Gain-of-Sensitivity Mutations in a Trim5-Resistant Primary Isolate of Pathogenic SIV Identify Two Independent Conserved Determinants of Trim5α Specificity

Retroviral capsid recognition by Trim5 blocks productive infection. Rhesus macaques harbor three functionally distinct Trim5 alleles: Trim5αQ, Trim5αTFP and Trim5CypA. Despite the high degree of amino acid identity between Trim5αQ and Trim5αTFP alleles, the Q/TFP polymorphism results in the differential restriction of some primate lentiviruses, suggesting these alleles differ in how they engage these capsids. Simian immunodeficiency virus of rhesus macaques (SIVmac) evolved to resist all three alleles. Thus, SIVmac provides a unique opportunity to study a virus in the context of the Trim5 repertoire that drove its evolution in vivo. We exploited the evolved rhesus Trim5α resistance of this capsid to identify gain-of-sensitivity mutations that distinguish targets between the Trim5αQ and Trim5αTFP alleles. While both alleles recognize the capsid surface, Trim5αQ and Trim5αTFP alleles differed in their ability to restrict a panel of capsid chimeras and single amino acid substitutions. When mapped onto the structure of the SIVmac239 capsid N-terminal domain, single amino acid substitutions affecting both alleles mapped to the β-hairpin. Given that none of the substitutions affected Trim5αQ alone, and the fact that the β-hairpin is conserved among retroviral capsids, we propose that the β-hairpin is a molecular pattern widely exploited by Trim5α proteins. Mutations specifically affecting rhesus Trim5αTFP (without affecting Trim5αQ) surround a site of conservation unique to primate lentiviruses, overlapping the CPSF6 binding site. We believe targeting this site is an evolutionary innovation driven specifically by the emergence of primate lentiviruses in Africa during the last 12 million years. This modularity in targeting may be a general feature of Trim5 evolution, permitting different regions of the PRYSPRY domain to evolve independent interactions with capsid.Trim5蛋白对逆转录病毒衣壳的识别可阻断病毒的有效感染。恒河猴携带三种功能迥异的Trim5等位基因：Trim5αQ、Trim5αTFP与Trim5CypA。尽管Trim5αQ与Trim5αTFP等位基因的氨基酸同源性极高，但Q/TFP多态性导致二者对部分灵长类慢病毒的限制活性存在显著差异，提示这两种等位基因与病毒衣壳的结合机制存在区别。恒河猴源猴免疫缺陷病毒（Simian Immunodeficiency Virus of Rhesus Macaques, SIVmac）已进化出可抵御上述三种等位基因的能力，因此SIVmac为在驱动其体内进化的Trim5基因库背景下开展病毒研究提供了独特契机。本研究利用该衣壳对恒河猴Trim5α的天然抗性，筛选出可区分Trim5αQ与Trim5αTFP等位基因靶标的敏感性获得突变体。尽管两种等位基因均可识别衣壳表面，但Trim5αQ与Trim5αTFP在限制一组嵌合衣壳及单氨基酸替换病毒的能力上存在差异。将影响两种等位基因的单氨基酸替换位点映射至SIVmac239衣壳N端结构域的三维结构后发现，这些位点均集中于β发夹结构。鉴于所有替换均不会仅影响Trim5αQ，且β发夹结构在逆转录病毒衣壳中高度保守，我们提出β发夹结构是Trim5α蛋白广泛利用的一类分子模式。特异性影响恒河猴Trim5αTFP（不影响Trim5αQ）的突变位点环绕着一个灵长类慢病毒特有的保守位点，该位点与CPSF6结合位点重叠。我们认为，靶向该位点是过去1200万年间非洲灵长类慢病毒出现所驱动的一项进化创新。这种靶向模块化特性可能是Trim5进化的普遍特征，允许PRYSPRY结构域的不同区域独立进化出与衣壳的互作结合能力。