Evolution of a TRIM5-CypA Splice Isoform in Old World Monkeys

The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5α has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3′ splice site upstream of exon 7, which may prevent or reduce expression of the α-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares ∼80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5–10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.

TRIM家族蛋白共享保守的相邻结构域排布：N端的RING结构域（RING domain），随后是1~2个B-box结构域以及卷曲螺旋（coiled-coil），这三者构成了该家族命名来源的三价基序（tripartite-motif）。然而，TRIM蛋白的C端区域差异显著，包含至少9个进化上独立、无同源性的蛋白质结构域。抗病毒限制因子TRIM5α的C端带有B30.2/SPRY结构域，这是决定病毒靶标特异性的主要区域。本研究阐述了亚洲猕猴TRIM5基因座下游编码亲环蛋白A（cyclophilin-A, CypA）的外显子的进化历程。可变剪接可产生嵌合转录本，编码与C端CypA结构域融合的TRIM基序，即TRIM5-CypA。我们在两种猕猴的原代淋巴细胞中检测到了TRIM5-CypA嵌合转录本。这类转录本部分来源于TRIM5基因座内的CypA假基因，其与此前在枭猴TRIM5中发现的CypA插入序列截然不同。该CypA插入序列与第7外显子上游的3'剪接位点突变相关，该突变可能会阻碍或降低α亚型的表达。所有被筛查的猪尾猕猴（Macaca nemestrina）均为CypA插入序列的纯合子。与之相反，在101只恒河猴（Macaca mulatta）中，携带CypA插入等位基因的个体占比为17%（17/101）。携带TRIM5-CypA等位基因的动物淋巴细胞中，HIV-1感染阻断效应弱于野生型动物细胞。尽管HIV-1的感染性仍显著低于SIV，但环孢素A（cyclosporine A, CsA）处理无法挽救该感染缺陷。因此，与枭猴TRIMCyp不同，猕猴TRIM5-CypA亚型的表达并不会增强对HIV-1的限制作用。尽管进化起源截然不同，但猕猴TRIM5-CypA的结构域排布与枭猴的TRIMCyp蛋白相似，氨基酸序列一致性约为80%。两个灵长类支系独立演化出TRIM5-CypA嵌合蛋白，这是趋同进化的一个典型案例。基于CypA插入序列在不同猕猴支系中的存在情况以及其在乌白眉猴（sooty mangabeys）中的缺失情况，我们估算猕猴TRIM5-CypA变体出现于500万~1000万年前的亚洲猕猴共同祖先中。通过可变剪接形成新基因是否在TRIM家族的进化中发挥了更广泛的作用，仍有待进一步研究。