Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 Nov;12(11):4433–4438. doi: 10.1002/j.1460-2075.1993.tb06128.x

Two defective forms of reverse transcriptase can complement to restore retroviral infectivity.

A Telesnitsky 1, S P Goff 1
PMCID: PMC413742  PMID: 7693456

Abstract

Retroviral DNA synthesis requires both the DNA polymerase and the RNaseH activities of reverse transcriptase (RT). To test whether two defective RTs--one carrying a mutation in the RNaseH domain and the other with a mutation in DNA polymerase--could work together to complete viral DNA synthesis, we generated phenotypically mixed virions of Moloney murine leukemia virus (M-MuLV) that contained two kinds of mutant RTs. One RNaseH catalytic site mutant complemented both tested DNA polymerase mutants and small amounts of intact viral DNA were generated. This demonstrates that retroviral DNA synthesis can be completed--albeit inefficiently--when DNA polymerase and RNaseH activities are provided by separate RT molecules. Other RNaseH mutants failed to complement, suggesting that some aspects of the RNaseH domain are essential to RT's DNA polymerase function. Phenotypically mixed virions were also used to demonstrate that RT and integrase (IN) can be provided by separate polyprotein precursors and complete the early stages of retroviral replication.

Full text

PDF
4438

Images in this article

4435Image
on p.4435
4436Image
on p.4436

Selected References

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

  1. Colicelli J., Goff S. P. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. doi: 10.1016/0022-2836(88)90378-6. [DOI] [PubMed] [Google Scholar]
  2. DeStefano J. J., Buiser R. G., Mallaber L. M., Myers T. W., Bambara R. A., Fay P. J. Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. J Biol Chem. 1991 Apr 25;266(12):7423–7431. [PubMed] [Google Scholar]
  3. Doolittle R. F., Feng D. F., Johnson M. S., McClure M. A. Origins and evolutionary relationships of retroviruses. Q Rev Biol. 1989 Mar;64(1):1–30. doi: 10.1086/416128. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Goff S. P. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr. 1990;3(8):817–831. [PubMed] [Google Scholar]
  6. Goff S., Traktman P., Baltimore D. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J Virol. 1981 Apr;38(1):239–248. doi: 10.1128/jvi.38.1.239-248.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Granowitz C., Colicelli J., Goff S. P. Analysis of mutations in the envelope gene of Moloney murine leukemia virus: separation of infectivity from superinfection resistance. Virology. 1991 Aug;183(2):545–554. doi: 10.1016/0042-6822(91)90983-i. [DOI] [PubMed] [Google Scholar]
  8. Hartman S. C., Mulligan R. C. Two dominant-acting selectable markers for gene transfer studies in mammalian cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8047–8051. doi: 10.1073/pnas.85.21.8047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  10. Hostomsky Z., Hostomska Z., Fu T. B., Taylor J. Reverse transcriptase of human immunodeficiency virus type 1: functionality of subunits of the heterodimer in DNA synthesis. J Virol. 1992 May;66(5):3179–3182. doi: 10.1128/jvi.66.5.3179-3182.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hostomsky Z., Hostomska Z., Hudson G. O., Moomaw E. W., Nodes B. R. Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1148–1152. doi: 10.1073/pnas.88.4.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hu S. C., Court D. L., Zweig M., Levin J. G. Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli. J Virol. 1986 Oct;60(1):267–274. doi: 10.1128/jvi.60.1.267-274.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hu W. S., Temin H. M. Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1556–1560. doi: 10.1073/pnas.87.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hu W. S., Temin H. M. Retroviral recombination and reverse transcription. Science. 1990 Nov 30;250(4985):1227–1233. doi: 10.1126/science.1700865. [DOI] [PubMed] [Google Scholar]
  15. Kanaya S., Kohara A., Miura Y., Sekiguchi A., Iwai S., Inoue H., Ohtsuka E., Ikehara M. Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis. J Biol Chem. 1990 Mar 15;265(8):4615–4621. [PubMed] [Google Scholar]
  16. Kati W. M., Johnson K. A., Jerva L. F., Anderson K. S. Mechanism and fidelity of HIV reverse transcriptase. J Biol Chem. 1992 Dec 25;267(36):25988–25997. [PubMed] [Google Scholar]
  17. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  18. Le Grice S. F., Naas T., Wohlgensinger B., Schatz O. Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. EMBO J. 1991 Dec;10(12):3905–3911. doi: 10.1002/j.1460-2075.1991.tb04960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lowe D. M., Parmar V., Kemp S. D., Larder B. A. Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. FEBS Lett. 1991 May 6;282(2):231–234. doi: 10.1016/0014-5793(91)80484-k. [DOI] [PubMed] [Google Scholar]
  20. Luo G. X., Taylor J. Template switching by reverse transcriptase during DNA synthesis. J Virol. 1990 Sep;64(9):4321–4328. doi: 10.1128/jvi.64.9.4321-4328.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Oyama F., Kikuchi R., Crouch R. J., Uchida T. Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease. J Biol Chem. 1989 Nov 5;264(31):18808–18817. [PubMed] [Google Scholar]
  22. Peliska J. A., Benkovic S. J. Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Science. 1992 Nov 13;258(5085):1112–1118. doi: 10.1126/science.1279806. [DOI] [PubMed] [Google Scholar]
  23. Repaske R., Hartley J. W., Kavlick M. F., O'Neill R. R., Austin J. B. Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase. J Virol. 1989 Mar;63(3):1460–1464. doi: 10.1128/jvi.63.3.1460-1464.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roth M. J., Schwartzberg P., Tanese N., Goff S. P. Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting. J Virol. 1990 Oct;64(10):4709–4717. doi: 10.1128/jvi.64.10.4709-4717.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schatz O., Mous J., Le Grice S. F. HIV-1 RT-associated ribonuclease H displays both endonuclease and 3'----5' exonuclease activity. EMBO J. 1990 Apr;9(4):1171–1176. doi: 10.1002/j.1460-2075.1990.tb08224.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schwartzberg P., Colicelli J., Goff S. P. Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection. Cell. 1984 Jul;37(3):1043–1052. doi: 10.1016/0092-8674(84)90439-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  29. Tanese N., Goff S. P. Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1777–1781. doi: 10.1073/pnas.85.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tanese N., Telesnitsky A., Goff S. P. Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function. J Virol. 1991 Aug;65(8):4387–4397. doi: 10.1128/jvi.65.8.4387-4397.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Telesnitsky A., Blain S. W., Goff S. P. Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H. J Virol. 1992 Feb;66(2):615–622. doi: 10.1128/jvi.66.2.615-622.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Telesnitsky A., Goff S. P. RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1276–1280. doi: 10.1073/pnas.90.4.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]
  34. Yang W., Hendrickson W. A., Crouch R. J., Satow Y. Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein. Science. 1990 Sep 21;249(4975):1398–1405. doi: 10.1126/science.2169648. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES