CONSTRUCTION OF A TYPE-1 HUMAN-IMMUNODEFICIENCY-VIRUS THAT MAINTAINS A PRIMER BINDING-SITE COMPLEMENTARY TO TRNA(HIS)

The initiation of human immunodeficiency virus type 1 reverse transcri ption occurs by extension of a tRNA(3)(Lys) primer bound near the 5' e nd of the viral RNA genome which is designated the primer binding site (PBS), Sequences within the viral genome upstream of the PBS which ar e complementary to the anticodon loop (USUU) and the T Psi C loop and arm (AGGGT(m) Psi) of tRNA(3)(Lys) are postulated to play a role in ma intaining the selective use of tRNA(3)(Lys) in reverse transcription, To investigate this possibility, proviral genomes which contain a PBS complementary to the 3'-terminal 18 nucleotides of tRNA(His) [pHXB2(Hi s)] as well as sequences upstream of this PBS which are complementary to either the anticodon loop [CCACAA; pHXB2(His-AC)] or T Psi C loop [ GACCGAGG; pHXB2(His-T Psi C)] of tRNA(His) were constructed. Infectiou s virus was recovered upon transfection into COS-1 cells of pHXB2(His) , pHXB2(His-AC), or pHXB2(His-T Psi C). The appearance of infectious v irus after cocultivation with SupT1 cells was delayed for the provirus es containing a PBS complementary to tRNA(His) compared with that obta ined by transfection of the wild-type provirus [pHXB2(WT)], However, b y several passages in SupT1 cells, the mutant viruses demonstrated rep lication kinetics similar to those of the wild-type virus, A DNA seque nce analysis of the PES region from integrated proviruses revealed tha t by day 15 of culture, the PBS of viruses derived from pHXB2(His) and pHXB2(His-T Psi C) reverted back to the wild-type PBS complementary t o tRNA(3)(Lys). In contrast, viruses derived from pHXB2(His-AC) mainta ined a PBS complementary to tRNA(His) for over 4 months in culture enc ompassing 12 serial passages, This study, then, is the first report of a stable human immunodeficiency virus type 1 which utilizes an altern ative tRNA primer and suggests that interactions between the primer tR NA anticodon loop and viral sequences upstream of the PBS contribute t o the specificity of the tRNA primer used in reverse transcription.