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

确定人猿免疫缺陷病毒体内连续传代后包膜蛋白中观察到的突变的功能意义

• 批准号: 10700374
• 负责人: Rajesh Thippeshappa
• 金额: $ 9.9万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700374
• 关键词: Acquired Immunodeficiency Syndrome; Animal Model; Animals; Antibody Response; Applications Grants; Binding; Biological Assay; Blood; CCR5 gene; CD4 Positive T Lymphocytes; CXCR4 gene; Cells; Cloning; Dependence; Development; Disease Progression; Genes; Genetic; Genome; HIV; HIV envelope protein; HIV-1; Immune response; Immunocompetent; Infection; Interferons; Macaca; Macaca nemestrina; Messenger RNA; Modeling; Modification; Molecular Cloning; Mutation; Names; Open Reading Frames; Pathogenesis; Pathogenicity; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Phenotype; Plasma; Predisposition; Protein Family; RNA Splicing; RNA-Directed DNA Polymerase; Resistance; Role; SIV; Sampling; Serum; Site; Standardization; Testing; Variant; Viral Load result; Viremia; Virion; Virus; Virus Diseases; Virus Replication; beta-Lactamase; cross-species transmission; env Gene Products; experimental study; improved; in vivo; insight; neutralizing antibody; nonhuman primate; nonsynonymous mutation; novel; prevent; simian human immunodeficiency virus; vaccine efficacy; vaccine trial; vif Gene Products; vif Genes; vpr Gene Products

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.

摘要/摘要： 常用的HIV-1动物模型包括猕猴感染猿猴免疫缺陷病毒 (SIV)或猿猴-人类免疫缺陷病毒(SIV)，含有HIV包膜(Env)或逆转录酶。 这些动物模型在了解艾滋病毒的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病毒之间的遗传差异 和siv，而其他HIV-1基因如gag、pol1、vif、vpr和nef的缺失限制了siv的应用。 疫苗研究中的这些模型。理想情况下，HIV-1感染/艾滋病的良好动物模型应该是 携带HIV-1病毒的猕猴。然而，由于逆转录病毒的存在，HIV-1不能在猕猴细胞中复制 制约因素。HIV-1可以通过用SIV基因替换其附属基因来进行复制，例如 VIF、VPX、VPR和NEF，它们可以中和干扰素诱导的猕猴细胞限制因子。人类-猿人 免疫缺陷病毒(HSIV)是人类免疫缺陷病毒(HIV-1NL4-3)的vif基因突变产物(HSIV-vifNL4-3)。 可以在辫尾猕猴(PTM)中持续复制的基因。然而，感染并不会导致高峰期病毒血症。 在SIV感染猕猴过程中观察到的设定病毒载量。PTMS的连续活体传代是 以增强HSIV的感染性或复制能力。三轮动物对动物的移植 用含有CXCR4-(HSIV-)混合物的起始初始接种在3个免疫活性PTM中感染血液 从以前感染的猕猴中恢复的vifNL4-3)和CCR5嗜性HSIV(基于PNL-VIF的HSIV-vif衍生物) AD8和Bru-Yu2)产生致病变异体。有趣的是，所有的猕猴都出现了顶峰 病毒血症接近或超过105拷贝/毫升，病毒复制持续20周以上。在体内追踪 传代后，从第3代猕猴(HSIV-P3IMCs)中获得3个感染性分子克隆(IMCs)。 对HSIV-P3IMC的测序显示，整个基因组中有几个有趣的突变，这可能表明 适应PTMS。这些突变可以帮助病毒克服限制因素，或更好地利用 宿主依赖因素，或者它们可以帮助病毒逃避宿主免疫反应。这笔赠款的重点 应用是确定观察到的包膜基因突变的功能意义。结果来自于 这项研究将为探讨包膜基因在HIV-1跨物种传播中的作用提供有价值的见解 扎着辫子的猕猴。