1887

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Volume 84, Issue 7

Characterization of the expression of the hepatitis C virus F protein Free

Abstract

Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is approximately 9·6 kb in length and encodes a polyprotein that is proteolytically cleaved to generate at least 10 mature viral protein products. Recently, a new protein, named F, has been described to be expressed through a ribosomal frameshift within the capsid-encoding sequence, a mechanism unique among members of the family . Here, expression of the F protein was investigated in an transcription/translation assay. Its expression in mammalian cells was confirmed using specific recombinant vaccinia viruses; under these conditions, protein expression is dependent on the HCV IRES. The F protein was tagged with firefly luciferase or the Myc epitope to facilitate its identification. Ribosomal frameshifting was dependent on the presence of mutations in the capsid-encoding sequence. No frameshifting was detected in the absence of any mutation. Furthermore, analysis of the F protein in time-course experiments revealed that the protein is very unstable and that its production can be stabilized by the proteasome inhibitor MG132. Finally, indirect immunofluorescence studies have localized the F protein in the cytoplasm, with notable perinuclear detection.

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2003-07-01
2024-05-01
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References

  1. Belcourt M. F., Farabaugh P. J. 1990; Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site. Cell 62:339–352
     [Google Scholar]
  2. Brierley I. 1995; Ribosomal frameshifting viral RNAs. J Gen Virol 76:1885–1892
     [Google Scholar]
  3. Chamorro M., Parkin N., Varmus H. E. 1992; An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA. Proc Natl Acad Sci U S A 89:713–717
     [Google Scholar]
  4. Choo Q.-L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. 1989; Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359–362
     [Google Scholar]
  5. Choo Q.-L., Richman K. H., Han J. H. 11 other authors 1991; Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A 88:2451–2455
     [Google Scholar]
  6. Dinman J. D. 1995; Ribosomal frameshifting in yeast viruses. Yeast 11:1115–1127
     [Google Scholar]
  7. Dubuisson J., Rice C. M. 1996; Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin. J Virol 70:778–786
     [Google Scholar]
  8. Feinstone S. M., Alter H. J., Dienes H. P., Shimizu Y., Popper H., Blackmore D., Sly D., London W. T., Purcell R. H. 1981; Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis 144:588–598
     [Google Scholar]
  9. Fenteany G., Standaert R. F., Lane W. S., Choi S., Corey E. J., Schreiber S. L. 1995; Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268:726–731
     [Google Scholar]
  10. Fournillier-Jacob A., Cahour A., Escriou N., Girard M., Wychowski C. 1996; Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells. J Gen Virol 77:1055–1064
     [Google Scholar]
  11. Fuerst T. R., Niles E. G., Studier F. W., Moss B. 1986; Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83:8122–8126
     [Google Scholar]
  12. Gesteland R. F., Atkins J. F. 1996; Recoding: dynamic reprogramming of translation. Annu Rev Biochem 65:741–768
     [Google Scholar]
  13. Herold J., Siddell S. G. 1993; An ‘elaborated’ pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA. Nucleic Acids Res 21:5838–5842
     [Google Scholar]
  14. Hussy P., Langen H., Mous J., Jacobsen H. 1996; Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology 224:93–104
     [Google Scholar]
  15. Ivanov I. P., Matsufuji S., Murakami Y., Gesteland R. F., Atkins J. F. 2000; Conservation of polyamine regulation by translational frameshifting from yeast to mammals. EMBO J 19:1907–1917
     [Google Scholar]
  16. Jacks T., Madhani H. D., Masiarz F. R., Varmus H. E. 1988; Signals for ribosomal frameshifting in the Rous sarcoma virus gag–pol region. Cell 55:447–458
     [Google Scholar]
  17. Kieny M.-P., Lathe R., Drillien R., Spehner D., Skory S., Schmitt D., Wiktor T., Koprowski H., Lecocq J.-P. 1984; Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature 312:163–166
     [Google Scholar]
  18. Lee D. H., Goldberg A. L. 1998; Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 8:397–403
     [Google Scholar]
  19. Lindenbach B. D., Rice C. M. 2001; Flaviviridae : the viruses and their replication. In Fields Virology , 4th edn. pp  991–1042 Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
     [Google Scholar]
  20. Liu Q., Tackney C., Bhat R. A., Prince A. M., Zhang P. 1997; Regulated processing of hepatitis C virus core protein is linked to subcellular localization. J Virol 71:657–662
     [Google Scholar]
  21. Lo S. Y., Selby M., Tong M., Ou J. H. 1994; Comparative studies of the core gene products of two different hepatitis C virus isolates: two alternative forms determined by a single amino acid substitution. Virology 199:124–131
     [Google Scholar]
  22. Lo S. Y., Masiarz F., Hwang S. B., Lai M. M., Ou J. H. 1995; Differential subcellular localization of hepatitis C virus core gene products. Virology 213:455–461
     [Google Scholar]
  23. 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
     [Google Scholar]
  24. Maillard P., Krawczynski K., Nitkiewicz J. 7 other authors 2001; Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients. J Virol 75:8240–8250
     [Google Scholar]
  25. Marczinke B., Bloys A. J., Brown T. D., Willcocks M. M., Carter M. J., Brierley I. 1994; The human astrovirus RNA-dependent RNA polymerase coding region is expressed by ribosomal frameshifting. J Virol 68:5588–5595
     [Google Scholar]
  26. McHutchison J. G., Poynard T. 1999; Combination therapy with interferon plus ribavirin for the initial treatment of chronic hepatitis C. Semin Liver Dis 19:57–65
     [Google Scholar]
  27. McHutchison J. G., Gordon S. C., Schiff E. R. 7 other authors 1998; Interferon α 2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. . N Engl J Med 339:1485–1492
     [Google Scholar]
  28. McLauchlan J. 2000; Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes. J Viral Hep 7:2–14
     [Google Scholar]
  29. McLauchlan J., Lemberg M. K., Hope G., Martoglio B. 2002; Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J 21:3980–3988
     [Google Scholar]
  30. Meunier J. C., Fournillier A., Choukhi A., Cahour A., Cocquerel L., Dubuisson J., Wychowski C. 1999; Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex. J Gen Virol 80:887–896
     [Google Scholar]
  31. Miller R. H., Purcell R. H. 1990; Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A 87:2057–2061
     [Google Scholar]
  32. Molinari E., Gilman M., Natesan S. 1999; Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo . EMBO J 18:6439–6447
     [Google Scholar]
  33. Naylor L. H. 1999; Reporter gene technology: the future looks bright. Biochem Pharmacol 58:749–757
     [Google Scholar]
  34. Parkin N. T., Chamorro M., Varmus H. E. 1992; Human immunodeficiency virus type 1 gag–pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo . J Virol 66:5147–5151
     [Google Scholar]
  35. Pietschmann T., Lohmann V., Rutter G., Kurpanek K., Bartenschlager R. 2001; Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J Virol 75:1252–1264
     [Google Scholar]
  36. Reil H., Kollmus H., Weidle U. H., Hauser H. 1993; A heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells. J Virol 67:5579–5584
     [Google Scholar]
  37. Rijnbrand R. C., Lemon S. M. 2000; Internal ribosome entry site-mediated translation in hepatitis C virus replication. Curr Top Microbiol Immunol 242:85–116
     [Google Scholar]
  38. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  39. Santolini E., Migliaccio G., La Monica N. 1994; Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol 68:3631–3641
     [Google Scholar]
  40. Schalm S. W., Weiland O., Hansen B. E. 9 other authors 1999; Interferon-ribavirin for chronic hepatitis C with and without cirrhosis: analysis of individual patient data of six controlled trials. Eurohep Study Group for Viral Hepatitis. Gastroenterology 117:408–413
     [Google Scholar]
  41. Stemmer W. P. C., Morris S. K. 1992; Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site directed mutagenesis. Biotechniques 13:214–220
     [Google Scholar]
  42. Takeuchi K., Kubo Y., Boonmar S. 7 other authors 1990; The putative nucleocapsid and envelope protein genes of hepatitis C virus determined by comparison of the nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carriers. J Gen Virol 71:3027–3033
     [Google Scholar]
  43. ten Dam E. B., Pleij C. W., Bosch L. 1990; RNA pseudoknots: translational frameshifting and readthrough on viral RNAs. Virus Genes 4:121–136
     [Google Scholar]
  44. Varaklioti A., Vassilaki N., Georgopoulou U., Mavromara P. 2002; Alternate translation occurs within the core coding region of the hepatitis C viral genome. J Biol Chem 277:17713–17721
     [Google Scholar]
  45. Walewski J. L., Keller T. R., Stump D. D., Branch A. D. 2001; Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA 7:710–721
     [Google Scholar]
  46. Wilson W., Braddock M., Adams S. E., Rathjen P. D., Kingsman S. M., Kingsman A. J. 1988; HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems. Cell 55:1159–1169
     [Google Scholar]
  47. Xu Z., Choi J., Yen T. S., Lu W., Strohecker A., Govindarajan S., Chien D., Selby M. J., Ou J. 2001; Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J 20:3840–3848
     [Google Scholar]
  48. Yasui K., Wakita T., Tsukiyama-Kohara K., Funahashi S. I., Ichikawa M., Kajita T., Moradpour D., Wands J. R., Kohara M. 1998; The native form and maturation process of hepatitis C virus core protein. J Virol 72:6048–6055
     [Google Scholar]
  49. Yeh C. T., Lo S. Y., Dai D. I., Tang J. H., Chu C. M., Liaw Y. F. 2000; Amino acid substitutions in codons 9–11 of hepatitis C virus core protein lead to the synthesis of a short core protein product. J Gastroenterol Hepatol 15:182–191
     [Google Scholar]
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Characterization of the expression of the hepatitis C virus F protein
J. Gen. Virol. 84, 1751 (2003); https://doi.org/10.1099/vir.0.19065-0
/content/journal/jgv/10.1099/vir.0.19065-0
/content/journal/jgv/10.1099/vir.0.19065-0
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