Biophysical Signatures in HIV-1 Env Correlating with Mother-to-Child Transmission

描述：HIV的特征之一是其极端的易变性，这导致病毒准种在感染后广泛多样化。在粘膜传播过程中，病毒也经历相反的现象，经历基因型多样性的严格缩小，因此大多数新感染是由准物种的单一传播的“创始人”（T/F）变体建立的。传播的物理决定因素仍然定义不清。虽然许多研究已经将T/F的特征追溯到介导感染的表面糖蛋白Env，但大多数研究都只研究了潜在差异的生物学后果，而无法解决一个Env与另一个Env的具体区别，因为经典方法不适合分析糖蛋白的结构变化。晶体学的要求使得有必要截断和去糖基化糖蛋白，去除使每个变体不同的特征，如可变环和糖基化，并可以调节核心保守区域内的结构。我们建议采用新的生物物理和结构方法来比较来自HIV T/F变体和来自供体变体的Env糖蛋白的结构，使用在一项具有良好特征的母婴传播（MTCT）研究中鉴定的传播对。我们开发了氢/氘交换质谱（HDX-MS）在天然条件下探测完整糖蛋白结构的方法。这种方法提供了识别变异特异性差异所需的灵敏度和分辨率，在整个糖蛋白中产生局部结构秩序和稳定性的指纹。在AIM 1中，我们将应用HDX-MS分析在内罗毕母乳喂养MTCT研究中发现的匹配传播对的Env。传播对提供了一种强有力的手段来查明可能与T/F变异传播相关的结构和表型差异相关的序列差异。我们假设T/F环境中的结构特性可能存在，包括更大的稳定性，赋予T/F变体选择优势。在AIM 2中，我们将使用感染性试验来测试T/F变异是否对热失活和低pH值和乳酸失活表现出增强的抵抗力，这些因素可能存在于传播部位，如婴儿胃肠道。Lee和Overbaugh实验室将结构生物学和病毒学专业知识相结合，用于检查匹配的传播对，可以为病毒传播性的物理决定因素提供重要的新见解，并提供与疫苗免疫原设计相关的结构细节。

DESCRIPTION: One of the hallmarks of HIV is its extreme mutability, which leads to broad diversification of the viral quasispecies following infection. During mucosal transmission, the virus experiences the opposite phenomenon as well, undergoing a stringent narrowing of genotypic diversity, such that the majority of new infections are established by a single transmitted, "founder" (T/F) variant from the quasispecies. The physical determinants of transmission remain poorly defined. While many studies have traced T/F signature traits to Env, the surface glycoprotein that mediates infection, most have examined the biological consequences of underlying differences without being able to address what specifically differentiates one Env from another because classical methods are poorly suited to analyze structural variation of glycoproteins. The requirements for crystallography make it necessary to truncate and deglycosylate the glycoprotein, removing the very features such as variable loops and glycosylation that make each variant distinct and that can modulate structure within core, conserved regions. We propose to apply novel biophysical and structural approaches to compare the structure of Env glycoproteins from T/F variants of HIV with Env from donor variants using transmission pairs identified in a well-characterized mother-to-child-transmission (MTCT) study. We have developed the use of hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to probe intact glycoprotein structure under native conditions. This approach provides the sensitivity and resolution necessary to identify variant-specific differences, producing a fingerprint of local structural order and stability throughout the glycoprotein. In AIM 1, we will apply HDX-MS to analyze Env from matched transmission pairs identified in a Nairobi Breastfeeding MTCT study. The transmission pairs provide a powerful means to pinpoint sequence differences that may be linked with structural and phenotypic differences relevant to transmission of T/F variants. We hypothesize that structural properties in T/F Env may exist including greater stability that confer a selective advantage to T/F variants. We anticipate that greater Env stability may result in complexes that are resistant to inactivation In AIM 2, using infectivity assays, we will test whether T/F variants exhibit enhanced resistance to heat inactivation and inactivation by low pH and lactic acid, factors that may be present at sites of transmission such as the infant gastrointenstinal tract. The combination of structural biology and virological expertise in the Lee and Overbaugh labs applied to examine the matched transmission pairs can provide significant new insights into the physical determinants of virus transmissibility and provide structural details relevant to vaccine immunogen design.