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.

中文翻译：

---

HIV-1 和 SIV 体内逆转录的低水平替代 tRNA 启动


背景 HIV 和 SIV 的逆转录 (RT) 是通过 tRNALys3 的受体茎与病毒 RNA 基因组的引物结合位点 (PBS) 的结合来启动的。先前的研究表明，tRNALys3 并不是唯一能够启动逆转录的分子，并且至少还有另一种赖氨酰 tRNA，即 tRNALys5，其受体茎序列与 tRNALys3 的受体茎序列仅存在一个过渡突变，从而导致了病毒基因组中 PBS 8 位的胸腺嘧啶 (T) 可以启动逆转录。结果我们采取了一种公正的方法，通过直接从 15 名人类和 11 只猕猴的血浆中对 HIV 和 SIV 进行深度测序来评估引物结合位点。我们发现，在人类中，病毒 RNA 基因组的水平较低，但可测量，其含有 PBS，其中第 8 位含有非规范 T (PBS-Lys5)，对应于 tRNAlys5 序列，平均占 0.52%（范围 0.07-1.6%）。病毒总数。该值与我们在 LANL HIV 数据库横断面评估中确定的 PBS-Lys5 比例 (0.51%) 非常一致。在长期感染 SIVmac239 的猕猴中，也检测到了 PBS-Lys5，但频率比 HIV 低 1 个对数，平均为 0.056%（范围 0.01-0.09%）。在这个比例下，PBS-Lys5与其他过渡突变相当，因此无法确定观察到的突变是使用非常低水平的tRNALys5作为RT引物的结果还是仅仅是体外cDNA合成/PCR错误的产物。我们还在 HIV 和 SIV 中鉴定了体内低水平的两个新的 PBS 序列，对应于 tRNALys6 和 tRNALys1,2，表明这些 tRNA 可能很少也用于引发 RT。 SIVmac239 中发现的 PBS-Lys5 的体内逆转速度很快，并在感染后 30 天达到背景水平。结论我们的结论是，虽然替代 tRNA 可以在体内启动 HIV 和 SIV 的逆转录，但它们对复制病毒群体的总体贡献是小的。