MUTATIONS IN BOTH THE U5 REGION AND THE PRIMER-BINDING SITE INFLUENCETHE SELECTION OF THE TRANSFER-RNA USED FOR THE INITIATION OF HIV-1 REVERSE TRANSCRIPTION

The initiation of HIV-I reverse transcription is primed by a cellular tRNA(Lys,3) molecule which is bound to a complementary sequence near t he 5' end of the viral RNA genome designated as the primer-binding sit e (PBS). Recent studies have suggested that sequences upstream of the PBS within U5 consisting of a stretch of adenine nucleotides (referred to as the A-loop) might be important in the selection and positioning of tRNA(Lys,3) primer used to initiate reverse transcription. To furt her explore the role that the A-loop plays in reverse transcription, w e have constructed proviral genomes in which the PBS was changed so as to be complementary to the 3'-terminal 18 nucleotides of tRNA(Ile), t RNA(Pro), or tRNA(Trp) [pHXB(Ile), pHXB(Pro), or pHXB(Trp), respective ly]; a second set of proviral genomes was constructed which contained additional mutations so that the A-loop regions were complementary to the anticodon region of tRNA(Ile) [pHXB(Ile-AC)], tRNA(Pro) [pHXB(Pro- AC)], or tRNA(Trp) [pHXB(Trp-AC)]. Transfection of the proviruses into COS-1 cells followed by coculture with SupT1 cells resulted in produc tion of infectious virus. PCR was used to amplify the PBS regions whic h were subcloned into M13mp18 followed by DNA sequence analysis. After short-term culture, the PBSs of proviruses derived from pHXB(Ile), pH XB(Pro), and pHXB(Trp) reverted to be complementary to tRNA(Lys,3). Th e PBSs of the viruses derived from pHXB(Ile-AC) also reverted to be co mplementary to tRNA(Lys,3); the A-loop region was still complementary to tRNA(Ile). In contrast, viruses derived from transfection of pHXB(P ro-AC) initially maintained a PBS complementary to tRNA(Pro). Upon ext ended culture, we identified proviruses which contained PBSs complemen tary to two additional tRNAs: tRNA(Ile) and tRNA(Lys,3). Furthermore, we found proviruses which contain two PBSs within the same genome: one complementary to tRNA(Lys,3) and a second complementary to tRNA(Pro) or tRNA(Ile). Viruses derived from transfection of pHXB(Trp-AC) were t he most delayed in appearance following transfection. Analysis of the PBS revealed that early after transfection, the majority of the PBSs w ere complementary to tRNA(Trp). After further in vitro culture, provir uses were identified with a PBS complementary to a new tRNA, tRNA(Met) . Finally, upon extended culture, the viruses derived from the transfe ction of pHXB(Ile-AC), pHXB(Pro-AC), and pHXB(Trp-AC) contained mutati ons upstream from the PBS in U5 that created a stretch of 3 adenine nu cleotides. The results of these studies then highlight the flexibility that exists with respect to the selection of the tRNA primer used to initiate HIV-I reverse transcription. (C) 1996 Academic Press. Inc.