Abstract
A transient protein expression system in COS-1 cells was used to study the role of hepatitis C virus (HCV)-encoded NS4A protein on HCV nonstructural polyprotein processing. By analyzing the protein expression and processing of a deletion mutant polypeptide, NS delta 4A, which encodes the entire putative HCV nonstructural polyprotein except the region encoding NS4A, the versatile functions of NS4A were revealed. Most of the NS3 processed from NS delta 4A was localized in the cytosol fraction and was degraded promptly. Coproduction of NS4A stabilizes NS3 and assists in its localization in the membrane. NS4A was found to be indispensable for cleavage at the 4B/5A site but not essential for cleavage at the 5A/5B site in NS delta 4A. The functioning of NS4A as a cofactor for cleavage at the 4B/5A site was also observed when 30 amino acids around this site was used as a substrate and a serine proteinase domain of 167 amino acids, from Gly-1049 to Ser-1215, was used as an enzyme protein, suggesting that possible domains for the interaction of NS4A were in those regions of the enzyme protein (NS3) and/or the substrate protein. Two proteins, p58 and p56, were produced from NS5A. For the production of p58, equal or excess molar amounts of NS4A relative to NS delta 4A were required. Deletion analysis of NS4A revealed a minimum functional domain of NS4A of 10 amino acids, from Gly-1678 to Ile-1687.
Full Text
The Full Text of this article is available as a PDF (389.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arias C. F., Preugschat F., Strauss J. H. Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology. 1993 Apr;193(2):888–899. doi: 10.1006/viro.1993.1198. [DOI] [PubMed] [Google Scholar]
- Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Kinetic and structural analyses of hepatitis C virus polyprotein processing. J Virol. 1994 Aug;68(8):5045–5055. doi: 10.1128/jvi.68.8.5045-5055.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. 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. 1993 Jul;67(7):3835–3844. doi: 10.1128/jvi.67.7.3835-3844.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chambers T. J., Grakoui A., Rice C. M. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites. J Virol. 1991 Nov;65(11):6042–6050. doi: 10.1128/jvi.65.11.6042-6050.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eckart M. R., Selby M., Masiarz F., Lee C., Berger K., Crawford K., Kuo C., Kuo G., Houghton M., Choo Q. L. The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor. Biochem Biophys Res Commun. 1993 Apr 30;192(2):399–406. doi: 10.1006/bbrc.1993.1429. [DOI] [PubMed] [Google Scholar]
- Failla C., Tomei L., De Francesco R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol. 1994 Jun;68(6):3753–3760. doi: 10.1128/jvi.68.6.3753-3760.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Falgout B., Pethel M., Zhang Y. M., Lai C. J. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol. 1991 May;65(5):2467–2475. doi: 10.1128/jvi.65.5.2467-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol. 1993 May;67(5):2832–2843. doi: 10.1128/jvi.67.5.2832-2843.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Shimotohno K. 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. 1991 Jul 1;88(13):5547–5551. doi: 10.1073/pnas.88.13.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hijikata M., Mizushima H., Akagi T., Mori S., Kakiuchi N., Kato N., Tanaka T., Kimura K., Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993 Aug;67(8):4665–4675. doi: 10.1128/jvi.67.8.4665-4675.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hijikata M., Mizushima H., Tanji Y., Komoda Y., Hirowatari Y., Akagi T., Kato N., Kimura K., Shimotohno K. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10773–10777. doi: 10.1073/pnas.90.22.10773. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaneko T., Tanji Y., Satoh S., Hijikata M., Asabe S., Kimura K., Shimotohno K. Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem Biophys Res Commun. 1994 Nov 30;205(1):320–326. doi: 10.1006/bbrc.1994.2667. [DOI] [PubMed] [Google Scholar]
- Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9524–9528. doi: 10.1073/pnas.87.24.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin C., Amberg S. M., Chambers T. J., Rice C. M. Cleavage at a novel site in the NS4A region by the yellow fever virus NS2B-3 proteinase is a prerequisite for processing at the downstream 4A/4B signalase site. J Virol. 1993 Apr;67(4):2327–2335. doi: 10.1128/jvi.67.4.2327-2335.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manabe S., Fuke I., Tanishita O., Kaji C., Gomi Y., Yoshida S., Mori C., Takamizawa A., Yosida I., Okayama H. Production of nonstructural proteins of hepatitis C virus requires a putative viral protease encoded by NS3. Virology. 1994 Feb;198(2):636–644. doi: 10.1006/viro.1994.1075. [DOI] [PubMed] [Google Scholar]
- Mizushima H., Hijikata M., Tanji Y., Kimura K., Shimotohno K. Analysis of N-terminal processing of hepatitis C virus nonstructural protein 2. J Virol. 1994 Apr;68(4):2731–2734. doi: 10.1128/jvi.68.4.2731-2734.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Preugschat F., Yao C. W., Strauss J. H. In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3. J Virol. 1990 Sep;64(9):4364–4374. doi: 10.1128/jvi.64.9.4364-4374.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
- Takamizawa A., Mori C., Fuke I., Manabe S., Murakami S., Fujita J., Onishi E., Andoh T., Yoshida I., Okayama H. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol. 1991 Mar;65(3):1105–1113. doi: 10.1128/jvi.65.3.1105-1113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tanji Y., Hijikata M., Hirowatari Y., Shimotohno K. Hepatitis C virus polyprotein processing: kinetics and mutagenic analysis of serine proteinase-dependent cleavage. J Virol. 1994 Dec;68(12):8418–8422. doi: 10.1128/jvi.68.12.8418-8422.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tanji Y., Hijikata M., Hirowatari Y., Shimotohno K. Identification of the domain required for trans-cleavage activity of hepatitis C viral serine proteinase. Gene. 1994 Aug 5;145(2):215–219. doi: 10.1016/0378-1119(94)90008-6. [DOI] [PubMed] [Google Scholar]
- Tomei L., Failla C., Santolini E., De Francesco R., La Monica N. NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol. 1993 Jul;67(7):4017–4026. doi: 10.1128/jvi.67.7.4017-4026.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]