Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV non-structural protein 3 (NS3) has many functions. It has protease and helicase activities, interacts with several host-cell proteins and plays a role in translation, replication and virus-particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6–NS3 yielded multiple spots. Individual protein spots were digested in gel and analysed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.


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  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. Bartenschlager, R.(1999). The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy. J Viral Hepat 6, 165–181.[CrossRef] [Google Scholar]
  3. Bartenschlager, R., Lohmann, V., Wilkinson, T. & Koch, J. O.(1995). Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation. J Virol 69, 7519–7528. [Google Scholar]
  4. Bartenschlager, R., Frese, M. & Pietschmann, T.(2004). Novel insights into hepatitis C virus replication and persistence. Adv Virus Res 63, 71–180. [Google Scholar]
  5. Borowski, P., Heiland, M., Oehlmann, K., Becker, B., Kornetzky, L., Feucht, H. & Laufs, R.(1996). Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase. Eur J Biochem 237, 611–618.[CrossRef] [Google Scholar]
  6. Borowski, P., Schulze zur Wiesch, J., Resch, K., Feucht, H., Laufs, R. & Schmitz, H.(1999). Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus. J Biol Chem 274, 30722–30728.[CrossRef] [Google Scholar]
  7. Duong, F. H., Christen, V., Berke, J. M., Penna, S. H., Moradpour, D. & Heim, M. H.(2005). Upregulation of protein phosphatase 2Ac by hepatitis C virus modulates NS3 helicase activity through inhibition of protein arginine methyltransferase 1. J Virol 79, 15342–15350.[CrossRef] [Google Scholar]
  8. 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]
  9. Failla, C., Tomei, L. & De, F. R.(1994). Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol 68, 3753–3760. [Google Scholar]
  10. Gallinari, P., Paolini, C., Brennan, D., Nardi, C., Steinkuhler, C. & De, F. R.(1999). Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A. Biochemistry 38, 5620–5632.[CrossRef] [Google Scholar]
  11. Ishido, S. & Hotta, H.(1998). Complex formation of the nonstructural protein 3 of hepatitis C virus with the p53 tumor suppressor. FEBS Lett 438, 258–262.[CrossRef] [Google Scholar]
  12. Jakubiec, A. & Jupin, I.(2007). Regulation of positive-strand RNA virus replication: the emerging role of phosphorylation. Virus Res 129, 73–79.[CrossRef] [Google Scholar]
  13. Jennings, T. A., Chen, Y., Sikora, D., Harrison, M. K., Sikora, B., Huang, L., Jankowsky, E., Fairman, M. E., Cameron, C. E. & Raney, K. D.(2008). RNA unwinding activity of the hepatitis C virus NS3 helicase is modulated by the NS5B polymerase. Biochemistry 47, 1126–1135.[CrossRef] [Google Scholar]
  14. Kaul, A., Worz, I. & Bartenschlager, R.(2009). Adaptation of the hepatitis C virus to cell culture. Methods Mol Biol 510, 361–372. [Google Scholar]
  15. Kim, D. W., Gwack, Y., Han, J. H. & Choe, J.(1995). C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 215, 160–166.[CrossRef] [Google Scholar]
  16. Kim, S. J., Kim, J. H., Kim, Y. G., Lim, H. S. & Oh, J. W.(2004). Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation. J Biol Chem 279, 50031–50041.[CrossRef] [Google Scholar]
  17. Lindenbach, B. D., Pragai, B. M., Montserret, R., Beran, R. K., Pyle, A. M., Penin, F. & Rice, C. M.(2007). The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication. J Virol 81, 8905–8918.[CrossRef] [Google Scholar]
  18. Lohmann, V., Korner, F., Koch, J., 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]
  19. Lutter, P., Meyer, H. E., Langer, M., Witthohn, K., Dormeyer, W., Sickmann, A. & Bluggel, M.(2001). Investigation of charge variants of rViscumin by two-dimensional gel electrophoresis and mass spectrometry. Electrophoresis 22, 2888–2897.[CrossRef] [Google Scholar]
  20. Ma, Y., Yates, J., Liang, Y., Lemon, S. M. & Yi, M.(2008). NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly. J Virol 82, 7624–7639.[CrossRef] [Google Scholar]
  21. Mann, M. & Jensen, O. N.(2003). Proteomic analysis of post-translational modifications. Nat Biotechnol 21, 255–261.[CrossRef] [Google Scholar]
  22. Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., Date, T., Matsuura, Y., Miyamura, T. & other authors(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.[CrossRef] [Google Scholar]
  23. McBride, A. E. & Silver, P. A.(2001). State of the arg: protein methylation at arginine comes of age. Cell 106, 5–8.[CrossRef] [Google Scholar]
  24. Meurs, E. F. & Breiman, A.(2007). The interferon inducing pathways and the hepatitis C virus. World J Gastroenterol 13, 2446–2454.[CrossRef] [Google Scholar]
  25. Moradpour, D., Penin, F. & Rice, C. M.(2007). Replication of hepatitis C virus. Nat Rev Microbiol 5, 453–463.[CrossRef] [Google Scholar]
  26. Otsuka, M., Kato, N., Moriyama, M., Taniguchi, H., Wang, Y., Dharel, N., Kawabe, T. & Omata, M.(2005). Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses. Hepatology 41, 1004–1012.[CrossRef] [Google Scholar]
  27. Rho, J., Choi, S., Seong, Y. R., Choi, J. & Im, D. S.(2001). The arginine-1493 residue in QRRGRTGR1493G motif IV of the hepatitis C virus NS3 helicase domain is essential for NS3 protein methylation by the protein arginine methyltransferase 1. J Virol 75, 8031–8044.[CrossRef] [Google Scholar]
  28. Sarioglu, H., Lottspeich, F., Walk, T., Jung, G. & Eckerskorn, C.(2000). Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins. Electrophoresis 21, 2209–2218.[CrossRef] [Google Scholar]
  29. Schweppe, R. E., Haydon, C. E., Lewis, T. S., Resing, K. A. & Ahn, N. G.(2003). The characterization of protein post-translational modifications by mass spectrometry. Acc Chem Res 36, 453–461.[CrossRef] [Google Scholar]
  30. Suzich, J. A., Tamura, J. K., Palmer-Hill, F., Warrener, P., Grakoui, A., Rice, C. M., Feinstone, S. M. & Collett, M. S.(1993). Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. J Virol 67, 6152–6158. [Google Scholar]
  31. Tanji, Y., Hijikata, M., Satoh, S., Kaneko, T. & Shimotohno, K.(1995). Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing. J Virol 69, 1575–1581. [Google Scholar]
  32. Tellinghuisen, T. L., Foss, K. L. & Treadaway, J.(2008). Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog 4, e1000032[CrossRef] [Google Scholar]
  33. Thingholm, T. E., Jensen, O. N. & Larsen, M. R.(2009). Analytical strategies for phosphoproteomics. Proteomics 9, 1451–1468.[CrossRef] [Google Scholar]
  34. Yamagata, A., Kristensen, D. B., Takeda, Y., Miyamoto, Y., Okada, K., Inamatsu, M. & Yoshizato, K.(2002). Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase. Proteomics 2, 1267–1276.[CrossRef] [Google Scholar]
  35. Yao, N., Reichert, P., Taremi, S. S., Prosise, W. W. & Weber, P. C.(1999). Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase. Structure 7, 1353–1363.[CrossRef] [Google Scholar]
  36. Yi, M. & Lemon, S. M.(2004). Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells. J Virol 78, 7904–7915.[CrossRef] [Google Scholar]
  37. Yu, G. Y., Lee, K. J., Gao, L. & Lai, M. M.(2006). Palmitoylation and polymerization of hepatitis C virus NS4B protein. J Virol 80, 6013–6023.[CrossRef] [Google Scholar]
  38. Zhang, C., Cai, Z., Kim, Y. C., Kumar, R., Yuan, F., Shi, P. Y., Kao, C. & Luo, G.(2005). Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase. J Virol 79, 8687–8697.[CrossRef] [Google Scholar]

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