BACKGROUND After viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. Previously we have utilized the CsA washout assay, in which TRIM-CypA mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of HIV-1 uncoating in owl monkey kidney (OMK) and HeLa cells. Here we have extended this analysis to the human microglial cell lines CHME3 and C20 to characterize uncoating in a cell type that is a natural target of HIV infection. METHODS The CsA washout was used to characterize uncoating of wildtype and capsid mutant viruses in CHME3 and C20 cells. Viral fusion assays and nevirapine addition assays were performed to relate the kinetics of viral fusion and reverse transcription to uncoating. RESULTS We found that uncoating initiated within the first hour after viral fusion and was facilitated by reverse transcription in CHME3 and C20 cells. The capsid mutation A92E did not significantly alter uncoating kinetics. Viruses with capsid mutations N74D and E45A decreased the rate of uncoating in CHME3 cells, but did not alter reverse transcription. Interestingly, the second site suppressor capsid mutation R132T was able to rescue the uncoating kinetics of the E45A mutation, despite having a hyperstable capsid. CONCLUSIONS These results are most similar to previously observed characteristics of uncoating in HeLa cells and support the model in which uncoating is initiated by early steps of reverse transcription in the cytoplasm. A comparison of the uncoating kinetics of CA mutant viruses in OMK and CHME3 cells reveals the importance of cellular factors in the process of uncoating. The E45A/R132T mutant virus specifically suggests that disrupted interactions with cellular factors, rather than capsid stability, is responsible for the delayed uncoating kinetics seen in E45A mutant virus. Future studies aimed at identifying these factors will be important for understanding the process of uncoating and the development of interventions to disrupt this process.

中文翻译：

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人类小胶质细胞系中HIV-1脱膜的特征。

背景技术病毒与细胞膜融合后，HIV-1的圆锥形衣壳会通过称为脱壳的过程进行分解。以前，我们利用CsA洗脱试验，其中TRIM-CypA介导的病毒复制限制用于检测病毒衣壳的状态，以研究猫头鹰猴肾（OMK）和HeLa细胞中HIV-1脱膜的动力学。在这里，我们已经将此分析扩展到人小胶质细胞系CHME3和C20，以表征作为HIV感染天然靶标的细胞类型的脱膜特征。方法用CsA冲洗液表征CHME3和C20细胞中野生型和衣壳突变病毒的脱膜情况。进行病毒融合测定和奈韦拉平添加测定以使病毒融合和逆转录动力学与脱膜相关。结果我们发现，脱膜是在病毒融合后的第一小时内开始的，并通过在CHME3和C20细胞中的逆转录而得以促进。衣壳突变A92E没有显着改变脱膜动力学。衣壳突变N74D和E45A的病毒降低了CHME3细胞的脱膜率，但没有改变逆转录。有趣的是，尽管具有高稳定的衣壳，第二位点抑制子衣壳突变R132T仍能挽救E45A突变的脱壳动力学。结论这些结果与以前在HeLa细胞中观察到的脱膜特征最相似，并且支持了其中脱膜是由细胞质中逆转录的早期步骤引发的模型。CA突变病毒在OMK和CHME3细胞中脱壳动力学的比较揭示了在脱壳过程中细胞因子的重要性。E45A / R132T突变病毒特别表明，与细胞因子的相互作用而不是衣壳稳定性受到破坏是造成E45A突变病毒中延迟的脱膜动力学的原因。旨在识别这些因素的未来研究对于理解脱衣过程和开发干扰该过程的干预措施将非常重要。