Nucleic Acids Research, 2000, Vol. 28, No. 8 1724-1729
© 2000 Oxford University Press
The effect of mutations in the HIV-1 nucleocapsid protein on strand transfer in cell-free reverse transcription reactions
1McGill University AIDS Centre, Jewish General Hospital, Montréal, Canada, 2Department of Microbiology and Immunology and 3Department of Medicine, McGill University, Montréal, Canada and 4Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM-UMR 8600 CNRS, UFR des Sciences Pharmaceutiques et Biologiques 4, 75270 Paris Cedex 06, France
Interactions between the nucleocapsid protein (NC) and reverse transcriptase of HIV-1 have been shown to promote the initiation of reverse transcription. We assayed the effect of NC on later events, using a strand transfer system with donor and acceptor HIV RNA templates and found that the presence of NC resulted in increased synthesis of full-length strand-transferred (FLST) DNA. This effect also occurred with mutated forms of NC that lacked both zinc fingers, or that contained a point mutation (histidine
cysteine) at amino acid 23. In contrast, NC-derived proteins containing only the proximal or distal zinc fingers, or lacking the N- and C-termini, were all unable to catalyze the synthesis of FLST DNA. Band-shift assays using both the mutated and wild-type forms of these proteins revealed that all the NC proteins promoted strand association between (–) strong-stop DNA [(–)ssDNA] and acceptor RNA. The zinc finger motifs were dispensable for full-length processive reverse transcription, and the N- and C-termini were required; however, all NC domains were dispensable for association of (–)ssDNA and acceptor RNA. This suggests that annealing is a less stringent reaction than DNA polymerization.
* To whom correspondence should be addressed at: McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, 3755 Chemin Côte Ste. Catherine, Montréal, Québec H3T 1E2, Canada. Tel: +1 514 340 8260; Fax: +1 514 340 7537; Email: mdwa@musica.mcgill.ca
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