Analysis of mutational lethality in highly conserved amino acids of the HIV-1 Gag

描述（由申请人提供）：艾滋病毒疫苗开发的一个主要障碍是个体之间和个体内部病毒株的巨大遗传变异。最近，人们提出了通用的HIV-1疫苗方法，专门针对病毒蛋白质组的高度保守区域，在这些区域，预计突变会严重损害病毒的生存能力。主要的假设是，在高度保守的氨基酸位点上的单个突变对感染病毒可能是致命的或几乎致命的，并且对这些突变的补偿很少发生，例如，补偿需要多个和/或降低适应性的二次突变。然而，高度保守的氨基酸位点突变的致死率尚未得到证实。此外，我们已经发现证据表明，高度保守位点突变的有害影响可能被可变位点的突变所补偿。我们将HIV-1 M组中保守性超过98%的氨基酸位点定义为高度保守位点（HCS），其他的定义为可变位点（VS）。我们建议进行一项试点探索性研究，以检验这种疫苗方法的主要假设。使用定点诱变和体外生长竞争试验，我们将检验在HIV-1亚型B感染受试者（PIC1362）中观察到的20个HCS（每个HCS有一个突变）中自然发生的Gag和Env突变的致死率。我们的目标是：在PIC1362中，1)确定Gag和Env蛋白中观察到的HCS突变的致死率，2)确定观察到的VS突变的代偿作用；3)在另外两名HIV-1感染受试者（PIC1113和PIC1693）中，确定PIC1362中观察到的Gag HCS突变的致死率，为我们在PIC1362中发现的普遍性提供初步证据。这些结果将为高度保守的氨基酸位点在HIV-1结构和功能中的关键作用提供新的见解，并有助于确定包含在保守元件HIV疫苗中的蛋白质组成分。公共卫生相关性：一种专门针对病毒蛋白质组高度保守区域的通用HIV-1疫苗设计可能为解决HIV-1病毒种群的巨大多样性提供一种新方法。这一建议验证了这种开发HIV-1疫苗方法的主要假设。

DESCRIPTION (provided by applicant): One major obstacle for HIV vaccine development is the enormous genetic variability of viral strains between and within individuals. Recently, universal HIV-1 vaccine approaches have been proposed that exclusively target the highly conserved regions of the viral proteome, in which mutations are predicted to severely compromise virus viability. The underlining hypothesis is that a single mutation at a highly conserved amino acid site can be lethal or nearly lethal to the infecting virus and that the occurrences of compensation for these mutations are rare, e.g., multiple and/or fitness-reducing secondary mutations are required for compensation. However, the lethality of mutations at highly conserved amino acid sites has yet to be shown. In addition, we have found evidence indicating that the deleterious effect of mutations at highly conserved sites may be compensated for by mutations at variable sites. We define amino acid sites that are over 98% conserved in HIV-1 group M as highly conserved sites (HCS) and others as variable sites (VS). We propose a pilot exploratory study to test the underlining hypothesis of this vaccine approach. Using site-directed mutagenesis and in vitro growth competition assays, we will examine the lethality of naturally occurring Gag and Env mutations at 20 HCS (one mutation at each HCS) observed in an HIV-1 subtype B infected subject (PIC1362). Our objectives are: in PIC1362, 1) determine the lethality of the observed HCS mutations in the Gag and Env proteins, 2) determine the compensatory effect of the observed VS mutations; and 3) in two other HIV-1 infected subjects (PIC1113 and PIC1693), determine the lethality of Gag HCS mutations observed in PIC1362, to provide initial evidence of the generality of our findings in PIC1362. The results will provide new insights into the critical role of highly conserved amino acid sites in HIV-1 structure and function and help define the proteome constituents for inclusion in a conserved-elements HIV vaccine. PUBLIC HEALTH RELEVANCE: A universal HIV-1 vaccine design exclusively targeting the highly conserved regions of the viral proteome may present a novel way to tackle the enormous diversity in the HIV-1 viral population. This proposal tests the underline hypothesis of this approach towards HIV-1 vaccine development.