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 conve rsion of viral RNA to double-stranded DNA. To better understand the mo lecular mechanisms of this process, we examined viral DNA synthesis in a simple cell-free system that uses the activities of HIV-1 reverse t ranscriptase to convert regions of single-stranded HIV-1 RNA to double -stranded DNA in a single incubation. This system recapitulated severa l of the required intermediate steps of viral DNA synthesis: RNA-templ ated 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 bath in the cell-free system and in cul tured virus. Direct comparison of viral and cell-free products reveale d differences in the precision and selectivity of plus-strand initiati on, suggesting that the cell-free system lacks one or more essential r eplication components. These studies provide clues about mechanisms of plus-strand initiation and serve as a starting point for the developm ent of more complex multicomponent cell-free systems.