Division of Clinical Research, Department of Medical Research, Taipei Veterans General Hospital , Taipei, Taiwan.
Institute of Clinical Medicine, National Yang Ming Chiao Tung University , Taipei, Taiwan.
J Virol. 2023 Sep 28;97(9):e0094823. doi: 10.1128/jvi.00948-23. Epub 2023 Sep 6.
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.
人免疫缺陷病毒 1 型颗粒的蛋白水解加工由病毒蛋白酶（PR）介导，这对于获得病毒感染力至关重要。PR 嵌入 Gag-Pol 中的激活是由病毒组装过程中 Gag-Pol 二聚化引发的。我们之前报道过，在 RT 色氨酸重复基序处的氨基酸取代会破坏病毒相关 RT 的稳定性，并削弱依非韦伦（EFV，一种 RT 二聚化增强剂）增加 PR 介导的 Gag 切割效率的能力。此外，RT 中的单个氨基酸变化由于增强的 Gag 切割而显著降低病毒产量。这些数据提出了 RT 结构域通过促进 Gag-Pol 二聚化来促进 PR 激活的可能性。为了验证这一假设，我们研究了跨越 RT L282 到 L310 的疏水性亮氨酸重复基序（LRM）在 RT/RT 相互作用中的可能参与。我们发现 LRM 氨基酸取代导致 RT 不稳定，并且 RT 因此容易受到 PR 的降解。LRM 突变体表现出降低的 Gag 切割效率，同时削弱 EFV 增强的 Gag 切割。此外，W401A 二聚化缺陷突变体通过插入缺失的 Gag-Pol 区域的亮氨酸拉链（LZ）基序降低了增强的 Gag 切割。重要的是，RT 和整合酶结构域的存在未能抵消 LZ 对 Gag 切割的增强作用。EFV 和 W402A 的 Gag 切割增强因子的组合显著损害了 Gag 切割，表明在 EFV 与 W402A Gag-Pol 结合后，W402A Gag-Pol 二聚体的形成受到干扰。我们的结果支持这样的观点，即 RT 通过影响 Gag-Pol/Gag-Pol 相互作用来调节 PR 激活。重要的是，稳定的逆转录酶（RT）p66/51 异二聚体是 HIV-1 基因组在病毒进入宿主细胞后进行复制所必需的。病毒蛋白酶（PR）的激活介导病毒颗粒加工有助于病毒在细胞释放后获得感染力。RT 和 PR 似乎都是抑制 HIV-1 复制的主要靶点。我们发现 p66/51RT 稳定性受损与 PR 介导的 Gag 切割不足之间存在很强的相关性，这表明 RT/RT 相互作用对于通过促进足够的 Gag-Pol 二聚化来触发 PR 激活至关重要。因此，如果发现 RT/RT 相互作用能够同时阻断 PR 和 RT 酶活性，那么它可能是一种有前途的抗 HIV/AIDS 治疗方法。
