Hepatitis B viral polymerase fusion proteins are biologically active and can interact with the hepatitis C virus core protein in vivo
Tóm tắt
Hepadnaviruses and retroviruses are evolutionarily related families because they both require a process of reverse transcription for genome replication. However, hepadnaviruses produce polymerase (pol) and core proteins separately, while retroviruses synthesize a gag-pol fusion protein that is subsequently cleaved by a virally encoded protease to release a functional polymerase. To test whether an additional sequence at the N-terminus of pol in hepatitis B virus (HBV) interferes with its function, we created two plasmids expressing core-pol fusion proteins, core144-pol and core31-pol. Secreted particles obtained from HuH-7 cells, which were cotransfected with a core-pol fusion protein-expressing plasmid and a core-expressing plasmid, showed a positive signal of HBV DNA by the endogenous polymerase assay, indicating that the core-pol fusion proteins retain DNA priming, polymerization and RNase H activities. The fusion protein was detected in the cytoplasm of transfected cells and in secreted virions by immunoprecipitation. Furthermore, we found by immunofluorescence staining that the HBV core-pol fusion protein colocalized with the hepatitis C virus (HCV) core protein in cytoplasm and in lipid droplets. Immunoprecipitation studies showed that the anti-HCV core complex contained the HBV core-pol fusion protein while the anti-HBV pol complex contained the HCV core protein, which supports the hypothesis that the HCV core protein can form a complex with the HBV core-pol fusion protein.
Tài liệu tham khảo
Barba G, Harper F, Harada T, Kohara M, Goulinet S, Matsuura Y, Eder G, Schaff Z, Chapman MJ, Miyamura T, Brechot C. Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc Natl Acad Sci USA 94:1200–1205;1997.
Bartenschlager R, Kuhn C, Shaller H. Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associatedP-gene product by radiolabelling at newly introduced phosphorylation sites. Nucleic Acids Res 20:195–202;1992.
Beasley RP, Hwang LY, Lin CC, Chieh CS. Hepatocellular carcinoma and hepatitis B virus. Lancet ii:1129–1132;1981.
Birnbaum F, Nassal M. Hepatitis B virus nucleocapsid assembly: Primary structure requirements in the core protein. J Virol 64:3319–3330;1990.
Chiang PW, Hu CP, Su TS, Lo SJ, Chu MH, Schaller H, Chang C. Encapsidation of truncated human hepatitis B virus genomes throughtrans-complementation of the core protein and polymerase. Virology 176:355–361;1990.
Ganem D, Varmus HE. The molecular biology of the hepatitis B viruses. Annu Rev Biochem 56:651–693;1987.
Graham F, van der Eb A. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456–457;1973.
Gregorio GV, Choudhuri K, Ma Y, Vegnente A, Mielei-Vergani G, Vergani D. Mimicry between the hepatitis B virus DNA polymerase and the antigen targets of nuclear and smooth muscle antibodies in chronic hepatitis B virus infection. J Immunol 162:1802–1810;1999.
Hadziyannis SJ, Sherman M, Liberman HM, Shafritz DA. Liver disease activity and hepatitis B virus replication in chronic delta antigenpositive hepatitis B virus carriers. Hepatology 5:544–547;1995.
Hilleman MR. Comparative biology and pathogenesis of AIDS and hepatitis B viruses: Related but different. AIDS Res Hum Retroviruses 10:1409–1419;1994.
Hirsch RC, Loeb DD, Polack JR, Ganem D.cis-acting sequence required for encapsidation of duck hepatitis B virus pregenomic RNA. J Virol 65:3309–3316;1991.
Honda A, Nakamura T, Nishimura S. RNA signals for translation frameshift: Influence of stem size and slippery sequence. Biochem Biophys Res Commun 15:575–582;1995.
Hope RG, McLauchlan J. Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein. J Gen Virol 81:1913–1925;2000.
Huang HL, Jeng KS, Hu CP, Tsai CH, Lo SJ, Chang CM. Identification and characterization of a structural protein of hepatitis B virus: A polymerase and surface fusion protein encoded by a spliced RNA. Virology 275:398–410;2000.
Hui EKW, Yi YS, Lo SJ. Hepatitis B virus core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped. J Gen Virol 80:2647–2659;1999.
Hwang WL, Su TS. Translational regulation of hepatitis B virus polymerase gene by termination-reinitiation of an upstream minicistron in a length-dependent manner. J Gen Virol 79:2181–2189;1998.
Junker M, Galle P, Schaller H. Expression and replication of the hepatitis B virus genome under foreign promoter control. Nucleic Acids Res 15:10117–10132;1987.
Lillehoj EP, Salazar FH, Mervis RJ, Raum MG, Chan HW, Ahmad N, Venkatesan S. Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus. J Virol 62:3053–3058;1988.
Lin CG, Lo SJ. Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus pol gene from the viral pregenome RNA. Virology 188:342–352;1992.
Lott L, Beames B, Notvall L, Lanford RE. Interaction between hepatitis B virus core protein and reverse transcriptase. J Virol 74:11479–11489;2000.
Miller RH. Proteolytic self-cleavage of hepatitis B virus core protein may generate serum e antigen. Science 236:722–725;1987.
Miller RH. Close evolutionary relatedness of the hepatitis B virus and murine leukemia virus polymerase gene sequences. Virology 164:147–155;1988.
Miller RH, Robinson WS. Common evolutionary origin of hepatitis B virus and retroviruses. Proc Natl Acad Sci USA 83:2531–2535;1986.
Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer Res 42:3858–3863;1982.
Nassal M, Junker-Niepmann M, Schaller H. Translational inactivation of RNA function: Discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly. Cell 63:1357–1363;1990.
Nassal M, Schaller H. Hepatitis B virus replication. Trends Microbiol 1:221–228;1993.
Radziwill G, Tucker W, Schaller H. Mutational analysis of the hepatitis B virus P gene product: Domain structure and RNase H activity. J Virol 64:613–620;1990.
Robinson WS, Miller RH, Marion PL. Hepadnaviruses and retroviruses share genome homology and feature of replication. Hepatology 7(1 suppl):64S-73S;1987.
Sakuragi J, Sakai H, Kawamura M, Tokunaga K, Ueda S, Adachi A. Generation and characterization of a host cell-dependentgag gene mutant of human immunodeficiency virus type 1. Virology 10:251–254;1995.
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 1989.
Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitor. Proc Natl Acad Sci USA 74:5463–5467;1977.
Shih CM, Chen CM, Chen SY, Lee WYH. Modulation of thetrans-suppression activity of hepatitis C virus core protein by phosphorylation. J Virol 69:1160–1171;1995.
Shih CM, Lo SJ, Miyamura T, Chen SY, Lee WU YH. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells. J Virol 67:5823–5832;1993.
Smith AJ, Cho MI, Hammerskjold ML, Rekosh D. Human immunodeficiency virus type 1 Pr55gag and Pr160gag-pol expressed from simian virus 40 late-replacement vector are efficiently processed and assembled into virus-like particles. J Virol 64:2743–2750;1990.
Staschke KA, Colacino JM. Priming of duck hepatitis B virus reverse transcription in vitro: Premature termination of primer DNA induced by the 5′-triphosphate of fialuridine. J Virol 68:8265–8269;1994.
Summers J, Mason WS. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29:403–415;1982.
Tessmer U, Krausslich HG. Cleavage of human immunodeficiency virus type 1 proteinase from the N-terminally adjacent p6* protein is essential for efficient Gag polyprotein processing and viral infectivity. J Virol 72:3459–3463;1998.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354;1979.
Tritch RJ, Cheng YE, Yin FH, Erickson-Viitanen S. Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein. J Virol 65:922–930;1991.
Urban M, McMillan DJ, Canning G, Newell A, Brown E, Mills JS, Jupp R. In vitro activity of hepatitis B virus polymerase: Requirement for distinct metal ions and the viral epsilon stemloop. J Gen Virol 79:1121–1131;1998.
Wang GH, Seeger C. Novel mechanism for reverse transcription in hepatitis B viruses. J Virol 67:6507–6512;1993.
Will H, Salfeld J, Pfaff E, Manso C, Theilmann L, Schaler H. Putative reverse transcriptase intermediates of human hepatitis B virus in primary liver carcinomas. Science 231:594–596;1986.
Wu JC, Chen PJ, Kuo MYP, Lee SD, Chen DS, Ting LP. Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line. J Virol 65:1099–1104;1991.
Wu JC, Lee SD, Govindarajan S, Kung TW, Ting LP. Correlation of serum delta RNA with clinical course of acute hepatitis delta virus superinfection in Taiwan: A longitudinal study. J Infect Dis 161:1116–1120;1990.
zu Putlitz J, Lanford RE, Carlson RI, Notvall L, De La Monte SM, Wands JR. Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein. J Virol 73:4188–4196;1999.