PDF(Pubmed)
Abstract：
Proteolytic processing of human immunodeficiency virus type 1 particles mediated by viral protease (PR) is essential for acquiring virus infectivity. Activation of PR embedded in Gag-Pol is triggered by Gag-Pol dimerization during virus assembly. We previously reported that amino acid substitutions at the RT tryptophan repeat motif destabilize virus-associated RT and attenuate the ability of efavirenz (EFV, an RT dimerization enhancer) to increase PR-mediated Gag cleavage efficiency. Furthermore, a single amino acid change at RT significantly reduces virus yields due to enhanced Gag cleavage. These data raise the possibility of the RT domain contributing to PR activation by promoting Gag-Pol dimerization. To test this hypothesis, we investigated the putative involvement of a hydrophobic leucine repeat motif (LRM) spanning RT L282 to L310 in RT/RT interactions. We found that LRM amino acid substitutions led to RT instability and that RT is consequently susceptible to degradation by PR. The LRM mutants exhibited reduced Gag cleavage efficiencies while attenuating the EFV enhancement of Gag cleavage. In addition, an RT dimerization-defective mutant, W401A, reduced enhanced Gag cleavage via a leucine zipper (LZ) motif inserted at the deleted Gag-Pol region. Importantly, the presence of RT and integrase domains failed to counteract the LZ enhancement of Gag cleavage. A combination of the Gag cleavage enhancement factors EFV and W402A markedly impaired Gag cleavage, indicating a disruption of W402A Gag-Pol dimerization following EFV binding to W402A Gag-Pol. Our results support the idea that RT modulates PR activation by affecting Gag-Pol/Gag-Pol interaction. IMPORTANCE A stable reverse transcriptase (RT) p66/51 heterodimer is required for HIV-1 genome replication in host cells following virus entry. The activation of viral protease (PR) to mediate virus particle processing helps viruses acquire infectivity following cell release. RT and PR both appear to be major targets for inhibiting HIV-1 replication. We found a strong correlation between impaired p66/51RT stability and deficient PR-mediated Gag cleavage, suggesting that RT/RT interaction is critical for triggering PR activation via the promotion of adequate Gag-Pol dimerization. Accordingly, RT/RT interaction is a potentially advantageous method for anti-HIV/AIDS therapy if it is found to simultaneously block PR and RT enzymatic activity.
摘要：
由病毒蛋白酶（PR）介导的1型人类免疫缺陷病毒颗粒的蛋白水解加工对于获得病毒感染性至关重要。在病毒组装期间,由Gag-Pol二聚化触发嵌入Gag-Pol中的PR的激活。我们以前报道过，RT色氨酸重复基序的氨基酸取代会破坏与病毒相关的RT的稳定性，并削弱依非韦仑的能力（EFV，RT二聚化增强子)以增加PR介导的Gag切割效率。此外，由于增强的Gag切割,在RT下的单个氨基酸变化显著降低病毒产量。这些数据提高了RT结构域通过促进Gag-Pol二聚化而有助于PR活化的可能性。为了检验这个假设，我们调查了RT/RT相互作用中跨越RTL282至L310的疏水性亮氨酸重复基序（LRM）的推定参与。我们发现LRM氨基酸取代导致RT不稳定，因此RT易于被PR降解。LRM突变体表现出降低的Gag切割效率,同时减弱Gag切割的EFV增强。此外,RT二聚化缺陷突变体,W401A,通过插入缺失的Gag-Pol区的亮氨酸拉链（LZ）基序减少增强的Gag切割。重要的是,RT和整合酶结构域的存在未能抵消Gag裂解的LZ增强。Gag裂解增强因子EFV和W402A的组合显著损害Gag裂解,表明在EFV与W402AGag-Pol结合后W402AGag-Pol二聚化的破坏。我们的结果支持RT通过影响Gag-Pol/Gag-Pol相互作用来调节PR激活的观点。重要性病毒进入后宿主细胞中HIV-1基因组复制需要稳定的逆转录酶(RT)p66/51异二聚体。病毒蛋白酶(PR)介导病毒颗粒加工的活化有助于病毒在细胞释放后获得感染性。RT和PR似乎都是抑制HIV-1复制的主要目标。我们发现p66/51RT稳定性受损与PR介导的Gag切割缺陷之间存在很强的相关性，表明RT/RT相互作用对于通过促进充分的Gag-Pol二聚化来触发PR激活至关重要。因此，如果发现RT/RT相互作用同时阻断PR和RT酶活性,则它是抗HIV/AIDS治疗的潜在有利方法。
