The effect of mutations in the HIV-1 nucleocapsid protein on strand transfer in cell-free reverse transcription reactions

Interactions between the nucleocapsid protein (NC) and reverse transcriptas e of HIV-1 have been shown to promote the initiation of reverse transcripti on. We assayed the effect of NC on later events, using a strand transfer sy stem with donor and acceptor HIV RNA templates and found that the presence of NC resulted in increased synthesis of full-length strand-transferred (FL ST) DNA. This effect also occurred with mutated forms of NC that lacked bot h zinc fingers, or that contained a point mutation (histidine-->cysteine) a t amino acid 23, In contrast, NC-derived proteins containing only the proxi mal or distal zinc fingers, or lacking the N- and C-termini, were all unabl e to catalyze the synthesis of FLST DNA, Band-shift assays using both the m utated and wild-type forms of these proteins revealed that all the NC prote ins promoted strand association between (-) strong-stop DNA [(-)ssDNA] and acceptor RNA. The zinc finger motifs were dispensable for full-length proce ssive reverse transcription, and the N- and C-termini were required; howeve r, all NC domains were dispensable for association of (-)ssDNA and acceptor RNA. This suggests that annealing is a less stringent reaction than DNA po lymerization.