Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type 1-infected cells and in a partially reconstituted cell-free system.
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.
Klarmann
G J
GJ
Department of Biochemistry, Eccles Institute of Human Genetics and the Huntsman Cancer Institute, University of Utah, Salt Lake City 84112-5330, USA.
Yu
H
H
Chen
X
X
Dougherty
J P
JP
Preston
B D
BD
D001665:Binding Sites; D002474:Cell-Free System; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans
D004277:DNA, Single-Stranded; D004279:DNA, Viral