Abstract
BackgroundReverse transcription (RT) of HIV and SIV is initiated by the binding of the acceptor stem of tRNALys3 to the primer binding site (PBS) of the viral RNA genome. Previous studies have suggested that this tRNALys3 is not the only molecule capable of priming reverse transcription, and that at least one other lysyl tRNA, tRNALys5, which has an acceptor stem sequence varying from tRNALys3 by only a single transition mutation resulting in the integration of a thymine (T) at position 8 of the PBS in the viral genome, can prime reverse transcription.ResultsWe undertook an unbiased approach, evaluating the primer binding site by deep-sequencing of HIV and SIV directly from the plasma of 15 humans and 11 macaques. We found that in humans there are low but measurable levels of viral RNA genomes harboring a PBS containing the noncanonical T at position 8 (PBS-Lys5) corresponding to the tRNAlys5 sequence and representing an average of 0.52% (range 0.07–1.6%) of the total viral population. This value is remarkably consistent with the proportion of PBS-Lys5 we identified in a cross-sectional assessment of the LANL HIV database (0.51%). In macaques chronically infected with SIVmac239, the PBS-Lys5 was also detected but at a frequency 1-log less than seen for HIV, with an average of 0.056% (range 0.01–0.09%). At this proportion, PBS-Lys5 was comparable to other transition mutations, making it impossible to determine whether the mutation observed is a result of use of tRNALys5 as an RT primer at very low levels or merely the product of in vitro cDNA synthesis/PCR error. We also identified two novel PBS sequences in HIV and SIV at low levels in vivo corresponding to tRNALys6 and tRNALys1,2, suggesting that these tRNAs may rarely also be used to prime RT. In vivo reversion of the PBS-Lys5 found in SIVmac239 was rapid and reached background levels by 30 days post-infection.ConclusionsWe conclude that while alternative tRNAs can initiate reverse transcription of HIV and SIV in vivo, their overall contributions to the replicating viral population are small.
Highlights
Reverse transcription (RT) of HIV and simian immunodeficiency virus (SIV) is initiated by the binding of the acceptor stem of tRNALys3 to the primer binding site (PBS) of the viral RNA genome
When similar analyses were conducted on plasma samples from chronically infected rhesus macaques, we found that the level of PBS corresponding to tRNALys5 fell into the range of RT and PCR error, making it impossible to conclusively define a role for this tRNA in SIV reverse transcription
The PBS sequence that results in perfect complementarity to tRNALys3 (PBS-Lys3), the canonical tRNA molecule used for HIV/SIV reverse transcription, represented almost the entire population of the sample, with values ranging from 96.5 to 98.5% of all sequences analyzed per individual. 45 other sequence variants were detected using this approach, including all 18 transition mutations and 18 transversion mutations, as well as several sequences that contained more than one mutation (Fig. 1)
Summary
Reverse transcription (RT) of HIV and SIV is initiated by the binding of the acceptor stem of tRNALys to the primer binding site (PBS) of the viral RNA genome. Previous studies have suggested that this tRNALys is not the only molecule capable of priming reverse transcription, and that at least one other lysyl tRNA, tRNALys, which has an acceptor stem sequence varying from tRNALys by only a single transition mutation resulting in the integration of a thymine (T) at position 8 of the PBS in the viral genome, can prime reverse transcription. The cytosine (C) to thymine (T) point mutation in the SIVmac239 PBS identified by Alexander et al reverts to the canonical C, suggesting that alternate tRNAs such as tRNALys, which is fully complementary to the suboptimal PBS-Lys do not function well or are insufficiently expressed in host cells [3, 9]. Soderberg et al [12] describe reversion of this nucleotide in an SIVmac239 derivative in vitro and report drastically increased replicative fitness following this reversion
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