Determining the Functional Significance of Mutations Observed in Envelope Protein Following Serial In Vivo Passaging of Human-Simian Immunodeficiency Virus

摘要/总结： 常用的HIV-1动物模型包括用猿猴免疫缺陷病毒感染猕猴 (SIV)或含有HIV包膜（Env）或逆转录酶的猿猴-人类免疫缺陷病毒（SHIV）。 这些动物模型在理解HIV发病机制和疾病进展方面非常有用， 以及了解疫苗和药物的功效。然而，HIV-1和HIV-2之间的基因差异 和SIV，以及SHIV中其他HIV-1基因如gag、pol、vif、vpr和nef的缺失限制了 疫苗研究中的这些模型。理想地，HIV-1感染/AIDS的良好动物模型将是 携带HIV-1的猕猴然而，由于逆转录病毒的存在，HIV-1不会在猕猴细胞中复制 制约因素HIV-1可以通过用SIV基因取代其辅助基因来复制， VIF、Vpx、Vpr和Nef，它们可以抵消猕猴细胞中干扰素诱导的限制因子。类人猿 免疫缺陷病毒（HSIV）是一种HIV-1NL 4 - 3衍生物，具有SIV vif基因取代（命名为HSIV-vifNL 4 - 3） 可以在猪尾猕猴（PTM）中持续复制。然而，感染并不导致高峰病毒血症 和在猕猴SIV感染期间观察到的设定点病毒载量。PTM中的连续体内传代是 以增强HSIV的感染性或复制能力。三轮动物间转移 感染的血液在3个免疫活性PTM中，起始初始接种物含有CXCR4-（HSIV-）的混合物。 从先前感染的猕猴回收的vifNL 4 - 3）和CCR5-嗜性HSIV（基于pNL-3的HSIV-vif衍生物）。 AD8和Bru-Yu2）以产生致病性变体。有趣的是，所有的猕猴都表现出 病毒血症接近或高于105拷贝/ml，病毒复制持续超过20周。以下体内 传代，从第3代猕猴（HSIV-P3 IMC）中回收三个感染性分子克隆（IMC）。 对HSIV-P3 IMC的测序显示，在整个基因组中有几个有趣的突变，这可能表明 适应PTM。这些突变可以帮助病毒克服限制因素，或更好地利用 宿主依赖性因素，或者它们可以帮助病毒逃避宿主的免疫反应。本补助金的重点 应用是确定在包膜基因中观察到的突变的功能意义。的结果 这项研究将为了解包膜基因在HIV-1跨种属传播中的作用提供有价值的见解， 马尾猕猴

Abstract/Summary: Commonly used animal models of HIV-1 include infection of macaques with Simian Immunodeficiency Virus (SIV) or Simian-Human Immunodeficiency Virus (SHIV) containing HIV envelope (Env) or reverse transcriptase. These animal models have been extremely useful in understanding HIV pathogenesis and disease progression, as well as understanding the efficacy of vaccines and drugs. However, the genetic difference between HIV-1 and SIV, and the absence of other HIV-1 genes such as gag, pol, vif, vpr, and nef in SHIV limits the utility of these models in vaccine studies. Ideally, good animal model of HIV-1 infection/AIDS would be infection of macaques with HIV-1. However, HIV-1 does not replicate in macaque cells due to the presence of retroviral restriction factors. HIV-1 can be made to replicate by substituting its accessory genes with SIV genes such as vif, vpx, vpr, and nef, which can counteract interferon-induced restriction factors in macaque cells. Human-Simian Immunodeficiency Virus (HSIV) is an HIV-1NL4-3 derivative with SIV vif gene substitution (named HSIV-vifNL4-3) that can replicate persistently in pigtail macaques (PTMs). However, infection did not result in high peak viremia and setpoint viral loads as observed during SIV infection of macaques. Serial in vivo passaging in PTMs was performed to enhance infectivity or replicative capacity of HSIV. Three rounds of animal-to-animal transfer of infected blood in 3 immunocompetent PTMs with starting initial inoculum containing a mixture of CXCR4- (HSIV- vifNL4-3 recovered from previously infected macaque) and CCR5-tropic HSIV (HSIV-vif derivative based on pNL- AD8 and Bru-Yu2) was conducted to generate pathogenic variants. Interestingly, all the macaques showed peak viremia close to or above 105 copies/ml and virus replication persisted for more than 20 weeks. Following in vivo passaging, three infectious molecular clones (IMCs) were recovered from passage 3 macaque (HSIV-P3 IMCs). Sequencing of HSIV-P3 IMCs showed several interesting mutations throughout the genome, perhaps suggesting adaptation to PTMs. These mutations could help the virus in overcoming restriction factors, or better utilization of host dependency factors, or they could help the virus escape host immune responses. Focus of this grant application is to determine the functional significance of mutations observed in envelope gene. The results from this study will provide valuable insights into the role of envelope gene in cross-species transmission of HIV-1 to pigtailed macaques.