Identification of Candidate Gene Polymorphisms Associated with Infectious Diseas

我的实验室的主要目标是确定导致传染病和其他复杂疾病的宿主因素。传染病对全球健康的巨大影响，以及许多人类病原体与常见癌症的关联，要求采取多种研究策略来阐明感染机制和发病机制。我们的策略是寻找对感染率或发病过程有不同影响的基因变异，从而确定包含该变异的基因参与感染或发病过程，增加对发病机制的了解，并指出治疗干预的靶点。我们的重点是发现调节艾滋病毒-1、丙型肝炎病毒和乙肝病毒感染及相关疾病的遗传因素。为此，我们开展了国际合作，在中国和南非建立病例对照和队列研究，分别调查与EBV和HB病毒相关的艾滋病毒-1、乙肝病毒和丙型肝炎以及常见的癌症--鼻咽癌和肝癌。我们已经与博茨瓦纳哈佛伙伴关系建立了合作关系，以在一个受艾滋病毒-1 C亚型感染严重影响的地区调查艾滋病毒-1感染、进展和抗逆转录病毒治疗反应的遗传相关性。C亚型感染是全球艾滋病毒-1感染的主要原因。使用候选基因和全基因组关联方法，我们使用了高通量的基因分型技术，包括Illumina和Affymetrix，以发现与HIV-1相关性肾病和进展为艾滋病相关的基因。每当观察到显著的关联时，实验室就使用精细的图谱来识别假定的因果等位基因，并使用功能分析来评估对基因转录和蛋白质水平的影响。成就：APOBEC3G是人类对HIV-1的一种先天抵抗因子，它被整合到萌芽中的病毒粒子中。我们之前已经证明，在HIV-1感染者中，编码APOBEC3G的基因的多态性与艾滋病的进展速度和CD4+T细胞下降的轨迹有关。APOBEC3G在缺乏HIV-1编码的病毒感染性因子(VIF)的情况下，会导致新生cDNA超突变，有效地阻止病毒整合和有效感染。然而，在导致APOBEC3G降解的泛素化途径中，人类APOBEC3Gs的抗HIV-1活性可通过与包括cullin5(CUL5)在内的蛋白质相互作用而被HIV-1 vif解除。通过对不同临床结局的HIV-1自然病史队列的研究，我们发现CUL5的变异等位基因和单倍型簇影响CD4+T细胞耗尽率。一个改变转录结合效率的内含子SNP与2.5倍的艾滋病快速进展有关。这一发现突出了CUL5在APOBEC3G降解途径中的重要性，并表明CUL5可能是药物开发的靶点。TRIM5最近被确定为关键的逆转录病毒限制因子，在猕猴体内，在进入猕猴后但在前病毒整合之前，对HIV-1感染提供了完全的限制。这种限制的机制被认为是由于TRIM5蛋白对病毒衣壳的作用，导致其通过泛素化途径过早分解和降解。人类TRIM5在限制HIV-1方面的作用也不大。我们已经完成了对欧洲人、非裔美国人、亚洲人和南非科萨族人TRIM5基因遗传变异程度和单倍型结构的全面调查。我们还发现3个SNPs(1个启动子和2个非同义SNPs)与HIV-1感染的易感性改变有关。为了扩大我们对与病毒衣壳相互作用的宿主蛋白的研究，我们对编码亲环素A(CypA)的PPIA基因的多态程度进行了全面的遗传分析。CypA是亲环素家族的一员，具有肽基-脯氨基顺式-反式异构酶活性，在HIV-1病毒衣壳萌发之前被整合到衣壳中，通过促进衣壳去涂层促进HIV-1的感染性。在一项遗传关联研究中，我们发现PPIA S启动子区域的功能变异与更快的CD_4+T细胞丢失和进展相关，显然是通过上调PPIA的表达。我们还确定了TRIM5和PPIA之间不存在基因-基因相互作用，尽管这两个编码宿主因子都作用于HIV-1衣壳的分解。这些发现可能有助于合理设计一类针对或模拟先天逆转录病毒限制因子(如APOBEC3G或TRIM5)和宿主因子(如HIV-1为完成其生命周期而选择的CypA和CUL5)的新型抗HIV药物

The major objective of my laboratory is to identify host factors that contribute to infectious and other complex diseases. The tremendous impact of infectious diseases on global health, and the association of many human pathogens with common cancers, call for multiple research strategies to elucidate the mechanisms of infection and pathogenesis. Our strategy is to search for genetic variants that differentially affect rates of infection, or the course of pathogenesis, and which thereby identify the gene containing the variant as participating in the process of infection or pathogenesis, increasing knowledge of the mechanisms of pathogenesis and pointing to targets for therapeutic intervention. Our focus has been to discover genetic factors modulating HIV-1, HCV, and HBV infections and associated diseases. To this end, we have developed international collaborations to establish case-control and cohort studies in China and southern Africa to investigate HIV-1, HBV, and HCV as well as the common carcinomas, NPC and HCC, associated with the EBV and HB viruses, respectively. We have established a collaboration with the Botswana Harvard Partnership to investigate the genetic correlates of HIV-1 infection, progression, and response to antiretroviral therapy in a region severely impacted by HIV-1 subtype C infection, the subtype responsible for the majority of HIV-1 infections globally. Using both candidate gene and genome wide association approaches, we have employed high throughput genotyping technologies, including Illumina and Affymetrix, to discover genes associated with HIV-1-associated nephropathy and with progression to AIDS. Whenever a significant association is observed, the laboratory uses fine mapping to identify putative causal alleles and functional assays to assess effects on gene transcription and protein levels. Accomplishments: APOBEC3G is a human innate resistance factor to HIV-1 that is incorporated into budding virions. We previously have shown that polymorphism in the gene encoding APOBEC3G is associated with rate of progression to AIDS and trajectory of CD4+ T cell decline in HIV-1-infected persons. APOBEC3G, in the absence of HIV-1 encoded viral infectivity factor (vif), causes hypermutation of the nascent cDNA, effectively preventing viral integration and competent infection. Human APOBEC3Gs anti-HIV-1 activity is, however, disarmed by HIV-1 vif by interaction with proteins, including Cullin5 (CUL5), in the ubiquitination pathway leading to the degradation of APOBEC3G. Through a study of HIV-1 natural history cohorts with different clinical outcomes, we discovered that variant alleles and haplotype clusters in CUL5 influences the rate of CD4+ T-cell depletion. One intronic SNP that alters transcription binding efficiency is associated with 2.5-fold more rapid progression to AIDS. This finding highlights the importance of CUL5 in the degradation pathway of APOBEC3G and suggests that CUL5 may be a target for drug development. TRIM5 has recently been identified as a key retrovirus restriction factor and in the Rhesus macaque provides a complete restriction to HIV-1 infection post entry but before proviral integration. The mechanism for this restriction is believed to result from the action of TRIM5 protein on the viral capsid causing its premature disassemble and degradation via a ubiquitination pathway. Human Trim5 also has a modest effect on restricting HIV-1. We have completed a comprehensive survery of the extent of genetic variation and haplotype structure of the TRIM5 gene in European and African Americans, Asians, and the Xhosa from South African. We have also shown that three SNPs (1 promoter and 2 nonsynonymous SNPs) were associated with altered susceptibility to HIV-1 infection. To extend our investigation of host proteins that interact with viral capsid we did a comprehensive genetic analysis of the extent of polymorphism in PPIA, encoding cyclophilin A (CypA). CypA, a member of the family of cyclophillins that possess peptidyl-prolyl cis-trans isomerase activity, is incorporated into the HIV-1 viron capsid prior to budding and promotes HIV-1 infectivity by facilating capsid uncoating. In a genetic association study, we discovered that functional variants in the promoter region of PPIA s are associated with more rapid CD4+ T-cell loss and progression to AIDS, apparently by up-regulating PPIA expression. We also determined that there were no gene-gene interactions between TRIM5 and PPIA, although both encode host factors act on disassembly of the HIV-1 capsid. These findings may inform the rational design of a new class of anti-HIV therapeutics that target or mimic innate retroviral restriction factors such as APOBEC3G or TRIM5 and host factors such as CypA and CUL5 co-opted by HIV-1 for completion of its life cycle