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
. 1991 Jun 15;88(12):5262–5266. doi: 10.1073/pnas.88.12.5262

The p15 carboxyl-terminal proteolysis product of the human immunodeficiency virus type 1 reverse transcriptase p66 has DNA polymerase activity.

P Hafkemeyer 1, E Ferrari 1, J Brecher 1, U Hübscher 1
PMCID: PMC51852  PMID: 1711222

Abstract

The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity. The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa. The latter has been characterized as possessing RNase H activity [Hansen, J., Schultze, T., Mellert, W. & Moelling, K. (1988) EMBO J. 7, 239-243]. We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein. On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product. The two reverse transcriptases appear to have different behaviors on various template/primer systems tested. Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication.

Full text

PDF
5262

Images in this article

5263Image
on p.5263
5265Image
on p.5265
5265Image
on p.5265
5266Image
on p.5266

Selected References

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

  1. Bernad A., Blanco L., Lázaro J. M., Martín G., Salas M. A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell. 1989 Oct 6;59(1):219–228. doi: 10.1016/0092-8674(89)90883-0. [DOI] [PubMed] [Google Scholar]
  2. Bordier B., Tarrago-Litvak L., Sallafranque-Andreola M. L., Robert D., Tharaud D., Fournier M., Barr P. J., Litvak S., Sarih-Cottin L. Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). Nucleic Acids Res. 1990 Feb 11;18(3):429–436. doi: 10.1093/nar/18.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cullen B. R., Greene W. C. Functions of the auxiliary gene products of the human immunodeficiency virus type 1. Virology. 1990 Sep;178(1):1–5. doi: 10.1016/0042-6822(90)90373-y. [DOI] [PubMed] [Google Scholar]
  5. Gassmann M., Thömmes P., Weiser T., Hübscher U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 1990 May;4(8):2528–2532. doi: 10.1096/fasebj.4.8.1970792. [DOI] [PubMed] [Google Scholar]
  6. Goulian M., Herrmann S. M., Sackett J. W., Grimm S. L. Two forms of DNA polymerase delta from mouse cells. Purification and properties. J Biol Chem. 1990 Sep 25;265(27):16402–16411. [PubMed] [Google Scholar]
  7. Hansen J., Schulze T., Mellert W., Moelling K. Identification and characterization of HIV-specific RNase H by monoclonal antibody. EMBO J. 1988 Jan;7(1):239–243. doi: 10.1002/j.1460-2075.1988.tb02805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hübscher U., Kornberg A. The delta subunit of Escherichia coli DNA polymerase III holoenzyme is the dnaX gene product. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6284–6288. doi: 10.1073/pnas.76.12.6284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Lightfoote M. M., Coligan J. E., Folks T. M., Fauci A. S., Martin M. A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McCune J. M. HIV-1: the infective process in vivo. Cell. 1991 Jan 25;64(2):351–363. doi: 10.1016/0092-8674(91)90644-e. [DOI] [PubMed] [Google Scholar]
  12. Ottiger H. P., Hübscher U. Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3993–3997. doi: 10.1073/pnas.81.13.3993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Restle T., Müller B., Goody R. S. Dimerization of human immunodeficiency virus type 1 reverse transcriptase. A target for chemotherapeutic intervention. J Biol Chem. 1990 Jun 5;265(16):8986–8988. [PubMed] [Google Scholar]
  14. 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]
  15. Spanos A., Hübscher U. Recovery of functional proteins in sodium dodecyl sulfate gels. Methods Enzymol. 1983;91:263–277. doi: 10.1016/s0076-6879(83)91024-8. [DOI] [PubMed] [Google Scholar]
  16. Starnes M. C., Cheng Y. C. Human immunodeficiency virus reverse transcriptase-associated RNase H activity. J Biol Chem. 1989 Apr 25;264(12):7073–7077. [PubMed] [Google Scholar]
  17. Thömmes P., Hübscher U. Eukaryotic DNA replication. Enzymes and proteins acting at the fork. Eur J Biochem. 1990 Dec 27;194(3):699–712. doi: 10.1111/j.1432-1033.1990.tb19460.x. [DOI] [PubMed] [Google Scholar]
  18. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Weber J., Grosse F. Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA. Nucleic Acids Res. 1989 Feb 25;17(4):1379–1393. doi: 10.1093/nar/17.4.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wilson S., Abbotts J., Widen S. Progress toward molecular biology of DNA polymerase beta. Biochim Biophys Acta. 1988 Feb 28;949(2):149–157. doi: 10.1016/0167-4781(88)90078-4. [DOI] [PubMed] [Google Scholar]
  21. di Marzo Veronese F., Copeland T. D., DeVico A. L., Rahman R., Oroszlan S., Gallo R. C., Sarngadharan M. G. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science. 1986 Mar 14;231(4743):1289–1291. doi: 10.1126/science.2418504. [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