MINIMAL SEQUENCE REQUIREMENTS OF A FUNCTIONAL HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PRIMER BINDING-SITE

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3)(LYS) primer bound n ear the 5' end of the genomic RNA at a position termed the primer bind ing site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 geno me which is complementary to the 3'-terminal 18 nucleotides of the tRN A(3)(Lys). To investigate the sequence specificity of the interaction between tRNA(3)(Lys) and the PBS, we have constructed proviral genomes containing mutations in the PBS region. A mutant PBS was constructed in which the 18 nucleotides complementary to tRNA(3)(Lys) were substit uted with 18 nucleotides predicted to be complementary to the 3'-termi nal bases of a tRNA(Phe) molecule [pHXB2PBS(phe)]. A second proviral g enome was constructed in which the PBS complementary to tRNA(Phe) was changed such that the first six nucleotides correspond to the wild-typ e PBS [pHXB2PBS(pheC)]. In all models of reverse transcription, the co mplementarity between the minus- and plus-strand PBS DNA facilitates t he template switch and elongation Of plus-strand DNA, resulting in a c omplete proviral genome. To test this model, we have inserted a five-n ucleotide sequence 6 bp 3' of the mutant PBSs, which corresponds to th e last five nucleotides of the wild-type PBSs [pHXB2PBS(phe+5) and pHX B2PBS(pheC+5)]. Transfection of plasmids containing the wild-type or m utant proviral genomes into COS-1 cells resulted in similar levels of intracellular expression of HIV-1 gag and env gene products as determi ned by immunoprecipitation with sera from AIDS patients and release of virus as determined by p24 assay. Transfection of pHXB2PBS(phe) or pH XB2PBS(phe+5) did not result in the production of infectious virus, wh ile replication-competent viruses from cells transfected with pHXB2PBS (pheC) were detected very infrequently. Transfection of pHXB2PBS(pheC5), however, consistently resulted in the production of infectious vir us, although the appearance of the virus was delayed compared with tho se from cells transfected with pHXB2(wild type). Reinfection of SupT1 cells with equal amounts of p24 antigen resulted in similar kinetics o f replication. PCR was used to amplify the PBS, and individual DNA pro ducts were subcloned into M13mp18. Sequence analysis of the PBS region of integrated proviruses derived from transfection of pHXB2PBS(pheC+5 ) revealed that the 18-nucleotide PBS complementary to tRNA(3)(Lys) wa s regenerated with a deletion of 6 bp 3' to the PBS region in all phag e clones examined. These results demonstrate that a minimum of six nuc leotides in the PBS are required for the initiation of reverse transcr iption and that complementarity between the minus-strand DNA and plus- strand DNA PBSs facilitates the completion of reverse transcription of the viral genome.