ROLES OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NUCLEOCAPSID PROTEININ ANNEALING AND INITIATION VERSUS ELONGATION IN REVERSE TRANSCRIPTION OF VIRAL NEGATIVE-STRAND STRONG-STOP DNA

To study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein (NC7) to a nneal tRNA(3)(Lys) primer onto viral genomic RNA and have then elimina ted NC7 from this primer-template complex by digestion with proteinase K and phenol-chloroform extraction of residual protein. Our data show that saturating concentrations of NC7 resulted in the formation of an active tRNA-template complex that yielded enhanced production of full length negative-strand strong-stop DNA [(-)ssDNA] and that this compl ex remained active even after the elimination of NC7. While both of th e two Zn finger motifs found within NC7 were essential for efficient e longation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn fingers yielded primer-template c omplexes that could still be initiated in 1-base-extension assays. In contrast, the use of heat annealing to produce primer-template complex es resulted in proportions of full-length (-)ssDNA lower than those se en with NC protein, and the addition of NC protein to such preformed p rimer-template complexes was able to reverse this defect only to a mar ginal extent.