Analysis of the replication of HIV-1 forced to use tRNA(Met(i) )supports a link between primer selection, translation and encapsidation

BACKGROUND: Previous studies have suggested that the process of HIV-1 tRNA primer selection and encapsidation of genomic RNA might be coupled with viral translation. In order to further investigate this relationship, proviruses were constructed in which the primer-binding site (PBS) was altered to be complementary to elongator tRNA(Met )(tRNA(Met(e))) (HXB2-Met(e)) or initiator tRNA(Met )(tRNA(Met(i))) (HXB2-Met(i)). These tRNA(Met )not only differ with respect to the 3' terminal 18-nucleotides, but also with respect to interaction with host cell proteins during protein synthesis. RESULTS: Consistent with previous studies, HXB2-Met(e) were infectious and maintained this PBS following short-term in vitro culture in SupT1 cells. In contrast, transfection of HBX2-Met(i) produced reduced amounts of virus (as determined by p24) and did not establish a productive infection in SupT1 cells. The low infectivity of the virus with the PBS complementary to tRNA(Met(i) )was not due to differences in endogenous levels of cellular tRNA(Met(i) )compared to tRNA(Met(e)); tRNA(Met(i) )was also capable of being selected as the primer for reverse transcription as determined by the endogenous reverse transcription reaction. The PBS of HXB2-Met(i) contains an ATG, which could act as an upstream AUG and syphon scanning ribosomes thereby reducing initiation of translation at the authentic AUG of Gag. To investigate this possibility, a provirus with an A to G change was constructed (HXB2-Met(i)AG). Transfection of HXB2-Met(i)AG resulted in increased production of virus, similar to that for the wild type virus. In contrast to HXB2-Met(i), HXB2-Met(i)AG was able to establish a productive infection in SupT1 cells. Analysis of the PBS following replication revealed the virus favored the genome with the repaired PBS (A to G) even though tRNA(Met(i) )was continuously selected as the primer for reverse transcription. CONCLUSION: The results of these studies suggest that HIV-1 has access to both tRNA(Met )for selection as the replication primer and supports a co-ordination between primer selection, translation and encapsidation during virus replication.