HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NUCLEOCAPSID PROTEIN (NCP7) DIRECTS SPECIFIC INITIATION OF MINUS-STRAND DNA-SYNTHESIS PRIMED BY HUMAN TRNA(3)(LYS) IN-VITRO - STUDIES OF VIRAL-RNA MOLECULES MUTATED IN REGIONS THAT FLANK THE PRIMER BINDING-SITE

Retroviral reverse transcription starts near the 5' end of unspliced v iral RNA at a sequence called the primer binding site (PBS), where the tRNA primer anneals to the RNA template for initiation of DNA synthes is, We have investigated the roles of NCp7 in annealing of primer tRNA (3)(Lys) to the PBS and in reverse transcriptase (RT) activity, using a cell-free reverse transcription reaction mixture consisting of vario us 5' viral RNA templates, natural primer tRNA(3)(Lys) or synthetic pr imer, human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NCp7), and HIV-1 RT, In the presence of tRNA(3)(Lys), NCp7 was found to stimulate synthesis of minus-strand strong-stop DNA [(-)ssDNA], co nsistent with previous reports, However, specific DNA synthesis was ob served only at a NCp7/RNA ratio similar to that predicted to be presen t in virions. Moreover, at these concentrations, NCp7 inhibited the sy nthesis of nonspecific reverse-transcribed DNA products, which are ini tiated because of self-priming by RNA templates, In contrast to result s obtained with tRNA(3)(Lys) as primer, NCp7 inhibited the synthesis o f (-)ssDNA products primed by an 18-nucleotide (nt) ribonucleotide (rP R), complementary to the PBS, even though rPR can initiate synthesis o f such material in the absence of preannealing with NCp7, Primer place ment hand shift assays showed that NCp7 was necessary for efficient fo rmation of the tRNA-RNA complex, In contrast, NCp7 was found to preven t formation of the rPR-RNA complex, Since NCp7 appears to exert opposi te effects (annealing versus dissociation) on tRNA(3)(Lys) and rPR sub strates, the non-PBS binding regions of the tRNA(3)(Lys) molecule may play a role in the annealing of tRNA to the template, We also investig ated the roles of an A-rich loop upstream of the PBS, a 7-nt region im mediately downstream of the PBS, and a 54-nt deletion further downstre am of the PBS in interactions with tRNA(3)(Lys). We found that deletio ns in the 54-nt region that may prevent formation of the U5-leader ste m prevented tRNA(3)(Lys) placement and priming, while deletions in the A-rich loop or the 7-nt sequence had relatively minor effects in this regard.