| 其他摘要 | 宿主在与病毒千百万年的博弈中进化出天然限制因子（restriction factor），构成机体“固有免疫”（intrinsic immunity）防线。TRIM5α（tripartite motif 5 alpha）是一种宿主特异性的限制因子，能限制包括人免疫缺陷病毒I型（human immunodeficiency virus type 1, HIV-1）、N型鼠白血病病毒（N-tropic murine leukemia virus，N-MLV）、猴免疫缺陷病毒I型（simian immunodeficiency virus, SIV）、猫免疫缺陷病毒（feline immunodeficiency virus，FIV）以及马传染病贫血病毒（equine infectious anemia virus，EIAV）等在内的逆转录病毒的复制。TRIM-cyclophilinA（TRIMCyp）融合蛋白是TRIM5α的一种剪接异构体，由亲环素（CyclophilinA，CypA）经LINE-1介导逆转座于TRIM5基因形成TRIMCyp融合基因后，选择性剪接形成。现已在灵长类中发现3种独立起源的TRIMCyp，分别为鹰猴TRIMCyp模式、北平顶猴TRIMCyp模式和TRIMCypA3模式。TRIMCyp与TRIM5α一样以物种依赖的方式限制逆转录病毒，并以其基因型的不同影响宿主的感染进程。TRIMCyp在不同物种中的独立形成和进化揭示了宿主与病毒互作时产生的趋同进化现象，提示TRIMCyp的形成不是随机，而是在病毒压力下形成。因此，深度探究TRIMCyp的限制能力和机制对于拓展对限制因子的认识、了解宿主和病毒的共同进化和提供针对病毒的基因治疗策略具有重要意义。本实验室前期发现猕猴属的熊猴（Macaca assamensis）表达北平顶猴TRIMCyp模式的TRIMCyp融合基因，并以amTRIMCyp/amTRIM5α杂合子的形成存在。由于同一物种中的TRIMCyp和TRIM5α的相互作用及其对限制能力产生的影响研究尚未见报道，因此我们在检测amTRIMCyp和amTRIM5α各自限制逆转录病毒能力的基础上，更通过稳定共表达模拟两者在宿主细胞内的生理表达情况，以研究两者互作带来的影响。我们发现，amTRIMCyp和amTRIM5α均能有效限制HIV-1的复制，amTRIM5α而非amTRIMCyp可有效限制N-MLV-GFP。当amTRIMCyp和amTRIM5α共表达时，细胞限制HIV-1能力极大增强，是单转染细胞的50-100倍，表明两者对HIV-1的限制能力有协同作用。该协同作用发生在HIV-1复制的逆转录早期，与amTRIMCyp和amTRIM5α的自身泛素化能力无关，并部分独立于amTRIMCyp与HIV-1衣壳蛋白的结合。免疫共沉淀显示amTRIMCyp和amTRIM5α直接结合，突变amTRIM5α破坏两者的结合使协同作用减弱，提示amTRIMCyp和amTRIM5α的协同抗病毒作用依赖于amTRIMCyp与amTRIM5α的结合。与过表达实验结果对应的是，TRIMCyp/TRIM5α杂合子熊猴PBMC比TRIM5α纯合子熊猴PBMC更加不易感HIV-1。熊猴TRIMCyp和TRIM5α强大的协同限制作用拓展了对同源TRIMCyp和TRIM5α互作的认识，为动物模型的选择提供了理论基础，也为HIV-1的基因治疗提供了新的策略。此外，本实验室前期在非灵长类的树鼩（Tupaia belangeri）基因组中发现，树鼩不仅有5个TRIM5基因拷贝，还有TRIMCyp融合基因存在的现象。本研究集合了进化分析手段和功能验证的方法，针对该TRIMCyp的起源历程、进化机制、新功能产生机制等方面进行了细致研究。我们发现树鼩TRIMCyp融合基因的起源独立于其它3种灵长类TRIMCyp，是第4种新模式的TRIMCyp融合基因。该基因的形成集合了基因重复、逆转座和外显子重组3种重要的新基因产生模式，是一种罕见的新基因形成方式。CypA经逆转录作用插入树鼩TRIM5的第8外显子上游，选择性剪接时第2-7外显子与CypA逆转座子相连接，从而转录翻译为树鼩TRIMCyp融合蛋白。树鼩TRIMCyp受到强烈正向选择且高表达于免疫相关组织器官，并且显著高于其姐妹基因TRIM5-4。树鼩TRIMCyp不能限制HIV-1、SIVmac和N-MLV等病毒的复制，其不能限制HIV-1的原因不在于CypA提前终止，而在于其N端的RBCC结构域序列存在突变。人工构建的树鼩TRIMCyp原始基因则具有限制HIV-1的能力，提示该基因的形成也许不是偶然，而是在病毒压力下形成的。有趣的是，树鼩TRIMCyp而非TRIM5-4能负调控仙台病毒（SeV）介导的抗病毒天然免疫应答，提示树鼩TRIMCyp丧失了病毒限制功能而产生了新的免疫调控功能。本研究不仅扩展了对病毒限制因子TRIMCyp/TRIM5α的功能和机制的理解，也为研究新基因产生、新功能产生和姐妹基因功能分化等提供了良好的素材。; For millions of years, the co-evolutionary arms race between hosts and the invading pathogens has driven the generation of several types of restriction factors, which constitute the intrinsic immunity defense line. As a species-specific restriction factor, TRIM5α exhibits potent restriction against retroviruses such as human immunodeficiency virus type 1 (HIV-1)、N-tropic murine leukemia virus (N-MLV)、simian immunodeficiency virus (SIV)、Feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV). TRIM-cyclophilinA (TRIMCyp) is an alternatively spliced isoform of TRIM5α, arose via a LINE-1 mediated retrotransposition of cyclophilinA (CypA) inserting into TRIM5 locus, leading to the formation of TRIMCyp gene fusion. To date, there are three independently generated modes of TRIMCyp found in primates, i.e. owl monkey TRIMCyp (omTRIMCyp), northern pig-tailed macaque TRIMCyp (npmTRIMCyp) and TRIMCypA3. TRIMCyp restricts retroviruses in a species-specific manner, and modulates immunodeficiency virus control in Asian macaques. The independent generation and evolution of TRIMCyp in distinct species showed convergent evolution in the long-time arms race between hosts and pathogens, suggesting that under the pressure of outer threatens, genome favors the prepared genes. Therefore, investigating the restriction capacity and underlying mechanism of TRIMCyp/TRIM5α can not only help replenish the knowledge of restriction factors, shed new light on the co-evolutionary of hosts and viruses, but also provide new strategies to gene therapy of HIV-1.In our previous study, we have found that assam macaque (Macaca assamensis) express a npmTRIMCyp chimera (amTRIMCyp), along with a TRIM5α allele (amTRIM5α). As there is no research on how homologous TRIMCyp and TRIM5α work together up to date, we set out to investigate the antiviral activity of each of amTRIMCyp and amTRIM5α, as well as the interaction and joint work of them. We found that both of amTRIMCyp and amTRIM5α can potently restrict HIV-1; amTRIM5α is restrictive against N-MLV while amTRIMTCyp is not. Surprisingly, when amTRIMCyp and amTRIM5α were co-expressed, the restriction ability increased dramatically by 50~100 fold compared with the single plasmid-transfected cells, indicating that there is synergistic effect on HIV-1restriction. This synergistic effect occurred at the early reverse transcription stage of HIV-1, uncoupled with self-ubiquitylation of amTRIMCyp or amTRIM5α, and was independent of the binding of capsid with the CypA domain of amTRIMCyp. Co-immunoprecipitation assay showed that amTRIMCyp associates with amTRIM5α. Disruption of the interaction between amTRIMCyp and amTRIM5α attenuates the synergistic effect on HIV-1 restriction, indicating that the synergistic effect depends on the association between amTRIMCyp and amTRIM5α. In good agreement of over-expression assays, TRIMCyp/TRIM5α heterozygous PBMCs showed less susceptibility of HIV-1than TRIM5α homozygous PBMCs. The mighty anti-HIV-1 synergistic effect contributes to better understand how homologous TRIMCyp and TRIM5α work together, providing theoretical basis of utilizing animal models with various TRIMCyp/TRIM5α alleles, and enlightening novel gene therapy strategies to block HIV-1 replication.On the other hand, our previous study which conducted on the Chinese tree shrew (Tupaia belangeri) draft genome showed multiple TRIM5 copies (tsTRIM5) and a possible TRIMCyp chimera (tsTRIMCyp). In the present study, by using integrated evolutionary and functional analyses as well as comparative genomic analyses, we investigate the gene generation, evolutionary trajectory, selective pressure, expression pattern and function of tsTRIMCyp. We showed that tsTRIMCyp originated independently in comparison with previously reported TRIMCyps, making it the novel fourth mode of TRIMCyp gene fusion. Meanwhile, the formation of tsTRIMCyp is a rare example of novel gene origination resulting from a combination of gene duplication, retrotransposition and exon shuffling processes. To form tsTRIMCyp, CypA was retrotransposed dozens of base pairs downstream the beginning of exon 8 of TRIM5, resulting in exon 2-7 fused with CypA cDNA and therefore TRIMCyp fusion protein. tsTRIMCyp underwent strong positive selection, while no signal of positive selection was detected for other tsTRIM5 genes. By both evolutionary analyses and functional assays, we showed the likely evolutionary trajectory of tsTRIMCyp, i.e., tsTRIMCyp acquired the ability of restricting paleoviruses at the time of its birth. After that, a decrease in environmental virus threatens allowed for the accumulation of residue changes followed by residue alterations in the RBCC domain enabled tsTRIMCyp to obtain new function. Notably, Functional assay results suggest a functional divergence between tsTRIMCyp and its closest paralog TRIM5-4 in the regulation of SeV-mediated signaling pathway, which not only reflects different fates under diverse evolutionary forces, but also presents a new mode of function of TRIMCyps. Our findings not only extend our understanding of evolutionary trajectory and function of non-primate TRIMCyp/TRIM5α, but also provide a new paradigm to study genetic innovations and evolutionary fates of duplicated genes. |
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