Pathogenic Conversion of Attenuated SIV D nef

分子克隆的SIVmac155/T3利用CXCR4作为病毒辅助受体，快速诱导恒河猴严重的CD4+T细胞耗竭。SIVmac155/T3中nef基因184个碱基的缺失导致病毒在体外对U87 MG细胞的感染力丧失，体内病毒致病性减弱。然而，感染了nef缺失突变体(SIVmac155/T3 Delta nef)的恒河猴最终出现了严重的CD4+T细胞耗竭。疾病的进展与突变病毒的出现和选择有关，命名为SIVmac543。SIVmac543与SIVmac155/T3一样，具有全长nef基因，在表达CD4和CXCR的U87 MG细胞中高效复制。我们的数据表明，SIVmac543基因组3‘端的突变补偿了全长nef基因在U87 MG细胞中复制病毒的功能。SIVmac543基因组3‘端最显著的突变是nef基因的广泛缺失：除了在SIVmac155/T3 Delta nef中引入的184个碱基缺失外，SIVmac543在与3’LTR的U3重叠的nef中还存在299个碱基的缺失。然而，广泛的额外缺失将N端和C端的Nef多肽恢复到一个单一的翻译框架中，SIVmac543 nef被预测编码大约11 kDa的肉豆蔻化蛋白。我们推测，严重缺失的SIVmac543nef基因编码了一个新的Nef蛋白，该蛋白含有一个最小结构域(S)，这是病毒在体外复制和体内致病所必需的。SIVmac543 nef基因的进化和选择可能与体内减毒SIV Delta nef的致病转化有关。为了验证我们的假设，我们提出了三个具体的目标：在目标1中，我们将通过构建重组体和定点突变体来确定SIVmac543 nef基因的突变是否有助于病毒在U87 MG细胞中的复制。还将分析SIVmac543 nef基因对恒河猴CD4+T细胞中病毒复制、病毒粒子感染性和病毒表达的影响。在目标2中，SIVmac543Nef蛋白的生物学功能将通过检测其与CD4、MHC-1类、T细胞受体、PAK激酶和Src家族酪氨酸激酶的相互作用来确定。SIVmac543的严重缺失的nef基因对体内病毒致病性的意义将通过恒河猴感染研究来研究(目标3)。所提出的研究将对Nef蛋白的结构和功能提供新的认识，并可能确定病毒致病所需的Nef蛋白的最小结构域。

Molecularly cloned SIVmac155/T3 utilizes CXCR4 as a viral coreceptor and rapidly induces severe CD4+ T cell depletion in rhesus macaques. A 184-bp deletion of nef gene from SIVmac155/T3 results in a loss of virus infectivity in U87MG cells in vitro and an attenuation of viral pathogenicity in vivo. However, a rhesus macaque infected with the nef deletion mutant (SIVmac155/T3 delta nef) eventually developed severe CD4+ T cell depletion. The disease progression was associated with an emergence and selection of mutant virus, designated SIVmac543. SIVmac543, like SIVmac155/T3 having a full-length nef gene, productively replicated in U87MG cells expressing CD4 and CXCR. Our data indicated that mutations in the 3' region of SIVmac543 genome compensate for a function of full-length nef gene for virus replication in U87MG cells. The most remarkable mutation in the 3' region of SIVmac543 genome was an extensive deletion in the nef gene: In addition to the 184bp deletion introduced in SIVmac155/T3 delta nef, the SIVmac543 has a 299-bp deletion in the nef overlapping with U3 of the 3' LTR. The extensive additional deletion, however, restores the N- and C-terminal Nef polypeptides into a single translation frame, and the SIVmac543 nef is predicted to encode approximately 11 kDa myristylated protein. We hypothesize that the heavily deleted SIVmac543 nef gene encodes a novel Nef protein that contains a minimum domain(s) required for both virus replication in vitro and pathogenicity in vivo. The evolution and selection of the SIVmac543 nef gene might be responsible for the pathogenic conversion of attenuated SIV delta nef in vivo. To verify our hypothesis, three Specific Aims are proposed: In Aim 1, we will determine if the mutations in the SIVmac543 nef gene contribute virus replication in U87MG cells by construction of recombinants and site-specific mutants. The effect of the SIVmac543 nef gene on virus replication in rhesus CD4+ T cells, virion infectivity and viral expression will also be analyzed. In Aim 2, the biological functions of SIVmac543 Nef protein will be defined by examining its interactions with CD4, MHC-class 1, T-cell receptor, PAK kinases and Src-family tyrosine kinases. The significance of the heavily deleted nef gene of SIVmac543 for virus pathogenicity in vivo will be investigated using rhesus macaque infection studies (Aim 3). The proposed studies will shed a new light on the structure- function of Nef protein and potentially define the minimum domain of Nef protein required for virus pathogenicity.