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. 1988 Oct;62(10):3662–3667. doi: 10.1128/jvi.62.10.3662-3667.1988

Characterization of human immunodeficiency virus type 1 reverse transcriptase by using monoclonal antibodies: role of the C terminus in antibody reactivity and enzyme function.

M Tisdale 1, P Ertl 1, B A Larder 1, D J Purifoy 1, G Darby 1, K L Powell 1
PMCID: PMC253508  PMID: 2458486

Abstract

We describe the production of eight monoclonal antibodies reactive with human immunodeficiency virus type 1 reverse transcriptase (RT) by immunization of mice with purified recombinant RT. These antibodies were found to react with one or the other of two regions of the enzyme and were found to be useful in immunodeficiency purification of large amounts of the enzyme. One epitope located at the C terminus of the enzyme was of particular interest, since it was present in only the larger, 66-kilodalton (kDa) RT species and not its smaller, 51-kDa counterpart. To define this epitope, a series of mutants was made which synthesized C-terminally truncated RT. These mutants indicated that the same region of the enzyme, when deleted, both removed the C-terminal epitope and drastically reduced RT activity, indicating the importance of this region in the function of the enzyme; however, even the 51-kDa enzyme component had demonstrable activity.

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Selected References

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  1. Banks L. M., Vaughan P. J., Meredith D., Powell K. L. Monoclonal antibodies to herpes simplex virus thymidine kinase. J Gen Virol. 1984 Sep;65(Pt 9):1625–1630. doi: 10.1099/0022-1317-65-9-1625. [DOI] [PubMed] [Google Scholar]
  2. Banks L., Purifoy D. J., Hurst P. F., Killington R. A., Powell K. L. Herpes simplex virus non-structural proteins. IV. Purification of the virus-induced deoxyribonuclease and characterization of the enzyme using monoclonal antibodies. J Gen Virol. 1983 Oct;64(Pt 10):2249–2260. doi: 10.1099/0022-1317-64-10-2249. [DOI] [PubMed] [Google Scholar]
  3. Butterworth B. E., Hall L., Stoltzfus C. M., Rueckert R. R. Virus-specific proteins synthesized in encephalomyocarditis virus-infected HeLa cells. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3083–3087. doi: 10.1073/pnas.68.12.3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chandra A., Gerber T., Kaul S., Wolf C., Demirhan I., Chandra P. Serological relationship between reverse transcriptases from human T-cell lymphotropic viruses defined by monoclonal antibodies. Evidence for two forms of reverse transcriptases in the AIDS-associated virus, HTLV-III/LAV. FEBS Lett. 1986 May 12;200(2):327–332. doi: 10.1016/0014-5793(86)81162-0. [DOI] [PubMed] [Google Scholar]
  5. Chatterjee R., Rinaldo C. R., Jr, Gupta P. Immunogenicity of human immunodeficiency virus (HIV) reverse transcriptase: detection of high levels of antibodies to HIV reverse transcriptase in sera of homosexual men. J Clin Immunol. 1987 May;7(3):218–224. doi: 10.1007/BF00915727. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  9. Köhler G., Howe S. C., Milstein C. Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. Eur J Immunol. 1976 Apr;6(4):292–295. doi: 10.1002/eji.1830060411. [DOI] [PubMed] [Google Scholar]
  10. Larder B. A., Purifoy D. J., Powell K. L., Darby G. Site-specific mutagenesis of AIDS virus reverse transcriptase. 1987 Jun 25-Jul 1Nature. 327(6124):716–717. doi: 10.1038/327716a0. [DOI] [PubMed] [Google Scholar]
  11. Larder B., Purifoy D., Powell K., Darby G. AIDS virus reverse transcriptase defined by high level expression in Escherichia coli. EMBO J. 1987 Oct;6(10):3133–3137. doi: 10.1002/j.1460-2075.1987.tb02623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laurence J., Saunders A., Kulkosky J. Characterization and clinical association of antibody inhibitory to HIV reverse transcriptase activity. Science. 1987 Mar 20;235(4795):1501–1504. doi: 10.1126/science.2435004. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Michael N. L., Rothbard J. B., Shiurba R. A., Linke H. K., Schoolnik G. K., Clayton D. A. All eight unassigned reading frames of mouse mitochondrial DNA are expressed. EMBO J. 1984 Dec 20;3(13):3165–3175. doi: 10.1002/j.1460-2075.1984.tb02275.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Powell K. L., Courtney R. J. Polypeptide synthesized in herpes simplex virus type 2-infected HEp-2 cells. Virology. 1975 Jul;66(1):217–228. doi: 10.1016/0042-6822(75)90192-0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]

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