Abstract
The reading frame of the HIV-1 pol gene, encoding protease (PR) and reverse transcriptase (RT), including RNase H as well as integrase, was fused to the bacterialbeta-galactosidase gene and overexpressed in Escherichia coli cells. The resulting fusion protein was cleaved autocatalytically leading to PR, RT and integrase. Immunoprecipitations of bacterial crude extracts with anti-RT antibodies precipitated both RT and PR. Co-precipitation of PR and RT was also observed with anti-PR antibodies, strongly suggesting a physical interaction between fully processed RT and PR within the bacterial cell. Physical interactions were confirmed with purified components by means of an ELISA assay. Furthermore, purified PR inhibited the DNA synthesis activity of purified RT, while its RNase H activity remained unaffected. The type of inhibition was uncompetitive with respect to poly(rA).oligo(dT); the inhibition constant was 50-100 nM. A possible physiological significance of this type of interaction is discussed.
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- Arrigo S. J., Huffman K. Potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication by inducible expression of HIV-1 PR multimers. J Virol. 1995 Oct;69(10):5988–5994. doi: 10.1128/jvi.69.10.5988-5994.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Austermann S., Kruhøffer M., Grosse F. Inhibition of human immunodeficiency virus type 1 reverse transcriptase by 3'-blocked oligonucleotide primers. Biochem Pharmacol. 1992 Jun 23;43(12):2581–2589. doi: 10.1016/0006-2952(92)90147-b. [DOI] [PubMed] [Google Scholar]
- Baier M., Behr E., Kurth R., König H. Cloning and expression of the complete SIVagm pol region in E. coli. Purification and partial characterization of the reverse transcriptase. Arzneimittelforschung. 1990 Nov;40(11):1284–1287. [PubMed] [Google Scholar]
- Bauer G., Temin H. M. Radioimmunological comparison of the DNA polymerases of avian retroviruses. J Virol. 1980 Mar;33(3):1046–1057. doi: 10.1128/jvi.33.3.1046-1057.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dahbo Y., Eriksson S. On the mechanism of deoxyribonucleoside toxicity in human T-lymphoblastoid cells. Reversal of growth inhibition by addition of cytidine. Eur J Biochem. 1985 Aug 1;150(3):429–434. doi: 10.1111/j.1432-1033.1985.tb09038.x. [DOI] [PubMed] [Google Scholar]
- Debouck C., Gorniak J. G., Strickler J. E., Meek T. D., Metcalf B. W., Rosenberg M. Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8903–8906. doi: 10.1073/pnas.84.24.8903. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dornreiter I., Höss A., Arthur A. K., Fanning E. SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha. EMBO J. 1990 Oct;9(10):3329–3336. doi: 10.1002/j.1460-2075.1990.tb07533.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goobar-Larsson L., Larsson P. T., Debouck C., Towler E. M. HIV-1 RT enhances the activity of a tethered dimer of HIV-1 proteinase. Biochem Biophys Res Commun. 1996 Mar 7;220(1):203–207. doi: 10.1006/bbrc.1996.0381. [DOI] [PubMed] [Google Scholar]
- Goobar-Larsson L., Luukkonen B. G., Unge T., Schwartz S., Utter G., Strandberg B., Oberg B. Enhancement of HIV-1 proteinase activity by HIV-1 reverse transcriptase. Virology. 1995 Jan 10;206(1):387–394. doi: 10.1016/s0042-6822(95)80054-9. [DOI] [PubMed] [Google Scholar]
- Gowda S. D., Stein B. S., Engleman E. G. Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus. J Biol Chem. 1989 May 25;264(15):8459–8462. [PubMed] [Google Scholar]
- Hagemeier A., Grosse F. A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA-polymerase-alpha-primase complex. Eur J Biochem. 1989 Nov 20;185(3):621–628. doi: 10.1111/j.1432-1033.1989.tb15158.x. [DOI] [PubMed] [Google Scholar]
- Heidenreich O., Kruhøffer M., Grosse F., Eckstein F. Inhibition of human immunodeficiency virus 1 reverse transcriptase by 3'-azidothymidine triphosphate. Eur J Biochem. 1990 Sep 24;192(3):621–625. doi: 10.1111/j.1432-1033.1990.tb19268.x. [DOI] [PubMed] [Google Scholar]
- Kaplan A. H., Swanstrom R. Human immunodeficiency virus type 1 Gag proteins are processed in two cellular compartments. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4528–4532. doi: 10.1073/pnas.88.10.4528. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kramer R. A., Schaber M. D., Skalka A. M., Ganguly K., Wong-Staal F., Reddy E. P. HTLV-III gag protein is processed in yeast cells by the virus pol-protease. Science. 1986 Mar 28;231(4745):1580–1584. doi: 10.1126/science.2420008. [DOI] [PubMed] [Google Scholar]
- Kräusslich H. G., Schneider H., Zybarth G., Carter C. A., Wimmer E. Processing of in vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV) type 1 by HIV proteinase generated in Escherichia coli. J Virol. 1988 Nov;62(11):4393–4397. doi: 10.1128/jvi.62.11.4393-4397.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Lori F., di Marzo Veronese F., de Vico A. L., Lusso P., Reitz M. S., Jr, Gallo R. C. Viral DNA carried by human immunodeficiency virus type 1 virions. J Virol. 1992 Aug;66(8):5067–5074. doi: 10.1128/jvi.66.8.5067-5074.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Panet A., Kra-Oz Z. A competition immunoassay for characterizing the reverse transcriptase of mammalian RNA tumor viruses. Virology. 1978 Aug;89(1):95–101. doi: 10.1016/0042-6822(78)90043-0. [DOI] [PubMed] [Google Scholar]
- Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
- Scholtissek S., Grosse F. A plasmid vector system for the expression of a triprotein consisting of beta-galactosidase, a collagenase recognition site and a foreign gene product. Gene. 1988;62(1):55–64. doi: 10.1016/0378-1119(88)90579-3. [DOI] [PubMed] [Google Scholar]
- Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
- St Clair M. H., Richards C. A., Spector T., Weinhold K. J., Miller W. H., Langlois A. J., Furman P. A. 3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. Antimicrob Agents Chemother. 1987 Dec;31(12):1972–1977. doi: 10.1128/aac.31.12.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Zhang H., Bagasra O., Niikura M., Poiesz B. J., Pomerantz R. J. Intravirion reverse transcripts in the peripheral blood plasma on human immunodeficiency virus type 1-infected individuals. J Virol. 1994 Nov;68(11):7591–7597. doi: 10.1128/jvi.68.11.7591-7597.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang H., Dornadula G., Pomerantz R. J. Endogenous reverse transcription of human immunodeficiency virus type 1 in physiological microenviroments: an important stage for viral infection of nondividing cells. J Virol. 1996 May;70(5):2809–2824. doi: 10.1128/jvi.70.5.2809-2824.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang H., Zhang Y., Spicer T. P., Abbott L. Z., Abbott M., Poiesz B. J. Reverse transcription takes place within extracellular HIV-1 virions: potential biological significance. AIDS Res Hum Retroviruses. 1993 Dec;9(12):1287–1296. doi: 10.1089/aid.1993.9.1287. [DOI] [PubMed] [Google Scholar]
- Zybarth G., Kräusslich H. G., Partin K., Carter C. Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain. J Virol. 1994 Jan;68(1):240–250. doi: 10.1128/jvi.68.1.240-250.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]