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. 1991 May;65(5):2155–2163. doi: 10.1128/jvi.65.5.2155-2163.1991

Evidence that less-than-full-length pol gene products are functional in hepadnavirus DNA synthesis.

T T Wu 1, L D Condreay 1, L Coates 1, C Aldrich 1, W Mason 1
PMCID: PMC240562  PMID: 1707980

Abstract

Duck hepatitis B virus mutants containing frameshift or stop codon mutations in a portion of the viral pol gene separating the terminal protein and reverse transcriptase domains had a leaky phenotype and, depending on the location and type of mutation, synthesized up to 10% as much viral DNA as did the wild type. This region of the pol gene had previously been reported to be refractory to missense mutations; in fact, the leakiness of most of our mutants appeared attributable to translational suppression, which would also be expected to introduce amino acid changes. However, at least one mutant (pH1093 + 2), which was ca. 10% as active as the wild type, appeared to use a novel pathway to express the viral pol gene. Our analyses indicated that pH1093 + 2 synthesized the viral reverse transcriptase as a fusion protein with the amino-terminal portion of the pre-S envelope protein. Thus, in this case, the products of the terminal-protein and reverse transcriptase domains of the pol gene would function as separate protein species, though perhaps noncovalently joined in a dimeric structure during assembly of DNA replication complexes. Evidence was also obtained that was consistent with the idea that the wild-type pol gene may, at least in certain instances, be expressed as functional, subgenic polypeptides.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bartenschlager R., Schaller H. The amino-terminal domain of the hepadnaviral P-gene encodes the terminal protein (genome-linked protein) believed to prime reverse transcription. EMBO J. 1988 Dec 20;7(13):4185–4192. doi: 10.1002/j.1460-2075.1988.tb03315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bavand M. R., Laub O. Two proteins with reverse transcriptase activities associated with hepatitis B virus-like particles. J Virol. 1988 Feb;62(2):626–628. doi: 10.1128/jvi.62.2.626-628.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bavand M., Feitelson M., Laub O. The hepatitis B virus-associated reverse transcriptase is encoded by the viral pol gene. J Virol. 1989 Feb;63(2):1019–1021. doi: 10.1128/jvi.63.2.1019-1021.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bosch V., Bartenschlager R., Radziwill G., Schaller H. The duck hepatitis B virus P-gene codes for protein strongly associated with the 5'-end of the viral DNA minus strand. Virology. 1988 Oct;166(2):475–485. doi: 10.1016/0042-6822(88)90518-1. [DOI] [PubMed] [Google Scholar]
  5. Büscher M., Reiser W., Will H., Schaller H. Transcripts and the putative RNA pregenome of duck hepatitis B virus: implications for reverse transcription. Cell. 1985 Mar;40(3):717–724. doi: 10.1016/0092-8674(85)90220-x. [DOI] [PubMed] [Google Scholar]
  6. Calos M. P., Lebkowski J. S., Botchan M. R. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. doi: 10.1073/pnas.80.10.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang C., Enders G., Sprengel R., Peters N., Varmus H. E., Ganem D. Expression of the precore region of an avian hepatitis B virus is not required for viral replication. J Virol. 1987 Oct;61(10):3322–3325. doi: 10.1128/jvi.61.10.3322-3325.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang L. J., Pryciak P., Ganem D., Varmus H. E. Biosynthesis of the reverse transcriptase of hepatitis B viruses involves de novo translational initiation not ribosomal frameshifting. Nature. 1989 Jan 26;337(6205):364–368. doi: 10.1038/337364a0. [DOI] [PubMed] [Google Scholar]
  9. Chen P. J., Chen C. R., Sung J. L., Chen D. S. Identification of a doubly spliced viral transcript joining the separated domains for putative protease and reverse transcriptase of hepatitis B virus. J Virol. 1989 Oct;63(10):4165–4171. doi: 10.1128/jvi.63.10.4165-4171.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Condreay L. D., Aldrich C. E., Coates L., Mason W. S., Wu T. T. Efficient duck hepatitis B virus production by an avian liver tumor cell line. J Virol. 1990 Jul;64(7):3249–3258. doi: 10.1128/jvi.64.7.3249-3258.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gerlich W. H., Robinson W. S. Hepatitis B virus contains protein attached to the 5' terminus of its complete DNA strand. Cell. 1980 Oct;21(3):801–809. doi: 10.1016/0092-8674(80)90443-2. [DOI] [PubMed] [Google Scholar]
  12. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  13. Halpern M. S., England J. M., Deery D. T., Petcu D. J., Mason W. S., Molnar-Kimber K. L. Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4865–4869. doi: 10.1073/pnas.80.15.4865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Halpern M. S., England J. M., Flores L., Egan J., Newbold J., Mason W. S. Individual cells in tissues of DHBV-infected ducks express antigens crossreactive with those on virus surface antigen particles and immature viral cores. Virology. 1984 Sep;137(2):408–413. doi: 10.1016/0042-6822(84)90233-2. [DOI] [PubMed] [Google Scholar]
  15. Hirsch R. C., Lavine J. E., Chang L. J., Varmus H. E., Ganem D. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature. 1990 Apr 5;344(6266):552–555. doi: 10.1038/344552a0. [DOI] [PubMed] [Google Scholar]
  16. Horwich A. L., Furtak K., Pugh J., Summers J. Synthesis of hepadnavirus particles that contain replication-defective duck hepatitis B virus genomes in cultured HuH7 cells. J Virol. 1990 Feb;64(2):642–650. doi: 10.1128/jvi.64.2.642-650.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jacks T., Madhani H. D., Masiarz F. R., Varmus H. E. Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell. 1988 Nov 4;55(3):447–458. doi: 10.1016/0092-8674(88)90031-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kawaguchi T., Nomura K., Hirayama Y., Kitagawa T. Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Res. 1987 Aug 15;47(16):4460–4464. [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Li J. S., Cova L., Buckland R., Lambert V., Deléage G., Trépo C. Duck hepatitis B virus can tolerate insertion, deletion, and partial frameshift mutation in the distal pre-S region. J Virol. 1989 Nov;63(11):4965–4968. doi: 10.1128/jvi.63.11.4965-4968.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lien J. M., Aldrich C. E., Mason W. S. Evidence that a capped oligoribonucleotide is the primer for duck hepatitis B virus plus-strand DNA synthesis. J Virol. 1986 Jan;57(1):229–236. doi: 10.1128/jvi.57.1.229-236.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mack D. H., Bloch W., Nath N., Sninsky J. J. Hepatitis B virus particles contain a polypeptide encoded by the largest open reading frame: a putative reverse transcriptase. J Virol. 1988 Dec;62(12):4786–4790. doi: 10.1128/jvi.62.12.4786-4790.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mandart E., Kay A., Galibert F. Nucleotide sequence of a cloned duck hepatitis B virus genome: comparison with woodchuck and human hepatitis B virus sequences. J Virol. 1984 Mar;49(3):782–792. doi: 10.1128/jvi.49.3.782-792.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Molnar-Kimber K. L., Summers J., Taylor J. M., Mason W. S. Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus. J Virol. 1983 Jan;45(1):165–172. doi: 10.1128/jvi.45.1.165-172.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakabayashi H., Taketa K., Miyano K., Yamane T., Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982 Sep;42(9):3858–3863. [PubMed] [Google Scholar]
  26. Petcu D. J., Aldrich C. E., Coates L., Taylor J. M., Mason W. S. Suramin inhibits in vitro infection by duck hepatitis B virus, Rous sarcoma virus, and hepatitis delta virus. Virology. 1988 Dec;167(2):385–392. [PubMed] [Google Scholar]
  27. Pugh J. C., Sninsky J. J., Summers J. W., Schaeffer E. Characterization of a pre-S polypeptide on the surfaces of infectious avian hepadnavirus particles. J Virol. 1987 May;61(5):1384–1390. doi: 10.1128/jvi.61.5.1384-1390.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pugh J. C., Yaginuma K., Koike K., Summers J. Duck hepatitis B virus (DHBV) particles produced by transient expression of DHBV DNA in a human hepatoma cell line are infectious in vitro. J Virol. 1988 Sep;62(9):3513–3516. doi: 10.1128/jvi.62.9.3513-3516.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Radziwill G., Tucker W., Schaller H. Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. J Virol. 1990 Feb;64(2):613–620. doi: 10.1128/jvi.64.2.613-620.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Roychoudhury S., Shih C. cis rescue of a mutated reverse transcriptase gene of human hepatitis B virus by creation of an internal ATG. J Virol. 1990 Mar;64(3):1063–1069. doi: 10.1128/jvi.64.3.1063-1069.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schlicht H. J., Radziwill G., Schaller H. Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins. Cell. 1989 Jan 13;56(1):85–92. doi: 10.1016/0092-8674(89)90986-0. [DOI] [PubMed] [Google Scholar]
  32. Schlicht H. J., Salfeld J., Schaller H. The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation. J Virol. 1987 Dec;61(12):3701–3709. doi: 10.1128/jvi.61.12.3701-3709.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Seeger C., Maragos J. Molecular analysis of the function of direct repeats and a polypurine tract for plus-strand DNA priming in woodchuck hepatitis virus. J Virol. 1989 May;63(5):1907–1915. doi: 10.1128/jvi.63.5.1907-1915.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Su T. S., Lai C. J., Huang J. L., Lin L. H., Yauk Y. K., Chang C. M., Lo S. J., Han S. H. Hepatitis B virus transcript produced by RNA splicing. J Virol. 1989 Sep;63(9):4011–4018. doi: 10.1128/jvi.63.9.4011-4018.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Summers J., Mason W. S. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell. 1982 Jun;29(2):403–415. doi: 10.1016/0092-8674(82)90157-x. [DOI] [PubMed] [Google Scholar]
  36. Summers J., Smith P. M., Horwich A. L. Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J Virol. 1990 Jun;64(6):2819–2824. doi: 10.1128/jvi.64.6.2819-2824.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Suzuki T., Masui N., Kajino K., Saito I., Miyamura T. Detection and mapping of spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus genome. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8422–8426. doi: 10.1073/pnas.86.21.8422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tuttleman J. S., Pugh J. C., Summers J. W. In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus. J Virol. 1986 Apr;58(1):17–25. doi: 10.1128/jvi.58.1.17-25.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wilson W., Braddock M., Adams S. E., Rathjen P. D., Kingsman S. M., Kingsman A. J. HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems. Cell. 1988 Dec 23;55(6):1159–1169. doi: 10.1016/0092-8674(88)90260-7. [DOI] [PubMed] [Google Scholar]
  41. Wilson W., Malim M. H., Mellor J., Kingsman A. J., Kingsman S. M. Expression strategies of the yeast retrotransposon Ty: a short sequence directs ribosomal frameshifting. Nucleic Acids Res. 1986 Sep 11;14(17):7001–7016. doi: 10.1093/nar/14.17.7001. [DOI] [PMC free article] [PubMed] [Google Scholar]

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