REDUCED REPLICATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 MUTANTS THAT USE REVERSE TRANSCRIPTION PRIMERS OTHER THAN THE NATURAL TRNA(3)(LYS)
REDUCED REPLICATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 MUTANTS THAT USE REVERSE TRANSCRIPTION PRIMERS OTHER THAN THE NATURAL TRNA(3)(LYS)
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DOI：
10.1128/jvi.69.5.3090-3097.1995
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发表时间：
1995-05-01
影响因子：
5.4
通讯作者：
BERKHOUT, B
中科院分区：
文献类型：
--
作者：
DAS, AT;KLAVER, B;BERKHOUT, B
1 型人类免疫缺陷病毒 (HIV-1) 和其他逆转录病毒的复制涉及病毒 RNA 基因组逆转录为双链 DNA。该反应由细胞 tRNA(3)(Lys) 分子引发，该分子与病毒基因组中的互补序列（称为引物结合位点 (PBS)）结合。为了研究引物使用的特异性，我们构建了一组 HIV-1 突变体，其 PBS 位点发生了改变，对应于其他 tRNA 种类（tRNA(Ile)、tRNA(1,2)(Lys)、tRNA(Phe)、tRNA(Pro)、tRNA(Trp)）。这些突变病毒能够复制，尽管与野生型 HIV-1 相比复制动力学延迟。与基因组 RNA 相关的 tRNA 种类的鉴定证明了与新 PBS 位点互补的 tRNA 的结合。然而，这些新引物对突变 PBS 位点的占据减少了，并且与突变病毒的复制潜力密切相关。这些结果表明 PBS 序列不足以使 tRNA 引物退火。经过长时间培养，所有突变体都恢复为野生型 PBS3Lys 序列。 PBS 位点两侧核苷酸的微小序列变化表明，这些逆转是由野生型 tRNA(3)(Lys) 引物退火到突变型 PBS 位点上，然后在逆转录过程中复制部分 tRNA(3)(Lys) 序列所致。此外，发现不同 PBS 突变体的回复效率与其 tRNA(3)(Lys) 结合能力相关。 PBSPro 变体观察到显着的逆转途径（PBSPro --> PBSIle --> PBSwt）。该途径可以通过 tRNA(Ile) 与 PBSPro 的有效碱基配对，然后将 tRNA(3)(Lys) 退火到 PBSIle 中间体上来解释。这些结果表明，HIV-1 专用于 tRNA(3)(Lys) 引物，并且 PBS 序列以外的因素决定了该逆转录病毒的选择性引物使用。
Replication of the human immunodeficiency virus type 1 (HIV-1) and other retroviruses involves reverse transcription of the viral RNA genome into a double-stranded DNA. This reaction is primed by the cellular tRNA(3)(Lys) molecule, which binds to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). In order to study the specificity of primer usage, we constructed a set of HIV-1 mutants with altered PBS sites corresponding to other tRNA species (tRNA(Ile), tRNA(1,2)(Lys), tRNA(Phe), tRNA(Pro), tRNA(Trp)). These mutant viruses were able to replicate, although with delayed replication kinetics compared with wild-type HIV-1. Identification of the tRNA species associated with the genomic RNA demonstrated binding of tRNAs complementary to the new PBS sites. However, the occupancy of the mutant PBS sites by these new primers was reduced and correlated well with the replication potential of the mutant viruses. These results suggest that the PBS sequence is not sufficient for annealing of the tRNA primer. Upon prolonged culturing, all mutants reverted to the wild-type PBS3Lys sequence. Minor sequence changes in the nucleotides flanking the PBS site indicate that these reversions resulted from annealing of the wild-type tRNA(3)(Lys) primer onto the mutant PBS sites, followed by copying of part of the tRNA(3)(Lys) sequence during reverse transcription. Furthermore, the reversion efficiency of the different PBS mutants was found to correlate with their tRNA(3)(Lys) binding capacity. A remarkable reversion pathway was observed for the PBSPro variant (PBSPro --> PBSIle --> PBSwt). This pathway can be explained by efficient base pairing of tRNA(Ile) to PBSPro, followed by annealing of tRNA(3)(Lys) onto the PBSIle intermediate. These results demonstrate that HIV-1 is dedicated to the tRNA(3)(Lys) primer and that factors other than the PBS sequence determine the selective primer usage of this retrovirus.