Rhesus TRIM5α Disrupts the HIV-1 Capsid at the Inter­Hexamer Interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction.

TRIM蛋白（TRIM proteins）在针对包括人类免疫缺陷病毒1型（HIV-1）在内的逆转录病毒感染的先天免疫防御中发挥关键作用。恒河猴TRIM5α（TRIM5αrh）可靶向HIV-1衣壳，并在逆转录前的感染早期阶段阻断病毒感染。已有研究表明，TRIM5α与组装完成的衣壳结合是其发挥病毒限制性感染作用的必要条件，且该结合过程需要卷曲螺旋（coiled-coil）结构域与B30.2/SPRY结构域的参与，但目前其限制性作用的分子机制尚未完全阐明。本研究通过冷冻电子显微镜（cryoEM）结合诱变技术与化学交联手段，探究了HIV-1衣壳蛋白（CA）组装体与包含卷曲螺旋和SPRY结构域的纯化恒河猴TRIM5α（CC-SPRYrh）之间的直接相互作用。实验观察到，CC-SPRYrh与CA组装体的结合呈现浓度依赖性，而在同等实验条件下，人类同源蛋白则无结合现象。值得注意的是，CC-SPRYrh而非其人类同源蛋白，能够以非随机方式破坏CA管状物，释放由CA六聚体组成的原丝片段，且并未将其解离为单体。进一步研究发现，使用在衣壳组装体的CTD-CTD三聚体界面形成二硫键的P207C/T216C突变CA进行六聚体间交联，可阻断这种结构破坏效应；但通过A14C/E45C在NTD-NTD界面进行的六聚体内交联则无法阻断该破坏。TRIM5αrh对六聚体间界面的上述破坏效应，同样出现在纯化的完整HIV-1核心结构中。本研究结果为TRIM5α破坏病毒颗粒核心的机制提供了新的认知，并证实TRIM5α对病毒衣壳的结构损伤，可能是TRIM5α介导的HIV-1感染限制性机制的重要组成部分。