Hulme Amy E, Kelley Z, Foley Deirdre, Hope Thomas J
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA
J Virol. 2015 May;89(10):5350-61. doi: 10.1128/JVI.00476-15. Epub 2015 Mar 4.
During uncoating, the conical capsid of HIV disassembles by dissociation of the p24 capsid protein (CA). Uncoating is known to be required for HIV replication, but the mechanism is poorly defined. Here, we examined the timing and effect of two capsid binding drugs (PF74 and BI2) on infectivity and capsid integrity in HIV-1-infected cells. The virus remained susceptible to the action of PF74 and BI2 for hours after uncoating as defined in parallel drug addition and cyclosporine (CsA) washout assays to detect the kinetics of drug susceptibility and uncoating, respectively. Resistance mutations in CA decreased the potency of these compounds, demonstrating that CA is the target of drug action. However, neither drug altered capsid integrity in a fluorescence microscopy-based assay. These data suggest that PF74 and BI2 do not alter HIV-1 uncoating but rather affect a later step in viral replication. Because both drugs bind CA, we hypothesized that a residual amount of CA associates with the viral complex after the loss of the conical capsid to serve as a target for these drugs. Superresolution structured illumination microscopy (SIM) revealed that CA localized to viral complexes in the nuclei of infected cells. Using image quantification, we determined that viral complexes localized in the nucleus displayed a smaller amount of CA than complexes at the nuclear membrane, in the cytoplasm, or in controls. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating and that this residual CA is the target of PF74 and BI2.
The HIV-1 capsid is a target of interest for new antiviral therapies. This conical capsid is composed of monomers of the viral CA protein. During HIV-1 replication, the capsid must disassemble by a poorly defined process called uncoating. CA has also been implicated in later steps of replication, including nuclear import and integration. In this study, we used cell-based assays to examine the effect of two CA binding drugs (PF74 and BI2) on viral replication in infected cells. HIV-1 was susceptible to both drugs for hours after uncoating, suggesting that these drugs affect later steps of viral replication. High-resolution structured illumination microscopy (SIM) revealed that a subset of CA localized to viral complexes in the nuclei of cells. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating, which may facilitate later steps of viral replication and serve as a drug target.
在脱壳过程中，HIV的锥形衣壳通过p24衣壳蛋白（CA）的解离而解体。已知脱壳是HIV复制所必需的，但机制尚不清楚。在这里，我们研究了两种衣壳结合药物（PF74和BI2）对HIV-1感染细胞中感染性和衣壳完整性的时间和影响。在平行药物添加和环孢素（CsA）洗脱试验中分别检测药物敏感性和脱壳动力学，结果表明病毒在脱壳后数小时内仍对PF74和BI2的作用敏感。CA中的抗性突变降低了这些化合物的效力，表明CA是药物作用的靶点。然而，在基于荧光显微镜的试验中，这两种药物都没有改变衣壳的完整性。这些数据表明，PF74和BI2不会改变HIV-1的脱壳过程，而是影响病毒复制的后期步骤。由于这两种药物都与CA结合，我们推测在锥形衣壳丢失后，仍有少量CA与病毒复合物结合，作为这些药物的靶点。超分辨率结构光照显微镜（SIM）显示，CA定位于感染细胞核内的病毒复合物中。通过图像定量分析，我们确定定位于细胞核内的病毒复合物中CA的含量比位于核膜、细胞质或对照中的复合物少。总的来说，这些数据表明脱壳后有一部分CA仍与病毒复合物结合，并且这种残留的CA是PF74和BI2的靶点。
HIV-1衣壳是新型抗病毒疗法感兴趣的靶点。这种锥形衣壳由病毒CA蛋白的单体组成。在HIV-1复制过程中，衣壳必须通过一个定义不清的称为脱壳的过程解体。CA也与复制的后期步骤有关，包括核输入和整合。在这项研究中，我们使用基于细胞的试验来研究两种CA结合药物（PF74和BI2）对感染细胞中病毒复制的影响。HIV-1在脱壳后数小时内对这两种药物都敏感，这表明这些药物影响病毒复制的后期步骤。高分辨率结构光照显微镜（SIM）显示，一部分CA定位于细胞核内的病毒复合物中。总的来说，这些数据表明脱壳后有一部分CA仍与病毒复合物结合，这可能有助于病毒复制的后期步骤，并作为药物靶点。
