熊猴存在TRIM5/TRIMCyp杂合子基因型

缺乏合适的动物模犁是制约艾滋病研究取得重大突破的关键瓶颈之一.细胞内的抗病毒蛋白被称为限制因子.研究不同灵长类动物抗HIV-1宿主限制因子的存在形式及作用机制对建立合适AIDS灵长类动物模型有十分重要的意义.TRIM5α是哺乳动物细胞中一种重要和关键的限制因子,它以物种依赖的方式限制包括HIV-1在内的逆转录病毒的感染.TRIM5-CypA融合基因是存在于新大陆猴与旧大陆猴中的一种独特的TRIM5基因形式.为了研究不同灵长类动物TRIM5基因的存在方式,该文对熊猴、藏婀猴、红面猴及中闰恒河猴4个物种共110只灵长类动物进行了TRIM5-CypA融合模式的研究.首次发现熊猴也存在TRIM5-CypA基因融合现象.熊猴TRIMCyp融合基因形成模式类似于北平顺猴TRIMCyp融合基因模式,即CypA假基因的cDNA序列通过逆转座方式插入到TRIM5基凶的3'-UTR区域.基因序列分析表明,该基因与北平顶猴相应基因序列高度相似;并且其TRIM5内含子6的3'-剪接位点也相应存存G-to-T突变现象(G/T).这提示熊猴也极有可能像北平顶猴一样表达TRIM5-CypA融合蛋白,从而导致熊猴可能跟北平顶猴一样可能被HIV-1感染.因此,熊猴极有希望成为一种新的HIV/AIDS灵长类动物模型.     

中国圈养食蟹猴TrimCyp基因频率

TRIM 5α在绝大部分的旧大陆猴中扮演抗逆转录病毒的角色,能够限制HIV-1的活性。TRIMCyp融合基因是继TRIM 5α后的另一个抗HIV-1因子研究热点。旧大陆猴的TRIMCyp融合基因是由CypA假基因cDNA序列以逆转录转座的方式插入至TRIM5基因的3’非翻译区形成,而且TRIMCyp融合基因在不同灵长类动物中具有地域、基因频率、基因型以及抗逆转病毒效应的差异。虽然食蟹猴TRIMCyp基因的频率在东南亚几个国家或地区已经被初步调查,但是,中国大陆食蟹猴养殖场的TRIMCyp基因频率还没有明确阐明。该研究对中国5个省11个养殖场共1594个食蟹猴(Macaca fascicularis)繁殖种群随机样本的TRIMCyp基因频率进行了筛查研究,发现各场频率略有差异,从7.65%～19.79%不等,显著低于已报道的菲律宾、马来西亚和印度尼西亚来源食蟹猴的TRIMCyp基因频率(34.85%~100%)。该原因可能是由于后者是建立于1978年的封闭群。对带有TRIMCyp融合基因的个体CypA测序发现带有NE单倍型的食蟹猴个体很少,NE单倍型频率(4.93%)显著低于东南亚三个国家食蟹猴的NE单倍型频率(11.1%～14.3%)纯合子。该研究为进一步开展食蟹猴HIV-1动物模型和发病机制提供了基础信息。     

灵长目动物TRIM5基因的进化及其抗逆转录病毒的种属差异性

灵长目动物TRIM5基因能编码TRIM5α蛋白,它在逆转录病毒侵染宿主细胞的早期阶段抑制病毒复制。同时科学家也在灵长目动物中发现了TRIM5基因的另外一种融合蛋白基因型——TRIMCyp,由于发生的剪接机制不同,新、旧大陆猴分别形成了不同的融合蛋白,从而导致它们抗病毒活性的广泛差异。TRIM5基因在灵长目动物进化过程中产生了很多突变位点,这些突变位点也使灵长目动物抗逆转录病毒的活性存在种属差异。本文简述了TRIM5基因及编码蛋白的结构,从自然选择的角度阐述了灵长目动物TRIM5基因的进化,揭示了由TRIM5基因多样性所造成的抗病毒种属差异,从而为研究灵长目动物的进化以及人猴共患疾病模式生物的构建提供一定的指导。     

发现平顶猴易感HIV-1重要分子机制

在国家自然科学基金和“973”项目的支持下，中国科学院昆明动物研究所研究员郑永唐组和宿兵组合作，发现在旧大陆猴平顶猴对HIV-1易感的重要分子机制，并首次发现新型天然免疫分子mnTRIMCyp。此项研究进一步阐明了天然免疫分子TRIM5α限制HIV-1感染和复制的作用机制，也为国际HIV／AIDS研究广泛关注的HIV／AIDS非人灵长类动物模型建立提供了重要思路。     

平顶猴在HIV/AIDS动物模型中的应用及研究进展

非人灵长类动物模型在HIV-1致病机制研究以及抗AIDS药物和疫苗研发中具有重要作用。由于缺乏HIV-1直接感染的动物,SIV/SHIV猕猴模型是目前AIDS研究中应用最为广泛的动物模型。虽然SIV/SHIV猕猴模型与人AIDS具有一定的相似性,但SIV/SHIV与HIV-1间的遗传差异较大,致使SIV/SHIV猕猴模型存在很多局限性。创建合适的非人灵长类动物模型仍然是HIV/AIDS研究中的热点和难点。平顶猴是目前唯一可以被HIV-1感染的旧大陆猴,在HIV-1静脉传播和性传播模型研究中具有许多优势。该文综述了SIV、HIV、SHIV和HSIV通过静脉和黏膜途径感染平顶猴的特征,并简要介绍了病毒在平顶猴细胞中复制的分子机制以及建立平顶猴AIDS模型的限制因素和前景。     

打靶恒河猴CD4~+ T细胞的TRIM5α基因影响其感染HIV的能力

目的:探讨打靶恒河猴CD4~+ T细胞的TRIM5α基因对其感染HIV-1能力的影响。方法:从恒河猴的外周血中通过磁珠分选获得CD4~+ T细胞,并采用流式检测阳性率。构建打靶TRIM5α基因的TALEN质粒,通过电转导入CD4~+ T细胞,流式分选出转染TALEN质粒的细胞,提取基因组T7E1酶切检测打靶效率。HIV-1病毒感染打靶TRIM5α的CD4~+ T细胞,并通过ELASA检测病毒感染的情况。结果:成功地从恒河猴的外周血中分选出了CD4~+ T细胞,流式检测阳性率为99.5%。打靶TRIM5α基因的TALEN质粒转染CD4~+ T细胞的转染效率约为24.8%,并可成功打靶TRIM5α,打靶效率约为40%。ELASA检测结果表明打靶TRIM5α的恒河猴CD4~+ T细胞对HIV病毒的感染能力增强。结论打靶恒河猴CD4~+ T细胞的TRIM5α基因可使其易感HIV病毒,为进一步建立恒河猴HIV-1感染动物模型奠定基础。     

恒河猴TRIM5α的组织分布及不同刺激促进PBMC中TRIM5α转录水平的上调

TRIM5α(tripartite motif protein 5-alpha)蛋白是恒河猴体内一种非常重要的限制因子,能抑制人免疫缺陷病毒(HIV-1, human immunodeficiency virus type 1)、马感染性贫血病毒(EIAV, equine infectious anemia virus)和猫免疫缺陷病毒 (FIV, feline immunodeficiencyvirus)等逆转录病毒的复制。恒河猴TRIM5α的组织分布以及在受到外界刺激时TRIM5α mRNA表达量的变化研究还未见报道。本研究从中国恒河猴的各组织中提取总RNA,以β-actin基因作为内参照,通过半定量RT-PCR检测各组织中TRIM5α mRNA的表达。选择HIV-GFP-VSVG假病毒感染外周血单核细胞（peripheral blood mononuclear cell,PBMC）,非特异性刺激剂——佛波脂(Phorbol myfismte acetate,PMA)+离子霉素(ionomycin,Ion)及CD28抗体+CD49d抗体分别共刺激恒河猴PBMC,研究不同刺激对恒河猴TRIM5α mRNA表达水平的影响。结果表明：TRIM5α mRNA表达于所研究的恒河猴21种组织中,免疫系统和泌尿生殖系统组织中表达量最高,而神经系统组织,如大脑、脊髓中表达较少,其他组织中未见明显的表达差异;HIV-GFP-VSVG感染和用PMA+Ion、CD28抗体+CD49d抗体分别共刺激PBMC能促进 PBMC中TRIM5α mRNA的转录水平的上调。     

人天然免疫基因TRIM5α的序列及进化分析

目的：克隆人TRIM5α基因,对其编码序列进行分析,并进行分子进化研究。方法：以HeLa细胞cDNA为模板,用RT-PCR方法克隆人TRIM5α基因,与人基因组序列对比分析其结构;用BioEdit、Genedoc和MEGA4.1软件进行蛋白序列联配和进化分析。结果：扩增了长1482bp的人TRIM5α基因片段,序列分析表明其覆盖了完整编码框,编码由493个氨基酸残基组成的人TRIM5α蛋白;人TRIM5α蛋白与黑猩猩、大猩猩、猩猩、猕猴TRIM5α蛋白的相似度分别为98.2%、96.8%、93.7%和87.6%。结论：克隆了人TRIM5α基因,为进一步研究该基因的功能奠定了基础。     

发展平顶猴易感HIV-1重要分子机制

在国家自然科学基金和"973"项目的支持下,中国科学院昆明动物研究所研究员郑永唐组和宿兵组合作,发现在旧大陆猴平顶对HIV-1易感的重要分子机制,并首次发现新型天然免疫分子mnTRIMCyp.     

HIV-1限制因子及其基因多态性

HIV-1是造成世界艾滋病大流行的主要病毒,其在细胞中的复制过程大体分为:吸附,膜融合,脱衣壳,逆转录,入核,整合,转录翻译,病毒包装及出膜。针对这些过程,宿主细胞进化出各种细胞限制因子来限制HIV-1感染细胞。本文简要介绍TRIM5α、APOBEC3G和束缚蛋白(tetherin)等3个细胞限制因子及其基因多态性。     

The Effect of Exon 7 Deletion during the Evolution of TRIMCyp Fusion Proteins on Viral Restriction,Cytoplasmic Body Formation and Multimerization

TRIMCyp is a fusion protein consisting of the TRIM5 gene product and retrotransposed Cyclophilin A (CypA). Two primate TRIMCyp fusion proteins with varying anti-HIV-1 activities independently evolved in owl monkeys and Old World monkeys. In addition, Old World monkey TRIMCyps lack exon7, which encodes amino acids in the Linker2 region. Previous studies on TRIM5α indicated that this region affects anti-retroviral activity, cytoplasmic body formation, and multimerization. The effects of exon7 deletion on the functions of the TRIMCyp are unclear. In this study, we found that the cytoplasmic bodies and multimers of owl monkey TRIMCyp (omTRIMCyp) are different from those of northern pig-tailed macaque TRIMCyp (npmTRIMCyp). In addition, we demonstrated that exon7 deletion affected cytoplasmic body formation and multimerization. Moreover, we unexpectedly found two chimeric proteins of omTRIMCyp and npmTRIMCyp that failed to block HIV-1 replication, despite the presence of CypA in omTRIMCyp. Further studies indicated that the cytoplasmic bodies and spontaneous multimerization were not responsible for TRIMCyp anti-HIV-1 activity. Moreover, potent viral restriction is associated with higher amounts of monomeric TRIMCyp when the CypA domain is able to recognize and bind to the HIV-1 capsid. Our results suggested that the deletion of exon7 during the evolution of TRIMCyp affected its function.     

Conformational Adaptation of Asian Macaque TRIMCyp Directs Lineage Specific Antiviral Activity

TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5α but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations.     

Retrovirus restriction by TRIM5alpha variants from Old World and New World primates

The TRIM5alpha proteins of humans and some Old World monkeys have been shown to block infection of particular retroviruses following virus entry into the host cell. Infection of most New World monkey cells by the simian immunodeficiency virus of macaques (SIVmac) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5alpha orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5alpha proteins functionally resembled human TRIM5alpha, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5alpha proteins from several New World monkey species restricted infection by SIVmac and the SIV of African green monkeys, SIVagm. Spider monkey TRIM5alpha, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIVmac, SIVagm, HIV-1, and N-MLV. Tandem duplications in the TRIM5alpha B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5alpha proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5alpha proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.     

Disruption of human TRIM5alpha antiviral activity by nonhuman primate orthologues

TRIM5 is a determinant of species-specific differences in susceptibility to infection by retroviruses bearing particular capsids. Human immunodeficiency virus type 1 (HIV-1) infection is blocked by the alpha isoform of macaque TRIM5 (TRIM5alpha(rh)) or by the product of the owl monkey TRIM5-cyclophilin A gene fusion (TRIMCyp). Human TRIM5alpha potently restricts specific strains of murine leukemia virus (N-MLV) but has only a modest effect on HIV-1. The amino termini of TRIM5 orthologues are highly conserved and possess a coiled-coil domain that promotes homomultimerization. Here we show that heterologous expression of TRIM5alpha(rh) or TRIMCyp in human cells interferes with the anti-N-MLV activity of endogenous human TRIM5alpha (TRIM5alpha(hu)). Deletion of the cyclophilin domain from TRIMCyp has no effect on heteromultimerization or colocalization with TRIM5alpha(hu) but prevents interference with anti-N-MLV activity. These data demonstrate that TRIM5 orthologues form heteromultimers and indicate that C-terminal extensions alter virus recognition by multimers of these proteins.     

Evolution of the antiretroviral restriction factor TRIMCyp in Old World primates

The retroviral restriction factor TRIMCyp, which is a fusion protein derived from the TRIM5 gene, blocks replication at a post-entry step. Among Old World primates, TRIMCyp has been found in four species of Asian macaques, but not in African monkeys. To further define the evolutionary origin of Old World TRIMCyp, we examined two species of baboons (genus Papio) and three additional macaque species, including M. sylvanus, which is the only macaque species found outside Asia, and represents the earliest diverging branch of the macaque lineage. None of four P. cynocephalus anubis, one P. hamadryas, and 36 M. sylvanus had either TRIMCyp mRNA or the genetic features required for its expression. M. sylvanus genomic sequences indicated that the lack of TRIMCyp in this species was not due to genetic homogeneity among specimens studied and revealed the existence of four TRIM5α alleles, all distinct from M. mulatta and Papio counterparts. Together with existing data on macaque evolution, our findings indicate that TRIMCyp evolved in the ancestors of Asian macaques approximately 5-6 million years before present (ybp), likely as a result of a retroviral threat. TRIMCyp then became fixed in the M. nemestrina lineage after it diverged from M. nigra, approximately 2 million ybp. The macaque lineage is unique among primates studied so far due to the presence and diversity of both TRIM5 and TRIMCyp restriction factors. Studies of these antiviral proteins may provide valuable information about natural antiviral mechanisms, and give further insight into the factors that shaped the evolution of macaque species.     

Cyclophilin A renders human immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral activity

TRIM5alpha is an important mediator of antiretroviral innate immunity influencing species-specific retroviral replication. Here we investigate the role of the peptidyl prolyl isomerase enzyme cyclophilin A in TRIM5alpha antiviral activity. Cyclophilin A is recruited into nascent human immunodeficiency virus type 1 (HIV-1) virions as well as incoming HIV-1 capsids, where it isomerizes an exposed proline residue. Here we show that cyclophilin A renders HIV-1 sensitive to restriction by TRIM5alpha in cells from Old World monkeys, African green monkey and rhesus macaque. Inhibition of cyclophilin A activity with cyclosporine A, or reducing cyclophilin A expression with small interfering RNA, rescues TRIM5alpha-restricted HIV-1 infectivity. The effect of cyclosporine A on HIV-1 infectivity is dependent on TRIM5alpha expression, and expression of simian TRIM5alpha in permissive feline cells renders them able to restrict HIV-1 in a cyclosporine A-sensitive way. We use an HIV-1 cyclophilin A binding mutant (CA G89V) to show that cyclophilin A has different roles in restriction by Old World monkey TRIM5alpha and owl monkey TRIM-Cyp. TRIM-Cyp, but not TRIM5alpha, recruits its tripartite motif to HIV-1 capsid via cyclophilin A and, therefore, HIV-1 G89V is insensitive to TRIM-Cyp but sensitive to TRIM5alpha. We propose that cyclophilin A isomerization of a proline residue in the TRIM5alpha sensitivity determinant of the HIV-1 capsid sensitizes it to restriction by Old World monkey TRIM5alpha. In humans, where HIV-1 has adapted to bypass TRIM5alpha activity, the effects of cyclosporine A are independent of TRIM5alpha. We speculate that cyclophilin A alters HIV-1 sensitivity to a TRIM5alpha-independent innate immune pathway in human cells.