Pathogenic Conversion of Attenuated SIV D nef

分子克隆SIVmac155/T3利用CXCR4作为病毒辅助受体，在恒河猴体内快速诱导严重的CD4+ T细胞消耗。SIVmac155/T3基因中nef基因缺失184 bp导致病毒在体外感染U87MG细胞的能力丧失，在体内降低病毒的致病性。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细胞耗损。疾病进展与突变病毒SIVmac543的出现和选择有关。SIVmac543与SIVmac155/T3一样具有全长nef基因，可在表达CD4和CXCR的U87MG细胞中高效复制。我们的数据表明，SIVmac543基因组3'区突变补偿了全长nef基因在U87MG细胞中的复制功能。SIVmac543基因组3‘区最显著的突变是nef基因的广泛缺失：除了在SIVmac155/T3 delta nef中引入的184bp缺失外，SIVmac543在与3’ LTR的U3重叠的nef中有299 bp的缺失。然而，广泛的额外缺失将N端和c端nef多肽恢复到单个翻译框中，并且SIVmac543 nef预计编码约11 kDa的肉化蛋白。我们假设大量缺失的SIVmac543 nef基因编码一种新的nef蛋白，该蛋白含有病毒体外复制和体内致病性所需的最小结构域(s)。SIVmac543 nef基因的进化和选择可能是SIV减毒δ nef在体内致病转化的原因。为了验证我们的假设，我们提出了三个特定目标：在目标1中，我们将通过构建重组体和位点特异性突变体来确定SIVmac543 nef基因的突变是否有助于病毒在U87MG细胞中的复制。SIVmac543 nef基因对病毒在恒河猴CD4+ T细胞中的复制、病毒粒子的感染性和病毒表达的影响也将被分析。在Aim 2中，SIVmac543 Nef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定义。SIVmac543的大量缺失的nef基因对病毒体内致病性的意义将通过恒河猴感染研究进行研究（Aim 3）。这些研究将为Nef蛋白的结构功能提供新的线索，并有可能确定病毒致病性所需的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.