Genomic Determinants of Intrinsic Antiviral Host Defenses

描述（由申请人提供）：对HIV-1感染的易感性在个体之间差异很大，疾病进展率不仅取决于病毒特性，还取决于宿主因素。该提议的中心假设是APOBEC 3（A3）限制因子家族的广泛多样性导致了HIV-1体内复制的高度变异性。越来越多的证据表明，基因组变异-从单核苷酸多态性（SNP）到大的结构变异-影响宿主抗病毒防御的功效。事实上，我们有证据表明，天然存在的SNP调节A3 F和A3 H的抗病毒活性。此外，我们已经确定了A3基因座附近和内部的三个区域，这些区域显示了HapMap人群之间的拷贝数变异（基于来自西非，亚洲和欧洲血统的450名个体的数据）。这些拷贝数变体（CNV）与A3 B、A3 E和A3 F外显子以及APOBEC基因座的可能上游调控序列重叠。初步数据表明，来自HIV-1感染的长期非进展者（LTNP）的APOBEC基因座富含A3拷贝数变异，表明A3拷贝数变异的保护作用。相反，我们已经证明，A3-CNV 2拷贝丢失（A3 B基因完全缺失）纯合的细胞在体外更容易感染HIV-1。总的来说，我们的初步数据强烈支持的概念，即A3区的结构多态性和由此产生的A3变异体对HIV-1的复制产生关键影响。在具体目标1（CNV发现和定位）中，我们将使用尖端技术以非常高的分辨率询问A3基因家族簇，以捕获嵌入该位点的广泛的CNV。为此，我们将使用来自不同种族（18个不同群体）或表型选择（LTNP）人群的样本。在具体目标2（基因表达）中，我们将确定A3-CNV基因型特异性的mRNA表达模式，并鉴定与重排A3基因座组相关的蛋白质变体。我们还将比较LTNP和治疗进展者的淋巴细胞和树突状细胞中A3基因组变体的转录调控。在具体目标3（基因功能）中，将使用来自具有已知A3基因型个体的淋巴细胞以及表达代表自然循环A3基因组变体的基因产物的模型细胞系，检测A3-CNV和SNP对病毒限制的影响。这些实验将整合A3区域的基因组变异数据，了解A3脱氨酶的调节和抗病毒功能。A3基因组结构的高变异性的功能性后果的全面调查，应有助于确定新的生物标志物，在未来的逆转录病毒感染的流行病学研究进行测试。此外，了解内在防御因子的功能多样性有可能创造新的治疗策略，旨在长期抑制HIV/AIDS疾病。 公共卫生相关性：个人对HIV-1感染和艾滋病疾病进展的易感性差异很大。该提案的目标是确定APOBEC 3基因座内结构基因组变异的程度，并评估这些内在抗性基因的变异性如何影响HIV-1复制。

DESCRIPTION (provided by applicant): Susceptibility to HIV-1 infection varies greatly between individuals, with disease progression rates determined not only by viral properties but also host factors. The central hypothesis of this proposal is that the extensive diversification of the APOBEC3 (A3) family of restriction factors contributes to the high degree of variability in HIV-1 replication in vivo. There is growing evidence that genomic variation - ranging from single nucleotide polymorphisms (SNPs) to large structural variants - influences the efficacy of host antiviral defenses. Indeed, we have evidence that naturally occurring SNPs modulate the antiviral activity of A3F and A3H. Moreover, we have identified three regions near and within the A3 locus itself that show copy number variations among HapMap populations (based on data from 450 individuals of West-African, Asian and European ancestry). These copy number variants (CNV) overlap with A3B, A3E and A3F exons and possibly upstream regulatory sequences of the APOBEC locus. Preliminary data indicate that the APOBEC loci from HIV-1 infected long-term non-progressors (LTNP) are enriched for A3 copy number variants, suggesting a protective role of A3 copy number variation. Conversely, we have shown that cells homozygous for A3-CNV2 copy loss (complete deletion of the A3B gene) are more susceptible to HIV-1 infection in vitro. Collectively, our preliminary data strongly support the notion that A3 regions are structurally polymorphic and that the resulting A3 variants exert a key impact on HIV-1 replication. In specific aim 1 (CNV discovery and mapping), we will interrogate the A3 gene family cluster at very high resolution using cutting edge technologies to capture the wide range of CNVs embedded in this locus. For this purpose, we will use samples from ethnically diverse (18 different groups) or phenotypically selected (LTNP) populations. In specific aim 2 (gene expression), we will determine the mRNA expression pattern specific for A3-CNV genotypes and identify protein variants associated with the panel of rearranged A3 loci. We will also compare the transcriptional regulation of A3 genomic variants in lymphocytes and dendritic cells of LTNPs and treated progressors. In specific aim 3 (gene function), the impact of A3-CNVs and SNPs on viral restriction will be tested, using lymphocytes from individuals with known A3 genotypes, as well as model cell-lines expressing gene products representing naturally circulating A3 genomic variants. These experiments will integrate genomic variation data of the A3 region with an understanding of the regulation and antiviral functions of A3 deaminases. This comprehensive investigation into the functional consequences of the high variability in A3 genomic architecture should help identify new biomarkers to be tested in future epidemiological studies of retroviral infections. In addition, understanding the functional diversity of intrinsic defense factors has the potential to create novel treatment strategies aimed at a long- lasting suppression of HIV/AIDS disease. PUBLIC HEALTH RELEVANCE: Susceptibility to HIV-1 infection and AIDS disease progression varies greatly among individuals. The goal of this proposal is to determine the degree of structural genomic variation within the APOBEC 3 locus, and to assess how variability of these intrinsic resistance genes impacts on HIV-1 replication.