Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation
Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation
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DOI：
10.1128/jvi.71.5.3407-3414.1997
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发表时间：
1997-05-01
影响因子：
5.4
通讯作者：
Parslow, TG
中科院分区：
文献类型：
--
作者：
Clever, JL;Parslow, TG
每个逆转录病毒颗粒包含两个病毒基因组的拷贝，其形式是非共价连接的RNA二聚体。早期的研究已经在人类免疫缺陷病毒1型（HIV-1）基因组的5'端附近绘制了一个顺式作用区域，称为psi位点，它对于基因组二聚化的起始以及与HIV-1 Gag蛋白的相互作用至关重要，后者被认为可以靶向RNA进入新生病毒粒子。这个HIV-1 psi位点被认为组织在四个独立的RNA茎环中；至少有三个（SL1， SL3和SL4）含有Gag的结合位点，其中一个（SL1）通过接吻环机制参与二聚体的起始。在这项研究中，我们创造了含有psi突变的HIV-1前病毒，这些突变会影响体外Gag结合、RNA二聚化或两者兼而有之，我们已经通过单步感染试验表征了这些突变对病毒组装和传染性的影响。我们发现，消除SL1或SL3中Gag结合位点的各种突变会在基因组RNA包装和病毒传染性方面产生明显的缺陷，在每种情况下，突变病毒粒子的基因组含量减少与剪接病毒转录物含量增加有关，这表明SL1和SL3都有助于区分剪接和未剪接的RNA。SL1和SL3茎的结构，而不是特定序列，对RNA包装至关重要。通过病毒粒子衍生RNA的非变性凝胶电泳评估，SL1的主干断裂或缺失也会导致异常的基因组二聚化。已知在SL1环中携带较少广泛突变的病毒粒子可以阻止体外二聚化，尽管病毒粒子RNA含量正常，但其感染性受损。这表明RNA二聚化不是基因组包装的先决条件，而是在逆转录病毒感染周期中起独立作用。
Retrovirus particles each contain two copies of the viral genome in the form of a noncovalently linked RNA dimer. Earlier studies have mapped a cis-acting region near the 5' end of the human immunodeficiency virus type 1 (HIV-1) genome, termed the psi locus, which appears essential for initiation of genomic dimerization, as well as for interactions with the HIV-1 Gag protein that are thought to target the RNA into nascent virions. This HIV-1 psi locus is proposed to be organized in four independent RNA stem-loops; at least three (SL1, SL3, and SL4) contain binding sites for Gag, and one of these (SL1) is implicated in dimer initiation through a kissing-loop mechanism. In this study, we have created HIV-1 proviruses containing psi mutations that affect in vitro Gag binding, RNA dimerization, or both, and we have characterized the effects of these mutations on viral assembly and infectivity by using a single-step infectious assay. We find that various mutations which eliminate the Gag binding sites in SL1 or SL3 produce marked defects in genomic RNA packaging and viral infectivity, In each case, the reduced genomic content of the mutant virions is associated with an increased content of spliced viral transcripts, suggesting that both SL1 and SL3 contribute to the discrimination between spliced and unspliced RNAs, The structures, but not the specific sequences, of the SL1 and SL3 stems appear critical for RNA packaging. Disruption of the stem or deletion of SL1 also results in abnormal genomic dimerization, as assessed by nondenaturing gel electrophoresis of virion-derived RNA. Virions carrying less extensive mutations in the SL1 loop that are known to prevent in vitro dimerization have impaired infectivity despite normal virion RNA content. This suggests that RNA dimerization is not a prerequisite for genomic packaging but instead serves an independent function in the retroviral infectious cycle.