27–44 [CrossRef]
    [Google Scholar]
  18. Schenk C, Meyrath M, Warnken U, Schnölzer M, Mier W et al. Characterization of a threonine-rich cluster in hepatitis C virus nonstructural protein 5A and its contribution to hyperphosphorylation. J Virol 2018;92:e00737–00718 [CrossRef]
    [Google Scholar]
  19. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture. Science 2000;290:1972–1974 [CrossRef]
    [Google Scholar]
  20. Evans MJ, Rice CM, Goff SP. Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc Natl Acad Sci USA 2004;101:13038–13043 [CrossRef]
    [Google Scholar]
  21. Appel N, Pietschmann T, Bartenschlager R. Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J Virol 2005;79:3187–3194 [CrossRef]
    [Google Scholar]
  22. Lohmann V, Körner F, Koch J, Herian U, Theilmann L et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999;285:110–113 [CrossRef]
    [Google Scholar]
  23. 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]
  24. Atkinson NJ, Witteveldt J, Evans DJ, Simmonds P. The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication. Nucleic Acids Res 2014;42:4527–4545 [CrossRef]
    [Google Scholar]
  25. Ross-Thriepland D, Mankouri J, Harris M. Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes. J Virol 2015;89:3123–3135 [CrossRef]
    [Google Scholar]
  26. Harak C, Meyrath M, Romero-Brey I, Schenk C, Gondeau C et al. Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture. Nat Microbiol 2016;2:16247 [CrossRef]
    [Google Scholar]
  27. Yin C, Goonawardane N, Stewart H, Harris M. A role for domain I of the hepatitis C virus NS5A protein in virus assembly. PLoS Pathog 2018;14:e1006834 [CrossRef]
    [Google Scholar]
  28. Lim PJ, Chatterji U, Cordek D, Sharma SD, Garcia-Rivera JA et al. Correlation between NS5A dimerization and hepatitis C virus replication. J Biol Chem 2012;287:30861–30873 [CrossRef]
    [Google Scholar]
  29. Hsu SC, Lo CW, Pan TC, Lee KY, Yu MJ. Serine 235 is the primary NS5A hyperphosphorylation site responsible for hepatitis C virus replication. J Virol 2017;91:e00194–00117 [CrossRef]
    [Google Scholar]
  30. Hsu SC, Tsai CN, Lee KY, Pan TC, Lo CW et al. Sequential S232/S235/S238 phosphorylation of the hepatitis C virus nonstructural protein 5A. J Virol 2018;92:e01295–01218 [CrossRef]
    [Google Scholar]
  31. Shanmugam S, Nichols AK, Saravanabalaji D, Welsch C, Yi M. HCV NS5A dimer interface residues regulate HCV replication by controlling its self-interaction, hyperphosphorylation, subcellular localization and interaction with cyclophilin A. PLoS Pathog 2018;14:e1007177 [CrossRef]
    [Google Scholar]
  32. Li YP, Ramirez S, Humes D, Jensen SB, Gottwein JM et al. Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors. Gastroenterology 2014;146:e814812–821 [CrossRef]
    [Google Scholar]
  33. Ramirez S, Mikkelsen LS, Gottwein JM, Bukh J. Robust HCV genotype 3A infectious cell culture system permits identification of escape variants with resistance to sofosbuvir. Gastroenterology 2016;151:e972973–985 [CrossRef]
    [Google Scholar]
  34. Macdonald A, Crowder K, Street A, McCormick C, Saksela K et al. The hepatitis C virus NS5A protein inhibits activating protein-1 (AP1) function by perturbing Ras-ERK pathway signaling. J Biol Chem 2003;278:17775–17784 [CrossRef]
    [Google Scholar]
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