1887

Abstract

The hepatitis C virus non-structural 5A (NS5A) protein is highly phosphorylated and plays roles in both virus genome replication and assembly of infectious virus particles. NS5A comprises three domains separated by low complexity sequences (LCS). Mass spectrometry analysis of NS5A revealed the existence of a singly phosphorylated tryptic peptide corresponding to the end of LCS I and the beginning of domain II that contained a number of potential phosphorylatable residues (serines and threonines). Here we use a mutagenic approach to investigate the potential role of three of these threonine residues. Phosphomimetic mutations of two of these (T242E and T244E) resulted in significant reductions in virus genome replication and the production of infectious virus, suggesting that the phosphorylation of these residues negatively regulated virus RNA synthesis. Mutation of T245 had no effect, however when T245E was combined with the other two phosphomimetic mutations (TripleE) the inhibitory effect on replication was less pronounced. Effects of the mutations on the ratio of basally/hyperphosphorylated NS5A, together with the apparent molecular weight of the basally phosphorylated species were also observed. Lastly, two of the mutations (T245A and TripleE) resulted in a perinuclear restricted localization of NS5A. These data add further complexity to NS5A phosphorylation and suggest that this analysis be extended outwith the serine-rich cluster within LCS I.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000975
2017-11-15
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/jgv/99/1/62.html?itemId=/content/journal/jgv/10.1099/jgv.0.000975&mimeType=html&fmt=ahah

References

  1. Thrift AP, El-Serag HB, Kanwal F. Global epidemiology and burden of HCV infection and HCV-related disease. Nat Rev Gastroenterol Hepatol 2017;14:122–132 [CrossRef][PubMed]
    [Google Scholar]
  2. Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS et al. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 2015;61:77–87 [CrossRef][PubMed]
    [Google Scholar]
  3. 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][PubMed]
    [Google Scholar]
  4. Grakoui A, Wychowski C, Lin C, Feinstone SM, Rice CM. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 1993;67:1385–1395[PubMed]
    [Google Scholar]
  5. Grakoui A, McCourt DW, Wychowski C, Feinstone SM, Rice CM. A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci USA 1993;90:10583–10587 [CrossRef][PubMed]
    [Google Scholar]
  6. Nielsen SU, Bassendine MF, Burt AD, Bevitt DJ, Toms GL. Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver. J Gen Virol 2004;85:1497–1507 [CrossRef][PubMed]
    [Google Scholar]
  7. Romero-Brey I, Merz A, Chiramel A, Lee JY, Chlanda P et al. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog 2012;8:e1003056 [CrossRef][PubMed]
    [Google Scholar]
  8. Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol 2007;5:453–463 [CrossRef][PubMed]
    [Google Scholar]
  9. Romero-Brey I, Berger C, Kallis S, Kolovou A, Paul D et al. NS5A domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis C virus replication. MBio 2015;6:e00759 [CrossRef][PubMed]
    [Google Scholar]
  10. Brass V, Bieck E, Montserret R, Wölk B, Hellings JA et al. An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem 2002;277:8130–8139 [CrossRef][PubMed]
    [Google Scholar]
  11. 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][PubMed]
    [Google Scholar]
  12. 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][PubMed]
    [Google Scholar]
  13. 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][PubMed]
    [Google Scholar]
  14. Lauck M, Sibley SD, Lara J, Purdy MA, Khudyakov Y et al. A novel hepacivirus with an unusually long and intrinsically disordered NS5A protein in a wild Old World primate. J Virol 2013;87:8971–8981 [CrossRef][PubMed]
    [Google Scholar]
  15. Kapoor A, Simmonds P, Scheel TK, Hjelle B, Cullen JM et al. Identification of rodent homologs of hepatitis C virus and pegiviruses. MBio 2013;4:e00216-13 [CrossRef][PubMed]
    [Google Scholar]
  16. Tellinghuisen TL, Foss KL, Treadaway J. Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog 2008;4:e1000032 [CrossRef][PubMed]
    [Google Scholar]
  17. Reed KE, Gorbalenya AE, Rice CM. The NS5A/NS5 proteins of viruses from three genera of the family flaviviridae are phosphorylated by associated serine/threonine kinases. J Virol 1998;72:6199–6206[PubMed]
    [Google Scholar]
  18. 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][PubMed]
    [Google Scholar]
  19. 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][PubMed]
    [Google Scholar]
  20. Goonawardane N, Gebhardt A, Bartlett C, Pichlmair A, Harris M. Phosphorylation of serine 225 in hepatitis C virus NS5A regulates protein-protein interactions. J Virol 2017;91:e00805-17 [CrossRef][PubMed]
    [Google Scholar]
  21. 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][PubMed]
    [Google Scholar]
  22. 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][PubMed]
    [Google Scholar]
  23. 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][PubMed]
    [Google Scholar]
  24. 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][PubMed]
    [Google Scholar]
  25. Ross-Thriepland D, Amako Y, Harris M. 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 2013;94:1009–1018 [CrossRef][PubMed]
    [Google Scholar]
  26. Nordle Gilliver A, Griffin S, Harris M. Identification of a novel phosphorylation site in hepatitis C virus NS5A. J Gen Virol 2010;91:2428–2432 [CrossRef][PubMed]
    [Google Scholar]
  27. Ross-Thriepland D, Harris M. Insights into the complexity and functionality of hepatitis C virus NS5A phosphorylation. J Virol 2014;88:1421–1432 [CrossRef][PubMed]
    [Google Scholar]
  28. Hughes M, Griffin S, Harris M. Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles. J Gen Virol 2009;90:1329–1334 [CrossRef][PubMed]
    [Google Scholar]
  29. Targett-Adams P, McLauchlan J. Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. J Gen Virol 2005;86:3075–3080 [CrossRef][PubMed]
    [Google Scholar]
  30. Stewart H, Bartlett C, Ross-Thriepland D, Shaw J, Griffin S et al. A novel method for the measurement of hepatitis C virus infectious titres using the IncuCyte ZOOM and its application to antiviral screening. J Virol Methods 2015;218:59–65 [CrossRef][PubMed]
    [Google Scholar]
  31. 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][PubMed]
    [Google Scholar]
  32. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11:791–796 [CrossRef][PubMed]
    [Google Scholar]
  33. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9:676–682 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000975
Loading
/content/journal/jgv/10.1099/jgv.0.000975
Loading

Data & Media loading...

Most Cited This Month

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