PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex
PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex
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DOI：
10.1128/jvi.01741-18
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发表时间：
2019-03-01
影响因子：
5.4
通讯作者：
Dash, Chandravanu
中科院分区：
文献类型：
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作者：
Balasubramaniam, Muthukumar;Zhou, Jing;Dash, Chandravanu
HIV-1衣壳蛋白（CA）促进病毒的逆转录和核进入。然而，CA在核进入后步骤中的作用仍然是推测性的。我们描述了CA和整合之间的直接联系，采用衣壳抑制剂PF 74作为探针，再加上从急性感染细胞分离的HIV-1整合前复合物（PIC）的生化分析。在低微摩尔浓度下，PF 74有效抑制HIV-1感染而不影响逆转录。令人惊讶的是，PF 74由于抑制核进入和/或整合而显著降低前病毒整合。然而，2倍减少核进入PF 74没有定量相关的抗病毒活性的水平。针对整合酶抑制剂雷特格韦的PF 74滴定显示出附加的抗病毒作用，其依赖于在进入核后步骤的阻断。PF 74的抑制作用不是由于缺陷病毒DNA末端的形成或整合的延迟，这表明该化合物抑制PIC相关的整合活性。出乎意料的是，从在PF 74存在下感染的细胞中回收的PIC表现出升高的整合活性。PF 74对PIC活性的影响是CA特异性的，因为该化合物不增加PF 74抗性HIV-1 CA突变体的PIC的整合活性。基于蔗糖梯度的分馏研究表明，在PF 74存在下组装的PIC含有较低水平的CA，这表明CA和PIC相关的整合活性之间存在负相关。最后，添加CA特异性抗体或PF 74抑制PIC相关的整合活性。总的来说，我们的研究结果表明，PF 74的PIC相关CA的目标在受损的HIV-1 integration.IMPORTANCE抗逆转录病毒疗法（ART），使用各种组合的小分子抑制剂已经非常有效地控制艾滋病毒。然而，ART方案中使用的药物价格昂贵，会引起副作用，并面临病毒耐药性。HIV-1 CA在病毒生命周期中起着关键作用，是一个有吸引力的治疗靶点。虽然目前还没有基于CA的治疗，但正在开发高效的CA特异性抑制剂作为一类新的抗病毒药物。通过清楚地了解CA在HIV-1感染中的作用，可以帮助开发CA靶向治疗的努力。CA被很好地建立以协调病毒的逆转录和核进入。然而，CA在HIV-1感染的后核进入步骤中的作用知之甚少。我们发现，CA特异性药物PF 74抑制HIV-1整合，揭示了这种多功能病毒蛋白在HIV-1感染的后核进入步骤中的新作用。
The HIV-1 capsid protein (CA) facilitates reverse transcription and nuclear entry of the virus. However, CA's role in post-nuclear entry steps remains speculative. We describe a direct link between CA and integration by employing the capsid inhibitor PF74 as a probe coupled with the biochemical analysis of HIV-1 preintegration complexes (PICs) isolated from acutely infected cells. At a low micromolar concentration, PF74 potently inhibited HIV-1 infection without affecting reverse transcription. Surprisingly, PF74 markedly reduced proviral integration owing to inhibition of nuclear entry and/or integration. However, a 2-fold reduction in nuclear entry by PF74 did not quantitatively correlate with the level of antiviral activity. Titration of PF74 against the integrase inhibitor raltegravir showed an additive antiviral effect that is dependent on a block at the post-nuclear entry step. PF74's inhibitory effect was not due to the formation of defective viral DNA ends or a delay in integration, suggesting that the compound inhibits PIC-associated integration activity. Unexpectedly, PICs recovered from cells infected in the presence of PF74 exhibited elevated integration activity. PF74's effect on PIC activity is CA specific since the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient-based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, the addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74's targeting of PIC-associated CA results in impaired HIV-1 integration.IMPORTANCE Antiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well established to coordinate reverse transcription and nuclear entry of the virus. However, the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection.