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

描述（由申请人提供）：HIV疫苗开发的一个主要障碍是个体之间和个体内病毒株的巨大遗传变异性。最近，已经提出了专门靶向病毒蛋白质组的高度保守区域的通用HIV-1疫苗方法，其中预测突变会严重损害病毒活力。强调的假设是，在高度保守的氨基酸位点处的单个突变对感染病毒可以是致命的或几乎致命的，并且这些突变的补偿的发生是罕见的，例如，需要多重和/或适合度降低的次级突变来进行补偿。然而，在高度保守的氨基酸位点的突变的致死性尚未被证明。此外，我们已经发现的证据表明，在高度保守的位点突变的有害影响可能会补偿在可变位点的突变。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），将其他氨基酸位点定义为可变位点（VS）。我们提出了一个试点探索性研究，以测试这种疫苗的方法强调的假设。使用定点突变和体外生长竞争试验，我们将检查在HIV-1亚型B感染受试者（PIC 1362）中观察到的20个HCS（每个HCS一个突变）处天然存在的Gag和Env突变的致死性。我们的目标是：在PIC 1362中，1）确定在Gag和Env蛋白中观察到的HCS突变的致死性，2）确定观察到的VS突变的补偿效应;和3）在另外两个HIV-1感染受试者（PIC 1113和PIC 1693）中，确定在PIC 1362中观察到的Gag HCS突变的致死性，以提供我们在PIC 1362中发现的一般性的初步证据。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，并有助于定义包含在保守元件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病毒群体的巨大多样性提供了一种新方法。该提案测试了这种方法对HIV-1疫苗开发的强调假设。

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.