Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Dec;81(24):7885–7889. doi: 10.1073/pnas.81.24.7885

The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locus.

A T Panganiban, H M Temin
PMCID: PMC392257  PMID: 6083562

Abstract

We mutagenized cloned spleen necrosis virus DNA to identify a region of the retrovirus genome encoding a polypeptide required for integration of viral DNA. Five plasmids bearing different lesions in the 3' end of the pol gene were examined for the ability to integrate or replicate following transfection of chicken embryo fibroblasts. Transfection with one of these DNAs resulted in the generation of mutant virus incapable of integrating but able to replicate at low levels; this phenotype is identical to that of mutants bearing alterations in the cis-acting region, att. To determine whether the 3' end of the pol gene encodes a protein that interacts with att, we did a complementation experiment. Cells were first infected with an att- virus and then superinfected with the integration-deficient virus containing a lesion in the pol gene and a wild-type att site. The results showed that the att- virus provided a transacting function allowing integration of viral DNA derived from the mutant bearing a wild-type att site. Thus, the 3' end of the pol gene serves as an "int" locus and encodes a protein mediating integration of retrovirus DNA through interaction with att.

Full text

PDF
7885

Images in this article

7887Image
on p.7887
7887Image
on p.7887
7888Image
on p.7888

Selected References

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

  1. Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970 Jun 27;226(5252):1209–1211. doi: 10.1038/2261209a0. [DOI] [PubMed] [Google Scholar]
  2. Bandyopadhyay P. K., Temin H. M. Expression from an internal AUG codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector. Mol Cell Biol. 1984 Apr;4(4):743–748. doi: 10.1128/mcb.4.4.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbacid M., Hunter E., Aaronson S. A. Avian reticuloendotheliosis viruses: evolutionary linkage with mammalian type C retroviruses. J Virol. 1979 May;30(2):508–514. doi: 10.1128/jvi.30.2.508-514.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bauer G., Temin H. M. Specific antigenic relationships between the RNA-dependent DNA polymerases of avian reticuloendotheliosis viruses and mammalian type C retroviruses. J Virol. 1980 Apr;34(1):168–177. doi: 10.1128/jvi.34.1.168-177.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dhar R., McClements W. L., Enquist L. W., Vande Woude G. F. Nucleotide sequences of integrated Moloney sarcoma provirus long terminal repeats and their host and viral junctions. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3937–3941. doi: 10.1073/pnas.77.7.3937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donehower L. A., Huang A. L., Hager G. L. Regulatory and coding potential of the mouse mammary tumor virus long terminal redundancy. J Virol. 1981 Jan;37(1):226–238. doi: 10.1128/jvi.37.1.226-238.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duyk G., Leis J., Longiaru M., Skalka A. M. Selective cleavage in the avian retroviral long terminal repeat sequence by the endonuclease associated with the alpha beta form of avian reverse transcriptase. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6745–6749. doi: 10.1073/pnas.80.22.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fritsch E., Temin H. M. Formation and structure of infectious DNA of spleen necrosis virus. J Virol. 1977 Jan;21(1):119–130. doi: 10.1128/jvi.21.1.119-130.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gibson W., Verma I. M. Studies on the reverse transcriptase of RNA tumor viruses. Structural relatedness of two subunits of avian RNA tumor viruses. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4991–4994. doi: 10.1073/pnas.71.12.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Golomb M., Grandgenett D. P. Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus. J Biol Chem. 1979 Mar 10;254(5):1606–1613. [PubMed] [Google Scholar]
  11. Grandgenett D. P., Vora A. C., Schiff R. D. A 32,000-dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity. Virology. 1978 Aug;89(1):119–132. doi: 10.1016/0042-6822(78)90046-6. [DOI] [PubMed] [Google Scholar]
  12. Guntaka R. V., Richards O. C., Shank P. R., Kung H. J., Davidson N. Covalently closed circular DNA of avian sarcoma virus: purification from nuclei of infected quail tumor cells and measurement by electron microscopy and gel electrophoresis. J Mol Biol. 1976 Sep 15;106(2):337–357. doi: 10.1016/0022-2836(76)90090-5. [DOI] [PubMed] [Google Scholar]
  13. Hishinuma F., DeBona P. J., Astrin S., Skalka A. M. Nucleotide sequence of acceptor site and termini of integrated avian endogenous provirus ev1: integration creates a 6 bp repeat of host DNA. Cell. 1981 Jan;23(1):155–164. doi: 10.1016/0092-8674(81)90280-4. [DOI] [PubMed] [Google Scholar]
  14. Hughes S. H., Mutschler A., Bishop J. M., Varmus H. E. A Rous sarcoma virus provirus is flanked by short direct repeats of a cellular DNA sequence present in only one copy prior to integration. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4299–4303. doi: 10.1073/pnas.78.7.4299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hunter E., Bhown A. S., Bennett J. C. Amino-terminal amino acid sequence of the major structural polypeptides of avian retroviruses: sequence homology between reticuloendotheliosis virus p30 and p30s of mammalian retroviruses. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2708–2712. doi: 10.1073/pnas.75.6.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hurwitz J., Leis J. P. RNA-dependent DNA polymerase activity of RNA tumor viruses. I. Directing influence of DNA in the reaction. J Virol. 1972 Jan;9(1):116–129. doi: 10.1128/jvi.9.1.116-129.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ju G., Skalka A. M. Nucleotide sequence analysis of the long terminal repeat (LTR) of avian retroviruses: structural similarities with transposable elements. Cell. 1980 Nov;22(2 Pt 2):379–386. doi: 10.1016/0092-8674(80)90348-7. [DOI] [PubMed] [Google Scholar]
  18. Kopchick J. J., Harless J., Geisser B. S., Killam R., Hewitt R. R., Arlinghaus R. B. Endodeoxyribonuclease activity associated with Rauscher murine leukemia virus. J Virol. 1981 Jan;37(1):274–283. doi: 10.1128/jvi.37.1.274-283.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Majors J. E., Varmus H. E. Nucleotide sequences at host-proviral junctions for mouse mammary tumour virus. Nature. 1981 Jan 22;289(5795):253–258. doi: 10.1038/289253a0. [DOI] [PubMed] [Google Scholar]
  20. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  21. Mizutani S., Temin H. M. Lack of serological relationship among DNA polymerases of avian leukosis-sarcoma viruses, reticuloendotheliosis viruses, and chicken cells. J Virol. 1973 Sep;12(3):440–448. doi: 10.1128/jvi.12.3.440-448.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mölling K., Bolognesi D. P., Bauer H., Büsen W., Plassmann H. W., Hausen P. Association of viral reverse transcriptase with an enzyme degrading the RNA moiety of RNA-DNA hybrids. Nat New Biol. 1971 Dec 22;234(51):240–243. doi: 10.1038/newbio234240a0. [DOI] [PubMed] [Google Scholar]
  23. Nissen-Meyer J., Nes I. F. Purification and properties of DNA endonucleases associated with Friend leukemia virus. Nucleic Acids Res. 1980 Nov 11;8(21):5043–5055. doi: 10.1093/nar/8.21.5043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Omer C. A., Faras A. J. Mechanism of release of the avian rotavirus tRNATrp primer molecule from viral DNA by ribonuclease H during reverse transcription. Cell. 1982 Oct;30(3):797–805. doi: 10.1016/0092-8674(82)90284-7. [DOI] [PubMed] [Google Scholar]
  25. Panganiban A. T., Temin H. M. Circles with two tandem LTRs are precursors to integrated retrovirus DNA. Cell. 1984 Mar;36(3):673–679. doi: 10.1016/0092-8674(84)90347-7. [DOI] [PubMed] [Google Scholar]
  26. Panganiban A. T., Temin H. M. The terminal nucleotides of retrovirus DNA are required for integration but not virus production. Nature. 1983 Nov 10;306(5939):155–160. doi: 10.1038/306155a0. [DOI] [PubMed] [Google Scholar]
  27. Reddy E. P., Smith M. J., Aaronson S. A. Complete nucleotide sequence and organization of the Moloney murine sarcoma virus genome. Science. 1981 Oct 23;214(4519):445–450. doi: 10.1126/science.6170110. [DOI] [PubMed] [Google Scholar]
  28. Samuel K. P., Papas T. S., Chirikjian J. G. DNA endonucleases associated with the avian myeloblastosis virus DNA polymerase. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2659–2663. doi: 10.1073/pnas.76.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scott M. L., McKereghan K., Kaplan H. S., Fry K. E. Molecular cloning and partial characterization of unintegrated linear DNA from gibbon ape leukemia virus. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4213–4217. doi: 10.1073/pnas.78.7.4213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shank P. R., Hughes S. H., Kung H. J., Majors J. E., Quintrell N., Guntaka R. V., Bishop J. M., Varmus H. E. Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA. Cell. 1978 Dec;15(4):1383–1395. doi: 10.1016/0092-8674(78)90063-6. [DOI] [PubMed] [Google Scholar]
  31. Shank P. R., Varmus H. E. Virus-specific DNA in the cytoplasm of avian sarcoma virus-infected cells is a precursor to covalently closed circular viral DNA in the nucleus. J Virol. 1978 Jan;25(1):104–104. doi: 10.1128/jvi.25.1.104-104.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shimotohno K., Mizutani S., Temin H. M. Sequence of retrovirus provirus resembles that of bacterial transposable elements. Nature. 1980 Jun 19;285(5766):550–554. doi: 10.1038/285550a0. [DOI] [PubMed] [Google Scholar]
  33. Shimotohno K., Temin H. M. No apparent nucleotide sequence specificity in cellular DNA juxtaposed to retrovirus proviruses. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7357–7361. doi: 10.1073/pnas.77.12.7357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sutcliffe J. G., Shinnick T. M., Verma I. M., Lerner R. A. Nucleotide sequence of Moloney leukemia virus: 3' end reveals details of replications, analogy to bacterial transposons, and an unexpected gene. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3302–3306. doi: 10.1073/pnas.77.6.3302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Temin H. M., Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970 Jun 27;226(5252):1211–1213. doi: 10.1038/2261211a0. [DOI] [PubMed] [Google Scholar]
  36. Van Beveren C., Goddard J. G., Berns A., Verma I. M. Structure of Moloney murine leukemia viral DNA: nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3307–3311. doi: 10.1073/pnas.77.6.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Van Beveren C., van Straaten F., Galleshaw J. A., Verma I. M. Nucleotide sequence of the genome of a murine sarcoma virus. Cell. 1981 Nov;27(1 Pt 2):97–108. doi: 10.1016/0092-8674(81)90364-0. [DOI] [PubMed] [Google Scholar]
  38. Wang L. H., Duesberg P. H. DNA polymerase of murine sarcoma-leukemia virus: lack of detectable RNase H and low activity with viral RNA and natural DNA templates. J Virol. 1973 Dec;12(6):1512–1521. doi: 10.1128/jvi.12.6.1512-1521.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Watanabe S., Temin H. M. Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5986–5990. doi: 10.1073/pnas.79.19.5986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wilhelmsen K. C., Eggleton K., Temin H. M. Nucleic acid sequences of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the proto-oncogene c-rel. J Virol. 1984 Oct;52(1):172–182. doi: 10.1128/jvi.52.1.172-182.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yamamoto T., Jay G., Pastan I. Unusual features in the nucleotide sequence of a cDNA clone derived from the common region of avian sarcoma virus messenger RNA. Proc Natl Acad Sci U S A. 1980 Jan;77(1):176–180. doi: 10.1073/pnas.77.1.176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yoshimura F. K., Weinberg R. A. Restriction endonuclease cleavage of linear and closed circular murine leukemia viral DNAs: discovery of a smaller circular form. Cell. 1979 Feb;16(2):323–332. doi: 10.1016/0092-8674(79)90009-6. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES