Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1987 Sep;61(9):2843–2851. doi: 10.1128/jvi.61.9.2843-2851.1987

The role of Moloney murine leukemia virus RNase H activity in the formation of plus-strand primers.

A J Rattray, J J Champoux
PMCID: PMC255801  PMID: 3039172

Abstract

On the basis of earlier studies with both detergent-disrupted virions (the endogenous reaction) and an in vitro reconstructed reaction, the RNase H activity associated with Moloney murine leukemia virus reverse transcriptase has been implicated in the generation of plus-strand RNA primers during reverse transcription. Here we used an oligonucleotide extension assay to show that the RNA primers remaining bound to the plus DNA strands initiated at the normal origin in the in vitro reaction are heterogeneous in length. This result indicates that, although a precise cleavage generates the 3' end of the priming RNA, RNase H exhibits less specificity at other break sites. During the endogenous reaction, a kinetic analysis of the synthesis of plus strands corresponding to different regions of the genome suggested that additional sites for the initiation of plus-strand DNA existed upstream of the normal origin. Direct analysis of fragments produced in the endogenous reaction, as well as in the in vitro reaction, confirmed the existence of upstream plus-strand initiation sites. Several of these sites were mapped to the nucleotide level by the oligonucleotide extension method. A comparison of the nucleotide sequences surrounding the upstream initiation sites with the sequence at the normal plus-strand origin revealed common features, which suggests a mechanism for plus-strand priming based on sequence recognition by the RNase H/reverse transcriptase protein. Although primer removal by RNase H is highly efficient for DNA fragments initiated at the normal origin, the RNA primers were inefficiently removed from the fragments initiated at the upstream sites. This result suggests that primer removal, like primer generation, involves sequence recognition by the enzyme.

Full text

PDF
2843

Images in this article

2847Image
on p.2847
2847Image
on p.2847
2848Image
on p.2848

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. Baltimore D., Smoler D. F. Association of an endoribonuclease with the avian myeloblastosis virus deoxyribonucleic acid polymerase. J Biol Chem. 1972 Nov 25;247(22):7282–7287. [PubMed] [Google Scholar]
  3. Been M. D., Champoux J. J. Cutting of M13mp7 phage DNA and excision of cloned single-stranded sequences by restriction endonucleases. Methods Enzymol. 1983;101:90–98. doi: 10.1016/0076-6879(83)01007-1. [DOI] [PubMed] [Google Scholar]
  4. Boone L. R., Skalka A. M. Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. I. Kinetics of synthesis and size of minus- and plus-strand transcripts. J Virol. 1981 Jan;37(1):109–116. doi: 10.1128/jvi.37.1.109-116.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boone L. R., Skalka A. M. Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. II. Evidence for a strand displacement mechanism in plus-strand synthesis. J Virol. 1981 Jan;37(1):117–126. doi: 10.1128/jvi.37.1.117-126.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Correct integration of retroviral DNA in vitro. Cell. 1987 May 8;49(3):347–356. doi: 10.1016/0092-8674(87)90287-x. [DOI] [PubMed] [Google Scholar]
  7. Champoux J. J. Efficient misincorporation by avian myeloblastosis virus reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate. J Mol Appl Genet. 1984;2(5):454–464. [PubMed] [Google Scholar]
  8. Champoux J. J., Gilboa E., Baltimore D. Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase. J Virol. 1984 Mar;49(3):686–691. doi: 10.1128/jvi.49.3.686-691.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collett M. S., Dierks P., Parsons J. T., Faras A. J. RNase H hydrolysis of the 5' terminus of the avian sarcoma virus genome during reverse transcription. Nature. 1978 Mar 9;272(5649):181–184. doi: 10.1038/272181a0. [DOI] [PubMed] [Google Scholar]
  10. Darlix J. L., Bromley P. A., Spahr P. F. Extensive in vitro transcription of rous sarcoma virus RNA by avian myeloblastosis virus DNA polymerase and concurrent activation of the associated RNase H. J Virol. 1977 Sep;23(3):659–668. doi: 10.1128/jvi.23.3.659-668.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeLorbe W. J., Luciw P. A., Goodman H. M., Varmus H. E., Bishop J. M. Molecular cloning and characterization of avian sarcoma virus circular DNA molecules. J Virol. 1980 Oct;36(1):50–61. doi: 10.1128/jvi.36.1.50-61.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Finston W. I., Champoux J. J. RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro. J Virol. 1984 Jul;51(1):26–33. doi: 10.1128/jvi.51.1.26-33.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Friedrich R., Moelling K. Effect of viral RNase H on the avian sarcoma viral genome during early transcription in vitro. J Virol. 1979 Sep;31(3):630–638. doi: 10.1128/jvi.31.3.630-638.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gerard G. F. Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I). Biochemistry. 1981 Jan 20;20(2):256–265. doi: 10.1021/bi00505a005. [DOI] [PubMed] [Google Scholar]
  15. Gilboa E., Goff S., Shields A., Yoshimura F., Mitra S., Baltimore D. In vitro synthesis of a 9 kbp terminally redundant DNA carrying the infectivity of Moloney murine leukemia virus. Cell. 1979 Apr;16(4):863–874. doi: 10.1016/0092-8674(79)90101-6. [DOI] [PubMed] [Google Scholar]
  16. Gilboa E., Mitra S. W., Goff S., Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. doi: 10.1016/0092-8674(79)90357-x. [DOI] [PubMed] [Google Scholar]
  17. Grandgenett D. P., Gerard G. F., Green M. Ribonuclease H: a ubiquitous activity in virions of ribonucleic acid tumor viruses. J Virol. 1972 Dec;10(6):1136–1142. doi: 10.1128/jvi.10.6.1136-1142.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hsu T. W., Sabran J. L., Mark G. E., Guntaka R. V., Taylor J. M. Analysis of unintegrated avian RNA tumor virus double-stranded DNA intermediates. J Virol. 1978 Dec;28(3):810–818. doi: 10.1128/jvi.28.3.810-818.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hsu T. W., Taylor J. M. Single-stranded regions on unintegrated avian retrovirus DNA. J Virol. 1982 Oct;44(1):47–53. doi: 10.1128/jvi.44.1.47-53.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kotewicz M. L., D'Alessio J. M., Driftmier K. M., Blodgett K. P., Gerard G. F. Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli. Gene. 1985;35(3):249–258. doi: 10.1016/0378-1119(85)90003-4. [DOI] [PubMed] [Google Scholar]
  21. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  22. Mitra S. W., Chow M., Champoux J., Baltimore D. Synthesis of murine leukemia virus plus strong stop DNA initiates at a unique site. J Biol Chem. 1982 Jun 10;257(11):5983–5986. [PubMed] [Google Scholar]
  23. Mitra S. W., Goff S., Gilboa E., Baltimore D. Synthesis of a 600-nucleotide-long plus-strand DNA by virions of Moloney murine leukemia virus. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4355–4359. doi: 10.1073/pnas.76.9.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mizutani S., Temin H. M., Kodama M., Wells R. T. DNA ligase and exonuclease activities in virions of rous sarcoma virus. Nat New Biol. 1971 Apr 21;230(16):232–235. doi: 10.1038/newbio230232a0. [DOI] [PubMed] [Google Scholar]
  25. Montgomery D. L., Hall B. D., Gillam S., Smith M. Identification and isolation of the yeast cytochrome c gene. Cell. 1978 Jul;14(3):673–680. doi: 10.1016/0092-8674(78)90250-7. [DOI] [PubMed] [Google Scholar]
  26. Olsen J. C., Watson K. F. Reverse transcription of avian myeloblastosis virus 35S RNA. Early synthesis of plus strand DNA of discrete size in reconstructed reactions. Nucleic Acids Res. 1982 Feb 11;10(3):1009–1027. doi: 10.1093/nar/10.3.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Omer C. A., Resnick R., Faras A. J. Evidence for involvement of an RNA primer in initiation of strong-stop plus DNA synthesis during reverse transcription in vitro. J Virol. 1984 May;50(2):465–470. doi: 10.1128/jvi.50.2.465-470.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Resnick R., Omer C. A., Faras A. J. Involvement of retrovirus reverse transcriptase-associated RNase H in the initiation of strong-stop (+) DNA synthesis and the generation of the long terminal repeat. J Virol. 1984 Sep;51(3):813–821. doi: 10.1128/jvi.51.3.813-821.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rothenberg E., Donoghue D. J., Baltimore D. Analysis of a 5' leader sequence on murine leukemia virus 21S RNA: heteroduplex mapping with long reverse transcriptase products. Cell. 1978 Mar;13(3):435–451. doi: 10.1016/0092-8674(78)90318-5. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  35. Smith J. K., Cywinski A., Taylor J. M. Initiation of plus-strand DNA synthesis during reverse transcription of an avian retrovirus genome. J Virol. 1984 Jan;49(1):200–204. doi: 10.1128/jvi.49.1.200-204.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sorge J., Hughes S. H. Polypurine tract adjacent to the U3 region of the Rous sarcoma virus genome provides a cis-acting function. J Virol. 1982 Aug;43(2):482–488. doi: 10.1128/jvi.43.2.482-488.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Staskus K. A., Collett M. S., Faras A. J. Initiation of DNA synthesis by the avian oncornavirus RNA-directed DNA polymerase: structural and functional localization of the major species of primer RNA on the oncornavirus genome. Virology. 1976 May;71(1):162–168. doi: 10.1016/0042-6822(76)90102-1. [DOI] [PubMed] [Google Scholar]
  38. Taylor J. M., Cywinski A., Smith J. K. Discontinuities in the DNA synthesized by an avian retrovirus. J Virol. 1983 Dec;48(3):654–659. doi: 10.1128/jvi.48.3.654-659.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Taylor J. M., Illmensee R. Site on the RNA of an avian sarcoma virus at which primer is bound. J Virol. 1975 Sep;16(3):553–558. doi: 10.1128/jvi.16.3.553-558.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Varmus H. E. Form and function of retroviral proviruses. Science. 1982 May 21;216(4548):812–820. doi: 10.1126/science.6177038. [DOI] [PubMed] [Google Scholar]
  43. Varmus H. E., Heasley S., Kung H. J., Oppermann H., Smith V. C., Bishop J. M., Shank P. R. Kinetics of synthesis, structure and purification of avian sarcoma virus-specific DNA made in the cytoplasm of acutely infected cells. J Mol Biol. 1978 Mar 25;120(1):55–82. doi: 10.1016/0022-2836(78)90295-4. [DOI] [PubMed] [Google Scholar]
  44. Verma I. M., Meuth N. L., Baltimore D. Covalent linkage between ribonucleic Acid primer and deoxyribonucleic Acid product of the avian myeloblastosis virus deoxyribonucleic Acid polymerase. J Virol. 1972 Oct;10(4):622–627. doi: 10.1128/jvi.10.4.622-627.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Verma I. M. Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. J Virol. 1975 Apr;15(4):843–854. doi: 10.1128/jvi.15.4.843-854.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES