The role of Pr55gag in the annealing of tRNA443Lys to human immunodeficiency virus type 1 genomic RNA
The role of Pr55gag in the annealing of tRNA443Lys to human immunodeficiency virus type 1 genomic RNA
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DOI：
10.1128/jvi.73.5.4485-4488.1999
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发表时间：
1999-05-01
影响因子：
5.4
通讯作者：
Kleiman, L
中科院分区：
文献类型：
--
作者：
Cen, S;Huang, YE;Kleiman, L
在人类免疫缺陷病毒1型（HIV-1）组装过程中，逆转录酶催化合成负链强终止cDNA的引物tRNA（tRNA（3）（Lys））被选择性包装到病毒中，并退火到RNA基因组上的引物结合位点上。HIV-1中tRNA（3）（Lys）的退火不依赖于多蛋白加工，并在体外由p7核衣壳（NCp 7）促进。我们先前已经证明，NC序列中第一个Cys-His盒侧翼的碱性氨基酸簇中的突变抑制了体内tRNA（3）（Lys）的退火70%至80%。在这份报告中，我们调查了这些NC突变是否通过Pr 55（gag）或pr 160（gag-pol）起作用。在体外逆转录试验中，以总病毒RNA作为引物tRNA模板的来源，测量tRNA（3）（Lys）的体内位置。用编码突变Pr 55（gag）或突变pr 160（gag-pol）的质粒和含有HIV-1前病毒DNA的质粒共转染COS细胞，表明只有Pr 55（gag）中的NC突变抑制tRNA（3）（Lys）的放置。Pr 55（gag）中的NC突变使病毒感染性降低95%，并且是反式显性阴性的，即，即使在野生型Pr 55（gag）存在下，它们也抑制tRNA（3）（Lys）的基因组定位。这种显性表型可能表明突变体Pr 55（gag）破坏了负责tRNA（3）（Lys）与基因组RNA退火的有序Pr 55（gag）结构。
During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA(3)(Lys), is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA(3)(Lys) in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA(3)(Lys) in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55(gag) or pr160(gag-pol). In vivo placement of tRNA(3)(Lys) is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells,vith a plasmid coding for either mutant Pr55(gag) or mutant pr160(gag-pol), and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55(gag) inhibit tRNA(3)(Lys) placement. The NC mutations in Pr55(gag) reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA(3)(Lys) even in the presence of wild-type Pr55(gag). This dominant phenotype may indicate that the mutant Pr55(gag) is disrupting an ordered Pr55(gag) structure responsible for the annealing of tRNA(3)(Lys) to genomic RNA.