Pathogenic Conversion of Attenuated SIV D nef

分子克隆的SIVmac 155/T3利用CXCR 4作为病毒辅助受体，并在恒河猴中迅速诱导严重的CD 4 + T细胞耗竭。 SIVmac 155/T3 nef基因缺失184-bp导致病毒在U87 MG细胞中的体外感染性丧失和病毒在体内的致病性减弱。然而，感染nef缺失突变体（SIVmac 155/T3 delta nef）的恒河猴最终出现严重的CD 4 + T细胞耗竭。 疾病进展与命名为SIVmac 543的突变病毒的出现和选择相关。与具有全长nef基因的SIVmac 155/T3一样，SIVmac 543在表达CD 4和CXCR的U87 MG细胞中有效复制。我们的数据表明，SIVmac 543基因组3'区的突变补偿了全长nef基因在U87 MG细胞中的病毒复制功能。在SIVmac 543基因组的3'区域中最显著的突变是nef基因中的广泛缺失：除了在SIVmac 155/T3 delta nef中引入的184 bp缺失之外，SIVmac 543在与3' LTR的U3重叠的nef中具有299-bp缺失。 然而，广泛的额外缺失将N-和C-末端Nef多肽恢复到单个翻译框中，并且预测SIVmac 543 nef编码约11 kDa肉豆蔻基化蛋白。我们推测严重缺失的SIVmac 543 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。 SIVmac 543 nef基因的进化和选择可能是导致减毒SIVdelta nef在体内致病性转化的原因。 为了验证我们的假设，提出了三个具体的目的：在目的1中，我们将通过构建重组体和位点特异性突变体来确定SIVmac 543 nef基因中的突变是否有助于U87 MG细胞中的病毒复制。还将分析SIVmac 543 nef基因对恒河猴CD 4 + T细胞中病毒复制、病毒粒子感染性和病毒表达的影响。 在目的2中，SIVmac 543 Nef蛋白的生物学功能将通过检查其与CD 4、MHC-class 1、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。 将使用恒河猴感染研究来研究SIVmac 543的严重缺失的nef基因对于病毒体内致病性的意义（目的3）。 这些研究将为Nef蛋白的结构-功能研究提供新的思路，并有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.