Mutations in the TΨC Loop of E. coli tRNA(Lys,3 )Have Varied Effects on In Trans Complementation of HIV-1 Replication

BACKGROUND: Human immunodeficiency virus (HIV-1) exclusively selects and utilizes tRNA(Lys,3 )as the primer for initiation of reverse transcription. Several elements within the TΨC stem loop of tRNA(Lys,3 )are postulated to be important for selection and use in reverse transcription. The post-transcriptional modification at nucleotide 58 could play a role during plus-strand synthesis to stop reverse transcriptase from re-copying the tRNA primer. Nucleotides 53 and 54 within the TΨC stem loop of the tRNA have been shown to be important to form the complex between tRNA and the HIV-1 viral genome during initiation of reverse transcription. RESULTS: To further delineate the features of the TΨC stem loop of tRNA(Lys,3 )in reverse transcription, we have developed a complementation system in which E. coli tRNA(Lys,3 )is provided in trans to an HIV-1 genome in which the PBS is complementary to this tRNA. Successful selection and use of E. coli tRNA(Lys,3 )results in the production of infectious virus. We have used this single round infectious system to ascertain the effects that different mutants in the TΨC stem loop of tRNA(Lys,3 )have on complementation. Mutants were designed within the TΨC loop (nucleotide 58) and within the stem and loop of the TΨC loop (nucleotides 53 and 54). Analysis of the expression of E. coli tRNA(Lys,3 )mutants revealed differences in the capacity for aminoacylation, which is an indication of intracellular stability of the tRNA. Alteration of nucleotide 58 from A to U (A58U), T54G and TG5453CC all resulted in tRNA(Lys,3 )that was aminoacylated when expressed in cells, while a T54C mutation resulted in a tRNA(Lys,3 )that was not aminoacylated. Both the A58U and T54G mutated tRNA(Lys,3 )complemented HIV-1 replication similar to wild type E. coli tRNA(Lys,3). In contrast, the TG5453CC tRNA(Lys,3 )mutant did not complement replication. CONCLUSION: The results demonstrate that post-transcriptional modification of nucleotide 58 in tRNA(Lys,3 )is not essential for HIV-1 reverse transcription. In contrast, nucleotides 53 and 54 of tRNA(Lys,3 )are important for aminoacylation and selection and use of the tRNA(Lys,3 )in reverse transcription.