Preferential completion of human immunodeficiency virus type 1 proviruses initiated with tRNA3Lys rather than tRNA1,2Lys.

A distinguishing feature of retrovirus replication is the process by which the RNA genome is converted into a DNA form prior to integration into the host cell chromosome. The reverse transcription of the retroviral genome is accomplished by a virally encoded enzyme, reverse transcriptase (RT) (1, 25). The initiation of reverse transcription occurs at a region in the viral RNA genome designated as the primer binding site (PBS). The PBS is complementary to the 3′-terminal nucleotides of the cellular tRNA molecule used for initiation. The RT extends the 3′ OH of the cellular tRNA molecule bound at the PBS. Following inter- or intramolecular copying of the viral RNA genome, the DNA copy of the viral genome, termed the provirus, is integrated into the host cell chromosome (6, 18). During the process of reverse transcription, the RT copies the attached tRNA molecule used for initiation of reverse transcription. Thus, the sequence of the PBS from the integrated provirus reflects the tRNA primer used for initiation of reverse transcription. The tRNA primer used for initiation of reverse transcription varies between different retroviruses (4, 7, 19). For human immunodeficiency virus type 1 (HIV-1), tRNA3Lys is exclusively used for initiation of reverse transcription (20, 21). Previous studies have found tRNA3Lys is present within HIV-1 virions. The viral polyprotein precursor, Gag-Pol, is probably responsible for the enrichment of tRNA3Lys in HIV-1 virions (16). Previous studies, though, have demonstrated that similar amounts of tRNA1Lys and tRNA2Lys to that of tRNA3Lys were also present in the HIV-1 virions (9, 30). The sequence homology between tRNA1,2Lys and tRNA3Lys is approximately 80% (there are only 2-nucleotide [nt] differences between tRNA1Lys and tRNA2Lys) (24). How the virus distinguishes between these three isoacceptors for the exclusive use of tRNA3Lys to initiate reverse transcription is not clear. Previous studies from this laboratory and others have demonstrated that HIV-1 has the capacity to utilize several different tRNA primers, including tRNA1,2Lys for the initiation of reverse transcription (5, 10, 13, 28, 29). However, upon extended in vitro culture, all of these viruses reverted back to utilizing tRNA3Lys for the initiation of reverse transcription. Recently, chemical and enzymatic methodologies have been used to delineate additional regions of interaction between the HIV-1 RNA genome and tRNA3Lys (8). In particular, an A-rich region was identified which was found to interact with the anticodon loop of tRNA3Lys. This A-rich region is present at the end of a stem-loop RNA structure (hence A-loop) and has been shown in previous studies to be important for the efficient initiation of reverse transcription. Different A-loop–PBS combinations corresponding to nucleotide sequences complementary to the anticodon and 3′-terminal sequences of tRNAs other than tRNA3Lys have resulted in the production of HIV-1 which stably utilizes these alternative tRNAs for initiation of reverse transcription (11, 12, 26, 30). In this study, we have addressed the specificity of the wild-type HIV-1 for tRNA3Lys for the initiation of reverse transcription. Using a sensitive endogenous RT-PCR, we demonstrate that either tRNA1Lys, tRNA2Lys, or tRNA3Lys can be used for the initiation of reverse transcription. Analysis of integrated proviral DNA sequences isolated from cell clones derived from a single infection found only proviral genomes with a PBS complementary to tRNA3Lys. Using a mutant virus which would be predicted to promote the selection of those genomes initiated with tRNA1,2Lys, we were still unable to recover any clones which were initiated with tRNA1Lys or tRNA2Lys. In contrast, drug-resistant clones with a PBS complementary to tRNA1,2Lys were only recovered if the starting virus contained a PBS complementary to tRNA1,2Lys. The results of these studies demonstrate that in addition to the preference for tRNA3Lys at the initiation of reverse transcription, there exists a second process during HIV-1 reverse transcription which favors the integration of proviral DNA initiated with tRNA3Lys.