Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and rnase h activities

Anne Marie Szilvay, Svanhild Nornes, Ida R. Haugan, Litta Olsen, Vinayaka R. Prasad, Curt Endresen, Stephen P. Goff, Dag E. Heiland

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

Original languageEnglish (US)
Pages (from-to)647-657
Number of pages11
JournalJournal of Acquired Immune Deficiency Syndromes
Volume5
Issue number7
StatePublished - 1992

Fingerprint

Epitope Mapping
Ribonucleases
RNA-Directed DNA Polymerase
Ribonuclease H
Monoclonal Antibodies
Epitopes
Antibody Binding Sites
Human immunodeficiency virus 1 reverse transcriptase
Hybridomas
Fluorescent Antibody Technique
Amino Acid Sequence
Immunization
Proteins
Western Blotting
HIV
Escherichia coli
Peptides
Antibodies

Keywords

  • Epitope mapping
  • HIV-1
  • Inhibition
  • Monoclonal antibodies
  • Reverse transcriptase

ASJC Scopus subject areas

  • Infectious Diseases
  • Pharmacology (medical)
  • Virology
  • Immunology and Allergy

Cite this

Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and rnase h activities. / Szilvay, Anne Marie; Nornes, Svanhild; Haugan, Ida R.; Olsen, Litta; Prasad, Vinayaka R.; Endresen, Curt; Goff, Stephen P.; Heiland, Dag E.

In: Journal of Acquired Immune Deficiency Syndromes, Vol. 5, No. 7, 1992, p. 647-657.

Research output: Contribution to journalArticle

Szilvay, AM, Nornes, S, Haugan, IR, Olsen, L, Prasad, VR, Endresen, C, Goff, SP & Heiland, DE 1992, 'Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and rnase h activities', Journal of Acquired Immune Deficiency Syndromes, vol. 5, no. 7, pp. 647-657.
Szilvay, Anne Marie ; Nornes, Svanhild ; Haugan, Ida R. ; Olsen, Litta ; Prasad, Vinayaka R. ; Endresen, Curt ; Goff, Stephen P. ; Heiland, Dag E. / Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and rnase h activities. In: Journal of Acquired Immune Deficiency Syndromes. 1992 ; Vol. 5, No. 7. pp. 647-657.
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