Mapping domains of retroviral integrase responsible for viral DNA specificity and target site selection by analysis of chimeras between human immunodeficiency virus type 1 and visna virus integrases

Michael Katzman, Malgorzata Sudol

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59 Scopus citations

Abstract

Human immunodeficiency virus type 1 (HIV-1) and visna virus integrases were purified from a bacterial expression system and assayed on oligonucleotide substrates derived from each terminus of human immunodeficiency virus type 1 and visna virus linear DNA. Three differences between the proteins were identified, including levels of specific 3'-end processing, patterns of strand transfer, and target site preferences. To map domains of integrase (IN) responsible for viral DNA specificity and target site selection, we constructed and purified chimeric proteins in which the N- terminal, central, and C-terminal regions of these lentiviral integrases were exchanged. All six chimeric proteins were active for disintegration, demonstrating that the active site in the central region of each chimera maintained a functional conformation. Analysis of endonucleolytic processing activity indicated that the N terminus of IN does not contribute to viral DNA specificity; this function must reside in the central region or C terminus of IN. In the viral DNA integration assay, chimeric proteins gave novel patterns of strand transfer products which did not match that of either wild-type IN. Thus, target site selection with a viral DNA terminus as nucleophile could not be mapped to regions of IN defined by these boundaries and may involve interactions between regions. In contrast, when target site preferences were monitored with a new assay in which glycerol stimulates IN-mediated cleavage of nonviral DNA, chimeras clearly segregated between the two wild-type patterns. Target site selection for this nonspecific alcoholysis activity mapped to the central region of IN. This report represents the first detailed description of functional chimeras between any two retroviral integrases.

Original languageEnglish (US)
Pages (from-to)5687-5696
Number of pages10
JournalJournal of virology
Volume69
Issue number9
DOIs
StatePublished - Sep 1995

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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