1887

Abstract

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

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2006-01-01
2019-11-22
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References

  1. Appel, N., Pietschmann, T. & Bartenschlager, R. ( 2005; ). 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 79, 3187–3194.[CrossRef]
    [Google Scholar]
  2. Asabe, S. I., Tanji, Y., Satoh, S., Kaneko, T., Kimura, K. & Shimotohno, K. ( 1997; ). The N-terminal region of hepatitis C virus-encoded NS5A is important for NS4A-dependent phosphorylation. J Virol 71, 790–796.
    [Google Scholar]
  3. Bartenschlager, R. & Lohmann, V. ( 2000; ). Replication of hepatitis C virus. J Gen Virol 81, 1631–1648.
    [Google Scholar]
  4. Bartenschlager, R., Ahlborn-Laake, L., Mous, J. & Jacobsen, H. ( 1993; ). Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 67, 3835–3844.
    [Google Scholar]
  5. Barton, D. J., Morasco, B. J. & Flanegan, J. B. ( 1999; ). Translating ribosomes inhibit poliovirus negative-strand RNA synthesis. J Virol 73, 10104–10112.
    [Google Scholar]
  6. Blight, K. J., Kolykhalov, A. A. & Rice, C. M. ( 2000; ). Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974.[CrossRef]
    [Google Scholar]
  7. Bukh, J., Pietschmann, T., Lohmann, V. & 7 other authors ( 2002; ). Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci U S A 99, 14416–14421.[CrossRef]
    [Google Scholar]
  8. Coito, C., Diamond, D. L., Neddermann, P., Korth, M. J. & Katze, M. G. ( 2004; ). High-throughput screening of the yeast kinome: identification of human serine/threonine protein kinases that phosphorylate the hepatitis C virus NS5A protein. J Virol 78, 3502–3513.[CrossRef]
    [Google Scholar]
  9. Dhanak, D., Duffy, K. J., Johnston, V. K. & 23 other authors ( 2002; ). Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 277, 38322–38327.[CrossRef]
    [Google Scholar]
  10. Evans, M. J., Rice, C. M. & Goff, S. P. ( 2004; ). Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc Natl Acad Sci U S A 101, 13038–13043.[CrossRef]
    [Google Scholar]
  11. 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.[CrossRef]
    [Google Scholar]
  12. Friebe, P., Lohmann, V., Krieger, N. & Bartenschlager, R. ( 2001; ). Sequences in the 5′ nontranslated region of hepatitis C virus required for RNA replication. J Virol 75, 12047–12057.[CrossRef]
    [Google Scholar]
  13. Goodbourn, S., Didcock, L. & Randall, R. E. ( 2000; ). Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J Gen Virol 81, 2341–2364.
    [Google Scholar]
  14. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M. & Rice, C. M. ( 1993; ). Characterization of the hepatitis-C virus-encoded serine proteinase - determination of proteinase-dependent polyprotein cleavage sites. J Virol 67, 2832–2843.
    [Google Scholar]
  15. Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M. & Shimotohno, K. ( 1991; ). Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci U S A 88, 5547–5551.[CrossRef]
    [Google Scholar]
  16. Hijikata, M., Mizushima, H., Tanji, Y., Komoda, Y., Hirowatari, Y., Akagi, T., Kato, N., Kimura, K. & Shimotohno, K. ( 1993; ). Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A 90, 10773–10777.[CrossRef]
    [Google Scholar]
  17. Koch, J. O. & Bartenschlager, R. ( 1999; ). Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B. J Virol 73, 7138–7146.
    [Google Scholar]
  18. Kolykhalov, A. A., Mihalik, K., Feinstone, S. M. & Rice, C. M. ( 2000; ). Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo. J Virol 74, 2046–2051.[CrossRef]
    [Google Scholar]
  19. Krieger, N., Lohmann, V. & Bartenschlager, R. ( 2001; ). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75, 4614–4624.[CrossRef]
    [Google Scholar]
  20. Lohmann, V., Korner, F., Koch, J. O., Herian, U., Theilmann, L. & Bartenschlager, R. ( 1999; ). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113.[CrossRef]
    [Google Scholar]
  21. Lohmann, V., Korner, F., Dobierzewska, A. & Bartenschlager, R. ( 2001; ). Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75, 1437–1449.[CrossRef]
    [Google Scholar]
  22. Lohmann, V., Hoffmann, S., Herian, U., Penin, F. & Bartenschlager, R. ( 2003; ). Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol 77, 3007–3019.[CrossRef]
    [Google Scholar]
  23. Luo, G., Xin, S. & Cai, Z. ( 2003; ). Role of the 5′-proximal stem-loop structure of the 5′ untranslated region in replication and translation of hepatitis C virus RNA. J Virol 77, 3312–3318.[CrossRef]
    [Google Scholar]
  24. McCormick, C. J., Rowlands, D. J. & Harris, M. ( 2002; ). Efficient delivery and regulable expression of hepatitis C virus full- length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors. J Gen Virol 83, 383–394.
    [Google Scholar]
  25. McCormick, C. J., Challinor, L., Macdonald, A., Rowlands, D. J. & Harris, M. ( 2004; ). Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system. J Gen Virol 85, 429–439.[CrossRef]
    [Google Scholar]
  26. Moradpour, D., Evans, M. J., Gosert, R., Yuan, Z., Blum, H. E., Goff, S. P., Lindenbach, B. D. & Rice, C. M. ( 2004; ). Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J Virol 78, 7400–7409.[CrossRef]
    [Google Scholar]
  27. Neddermann, P., Clementi, A. & De Francesco, R. ( 1999; ). Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein. J Virol 73, 9984–9991.
    [Google Scholar]
  28. Neddermann, P., Quintavalle, M., Di Pietro, C., Clementi, A., Cerretani, M., Altamura, S., Bartholomew, L. & De Francesco, R. ( 2004; ). Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture. J Virol 78, 13306–13314.[CrossRef]
    [Google Scholar]
  29. Poynard, T., Yuen, M. F., Ratziu, V. & Lai, C. L. ( 2003; ). Viral hepatitis C. Lancet 362, 2095–2100.[CrossRef]
    [Google Scholar]
  30. Reed, K. E. & Rice, C. M. ( 2000; ). Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol 242, 55–84.
    [Google Scholar]
  31. Reed, K. E., Xu, J. & Rice, C. M. ( 1997; ). Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase. J Virol 71, 7187–7197.
    [Google Scholar]
  32. Robertson, M. E., Seamons, R. A. & Belsham, G. J. ( 1999; ). A selection system for functional internal ribosome entry site (IRES) elements: analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES. RNA 5, 1167–1179.[CrossRef]
    [Google Scholar]
  33. Teterina, N. L., Egger, D., Bienz, K., Brown, D. M., Semler, B. L. & Ehrenfeld, E. ( 2001; ). Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis. J Virol 75, 3841–3850.[CrossRef]
    [Google Scholar]
  34. Tsukiyama-Kohara, K., Iizuka, N., Kohara, M. & Nomoto, A. ( 1992; ). Internal ribosome entry site within hepatitis C virus RNA. J Virol 66, 1476–1483.
    [Google Scholar]
  35. Wang, C., Sarnow, P. & Siddiqui, A. ( 1993; ). Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J Virol 67, 3338–3344.
    [Google Scholar]
  36. Yi, M. & Lemon, S. M. ( 2003; ). 3′ Nontranslated RNA signals required for replication of hepatitis C virus RNA. J Virol 77, 3557–3568.[CrossRef]
    [Google Scholar]
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