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J. Virol., 12 1997, 9259-9269, Vol 71, No. 12
Copyright © 1997, American Society for Microbiology

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

GJ Klarmann, H Yu, X Chen, JP Dougherty and BD Preston
Department of Biochemistry, Eccles Institute of Human Genetics and the Huntsman Cancer Institute, University of Utah, Salt Lake City 84112- 5330, USA.

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.

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