Discontinuous Plus-strand DNA Synthesis in Human Immunodeficiency Virus Type 1-infected Cells and in a Partially Reconstituted Cell-free System

Overview

Journal J Virol

Publisher American Society for Microbiology

Date 1997 Nov 26

PMID 9371584

Citations 18

Authors

G J Klarmann

H Yu

X Chen

J P Dougherty

B D Preston

Affiliations

Soon will be listed here.

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.

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