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 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.
Hsu
M
M
McGill University AIDS Centre, Jewish General Hospital, Montréal, Canada.
Rong
L
L
de Rocquigny
H
H
Roques
B P
BP
Wainberg
M A
MA
D002474:Cell-Free System; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D019590:Nucleocapsid Proteins / Q000235:genetics*; D017354:Point Mutation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D016335:Zinc Fingers
D004277:DNA, Single-Stranded; D019590:Nucleocapsid Proteins; D012367:RNA, Viral