Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type 1-infected cells and in a partially reconstituted cell-free system

George J. Klarmann, Hong Yu, Xin Chen, Joseph P. Dougherty, Bradley D. Preston

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

Human immunodeficiency virus type 1 (HIV-1) replication requires conversion of viral RNA to double-stranded DNA. To better understand the molecular mechanisms of this process, we examined viral DNA synthesis in a simple cell-free system that uses the activities of HIV-1 reverse transcriptase to convert regions of single-stranded HIV-1 RNA to double- stranded DNA in a single incubation. This system recapitulated several of the required intermediate steps of viral DNA synthesis: RNA-templated minus- strand polymerization, preferential plus-strand initiation at the central and 3' HIV-1 polypurine tracts, and DNA-templated plus-strand polymerization. Secondary sites of plus-strand initiation were also observed at low frequency both in the cell-free system and in cultured virus. Direct comparison of viral and cell-free products revealed differences in the precision and selectivity of plus-strand initiation, suggesting that the cell-free system lacks one or more essential replication components. These studies provide clues about mechanisms of plus-strand initiation and serve as a starting point for the development of more complex multicomponent cell-free systems.

Original languageEnglish (US)
Pages (from-to)9259-9269
Number of pages11
JournalJournal of virology
Volume71
Issue number12
DOIs
StatePublished - Dec 1997
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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