EFFECTS OF ALTERATIONS OF PRIMER-BINDING SITE SEQUENCES ON HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REPLICATION
EFFECTS OF ALTERATIONS OF PRIMER-BINDING SITE SEQUENCES ON HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REPLICATION
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DOI：
10.1128/jvi.68.10.6198-6206.1994
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发表时间：
1994-10-01
影响因子：
5.4
通讯作者：
PARNIAK, MA
中科院分区：
文献类型：
--
作者：
LI, XG;MAK, J;PARNIAK, MA
人免疫缺陷病毒1型基因组RNA引物结合位点（PBS）序列包含18个核苷酸，其与复制起始引物tRNA（3）（Lys）3 ′末端的核苷酸互补。为了研究PBS在病毒复制中的作用，我们删除了原始的野生型PBS（与tRNA（3）（Ly 3）互补），或将其替换为与tRNA（1，2）（Lys）或tRNA（Phe）互补的DNA序列。用这样的分子构建体转染COS细胞产生类似水平的病毒子代，其在病毒蛋白和tRNA含量方面是不可区分的。从PBS缺失的分子克隆衍生的病毒颗粒对MT-4、Jurkat和CEM-T4细胞无感染性。然而，感染性病毒来源于其中PBS已被改变为与tRNA（1，2）（Lys）或tRNA（Phe）互补的序列的构建体，尽管突变形式与野生型相比在复制效率上显示出显著滞后。对突变病毒感染细胞中逆转录DNA的分子分析表明，tRNA（1，2）（Lys）和tRNA（Phe）在感染的早期阶段都可以作为逆转录的引物。感染后6天获得的全长前病毒DNA的测序揭示了突变的PBS，表明已经发生了完整的逆转录循环。在随后的几轮感染中，观察到突变的PBS回复到野生型序列，伴随着病毒基因产物的产生增加。通过使用不同引物对的特异性PCR分析和通过扩增片段的直接测序证实了野生型PBS序列的回复。我们还进行了内源性体外逆转录实验，其中负链强终止病毒DNA的合成从含有与各种tRNA isoceptor互补的PBS的合成RNA模板引发。这些结果表明，tRNA（3）（Lys）是比tRNA（1，2）（Lys）或tRNA（Phe）更有效的此类反应的引物。
The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3' end of the replication initiation primer tRNA(3)(Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3)(Ly3)) or replaced it with DNA sequences complementary to either tRNA(1,2)(Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2)(Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2)(Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3)(Lys) was a much more efficient primer of such reactions than either tRNA(1,2)(Lys) or tRNA(Phe).