Restoration of tRNA(3)(Lys)-primed (-)-strand DNA synthesis to an HIV-1 reverse transcriptase mutant with extended tRNAs - Implications for retroviral replication

The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51 Delta 13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA(3)(Lys), the cognate HIV primer. When the ability to support (-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs), However, it failed to do so from natural and synthetic versions of tRNA(3)(Lys). tRNA-primed (-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA(3)(Lys) with 81- and 107-nt tRNA-DNA chimeras, ie. tRNA(3)(Lys) extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs, These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive (-)-strand DNA synthesis. Alternatively, specific interactions between tRNA(3)(Lys) and HIV-1 RT may be necessary for efficient initiation of (-)-strand DNA synthesis.