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. 1985 May;54(2):247–255. doi: 10.1128/jvi.54.2.247-255.1985

Molecular cloning and partial sequencing of hepatitis A viral cDNA.

D L Linemeyer, J G Menke, A Martin-Gallardo, J V Hughes, A Young, S W Mitra
PMCID: PMC254791  PMID: 2985793

Abstract

Hepatitis A virus was purified from infected monkey kidney cell cultures, and the viral RNA was used to synthesize double-stranded cDNA. This cDNA was cloned either after insertion into a plasmid-primed synthesis system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which represented at least 97% of the viral genome, lacking ca. 220 bases from the 5' end of the genome. The clones were verified to be hepatitis A virus specific based on their positive hybridization to viral RNA and to total hepatitis A virus-infected cellular RNA from a heterologous marmoset host system. The nucleotide sequence of 3,054 base pairs of cDNA homologous to the 5' half of the viral genome was determined, and an open reading frame of 854 consecutive coding triplets was identified. In addition, sequences which encode the VP-1 and VP-3 viral structural proteins were located in the nucleotide sequence.

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Selected References

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

  1. Beck E., Forss S., Strebel K., Cattaneo R., Feil G. Structure of the FMDV translation initiation site and of the structural proteins. Nucleic Acids Res. 1983 Nov 25;11(22):7873–7885. doi: 10.1093/nar/11.22.7873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boothroyd J. C., Highfield P. E., Cross G. A., Rowlands D. J., Lowe P. A., Brown F., Harris T. J. Molecular cloning of foot and mouth disease virus genome and nucleotide sequences in the structural protein genes. Nature. 1981 Apr 30;290(5809):800–802. doi: 10.1038/290800a0. [DOI] [PubMed] [Google Scholar]
  3. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  4. Coulepis A. G., Locarnini S. A., Gust I. D. Iodination of hepatitis A virus reveals a fourth structural polypeptide. J Virol. 1980 Aug;35(2):572–574. doi: 10.1128/jvi.35.2.572-574.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coulepis A. G., Tannock G. A., Locarnini S. A., Gust I. D. Evidence that the genome of hepatitis A virus consists of single-stranded RNA. J Virol. 1981 Jan;37(1):473–477. doi: 10.1128/jvi.37.1.473-477.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dienstag J. L., Alaama A., Mosley J. W., Redeker A. G., Purcell R. H. Etiology of sporadic hepatitis B surface antigen-negative hepatitis. Ann Intern Med. 1977 Jul;87(1):1–6. doi: 10.7326/0003-4819-87-1-1. [DOI] [PubMed] [Google Scholar]
  7. Dienstag J. L. Hepatitis A virus: virologic, clinical, and epidemiologic studies. Hum Pathol. 1981 Dec;12(12):1097–1106. doi: 10.1016/s0046-8177(81)80330-9. [DOI] [PubMed] [Google Scholar]
  8. Dienstag J. L., Schulman A. N., Gerety R. J., Hoofnagle J. H., Lorenz D. E., Purcell R. H., Barker L. F. Hepatitis A antigen isolated from liver and stool: immunologic comparison of antisera prepared in guinea pigs. J Immunol. 1976 Sep;117(3):876–881. [PubMed] [Google Scholar]
  9. Gust I. D., Coulepis A. G., Feinstone S. M., Locarnini S. A., Moritsugu Y., Najera R., Siegl G. Taxonomic classification of hepatitis A virus. Intervirology. 1983;20(1):1–7. doi: 10.1159/000149367. [DOI] [PubMed] [Google Scholar]
  10. Hughes J. V., Stanton L. W., Tomassini J. E., Long W. J., Scolnick E. M. Neutralizing monoclonal antibodies to hepatitis A virus: partial localization of a neutralizing antigenic site. J Virol. 1984 Nov;52(2):465–473. doi: 10.1128/jvi.52.2.465-473.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jacobson M. F., Asso J., Baltimore D. Further evidence on the formation of poliovirus proteins. J Mol Biol. 1970 May 14;49(3):657–669. doi: 10.1016/0022-2836(70)90289-5. [DOI] [PubMed] [Google Scholar]
  12. Kitamura N., Semler B. L., Rothberg P. G., Larsen G. R., Adler C. J., Dorner A. J., Emini E. A., Hanecak R., Lee J. J., van der Werf S. Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature. 1981 Jun 18;291(5816):547–553. doi: 10.1038/291547a0. [DOI] [PubMed] [Google Scholar]
  13. Kozak M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature. 1984 Mar 15;308(5956):241–246. doi: 10.1038/308241a0. [DOI] [PubMed] [Google Scholar]
  14. Küpper H., Keller W., Kurz C., Forss S., Schaller H., Franze R., Strohmaier K., Marquardt O., Zaslavsky V. G., Hofschneider P. H. Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli. Nature. 1981 Feb 12;289(5798):555–559. doi: 10.1038/289555a0. [DOI] [PubMed] [Google Scholar]
  15. Lamb R. A., Lai C. J. Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus. Cell. 1980 Sep;21(2):475–485. doi: 10.1016/0092-8674(80)90484-5. [DOI] [PubMed] [Google Scholar]
  16. Locarnini S. A., Coulepis A. G., Westaway E. G., Gust I. D. Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies. J Virol. 1981 Jan;37(1):216–225. doi: 10.1128/jvi.37.1.216-225.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Provost P. J., Hilleman M. R. Propagation of human hepatitis A virus in cell culture in vitro. Proc Soc Exp Biol Med. 1979 Feb;160(2):213–221. doi: 10.3181/00379727-160-40422. [DOI] [PubMed] [Google Scholar]
  21. Racaniello V. R., Baltimore D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4887–4891. doi: 10.1073/pnas.78.8.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rakela J., Redeker A. G., Edwards V. M., Decker R., Overby L. R., Mosley J. W. Hepatits A virus infection in fulminant hepatitis and chronic active hepatitis. Gastroenterology. 1978 May;74(5 Pt 1):879–882. [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Siegl G., Frösner G. G. Characterization and classification of virus particles associated with hepatitis A. I. Size, density, and sedimentation. J Virol. 1978 Apr;26(1):40–47. doi: 10.1128/jvi.26.1.40-47.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  26. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ticehurst J. R., Racaniello V. R., Baroudy B. M., Baltimore D., Purcell R. H., Feinstone S. M. Molecular cloning and characterization of hepatitis A virus cDNA. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5885–5889. doi: 10.1073/pnas.80.19.5885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zimmern D., Kaesberg P. 3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4257–4261. doi: 10.1073/pnas.75.9.4257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. von der Helm K., Winnacker E. L., Deinhardt F., Frösner G., Gauss-Müller V., Bayerl B., Scheid R., Siegl G. Cloning of hepatitis A virus genome. J Virol Methods. 1981 Jul;3(1):37–43. doi: 10.1016/0166-0934(81)90020-3. [DOI] [PubMed] [Google Scholar]

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