Abstract
BackgroundHuman immunodeficiency virus (HIV-1) exclusively selects and utilizes tRNALys,3 as the primer for initiation of reverse transcription. Several elements within the TΨC stem loop of tRNALys,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.ResultsTo further delineate the features of the TΨC stem loop of tRNALys,3 in reverse transcription, we have developed a complementation system in which E. coli tRNALys,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 tRNALys,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 tRNALys,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 tRNALys,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 tRNALys,3 that was aminoacylated when expressed in cells, while a T54C mutation resulted in a tRNALys,3 that was not aminoacylated. Both the A58U and T54G mutated tRNALys,3 complemented HIV-1 replication similar to wild type E. coli tRNALys,3. In contrast, the TG5453CC tRNALys,3 mutant did not complement replication.ConclusionThe results demonstrate that post-transcriptional modification of nucleotide 58 in tRNALys,3 is not essential for HIV-1 reverse transcription. In contrast, nucleotides 53 and 54 of tRNALys,3 are important for aminoacylation and selection and use of the tRNALys,3 in reverse transcription.
Highlights
Human immunodeficiency virus (HIV-1) exclusively selects and utilizes tRNALys,3 as the primer for initiation of reverse transcription
Construction of E. coli tRNALys,3 mutants and HIV-1 proviral genomes In a recent study, we have described a complementation system which relies on the addition of E. coli tRNALys,3 in trans to complement an HIV-1 proviral genome in which the primer-binding site (PBS) was altered to be complementary to the 3' terminal 18-nucleotides of this tRNA [15]
We demonstrated that transfection of the proviral genome with a PBS to E. coli tRNALys,3 into mammalian cells required complementation by E. coli tRNALys,3cDNA in order to produce infectious virus [15]
Summary
Human immunodeficiency virus (HIV-1) exclusively selects and utilizes tRNALys, as the primer for initiation of reverse transcription. The posttranscriptional 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. The initiation of reverse transcription occurs at the 5' end of the viral genome at a site designated as the primer-binding site (PBS) [1]. The reverse transcriptase translocates to the 3' end of the viral RNA genome and proceeds to generate a complete minus-strand DNA copy of the viral RNA genome. During plus-strand synthesis, the reverse transcriptase copies the tRNA primer that is attached to the minusstrand DNA to generate a plus-strand copy of the PBS. Complementation between the plus- and minus-strand PBS facilitates the completion of the viral genome, designated as the provirus
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