SIV MOLECULAR CLONES IN MACAQUES

HIV和SIV的nef基因编码一种非必需的肉豆蔻化蛋白 用于病毒的体外复制。这是一个明确定义的功能 蛋白质在病毒复制中的作用尚未确定。体内研究 用SIV突变体分析恒河猴的感染情况 NEF对于维持高病毒载量和进展至关重要 献给西米亚艾滋病。我们发现这些病毒中的NEF与 一种细胞丝氨酸激酶。这种活性使两种蛋白质磷酸化， 免疫共沉淀法测定NEF的体外激酶活性。要确定 NEF区域(S)与细胞激酶活性相关， 不同的nef等位基因和突变体已经在激酶试验中被分析。 Nef的两个结构域是与细胞丝氨酸结合的关键。 激活剂。第一结构域是N处的膜靶向信号 末端；第二个包含一个中心的高度保守的区域 在所有灵长类慢病毒nef基因中。以确定该关联是否 NEF与细胞激酶的结合对于NEF在体内的功能是必不可少的， 一种激酶阴性的点突变被引入到致病的 分子克隆SIVMAC39。在突变体中，arg137arg138发生了改变 转到137卢比138卢比。该突变体nef也不下调细胞表面。 CD4抗原。将突变病毒接种给幼年猕猴以 分析活体内蛋白激酶联合功能的意义。 在感染的前四周，相对较低的病毒载量 观察到，但在8点后增加。病毒在4周和8周后恢复 接种后由nef基因序列的变异体组成 恢复为原型序列，即leu137、leu138的密码子具有 突变回了arg137 arg138。突变的NEF能够将 与细胞丝氨酸激酶和下调的细胞表面CD4 抗原。这些数据表明，对 NEF从激酶阴性表型向激酶阳性表型逆转 活体表型。这种逆转直接与病毒感染的增加有关 感染宿主的负担。

The nef gene of HIV and SIV encodes a myristylated protein dispensable for viral replication in vitro. A clearly defined function of this protein in viral replication has not been established. In vivo studies analyzing infection of rhesus macaques with mutants of siv demonstrated that nef is critical for maintenance of high virus load and progression to Simian AIDS. We have found that nef of these viruses associates with a cellular serine kinase. This activity phosphorylates two proteins that co-immunoprecipitate with nef in in vitro kinase assays. To identify the region(s) of nef important for association with cellular kinase activity, various nef alleles and mutants have been analyzed in the kinase assay. Two domains of nef are critical for association with the cellular serine kinase. The first domain is a membrane targeting signal at the N terminus; the second encompasses a central, highly conserved region found in all primate lentivirus nef genes. To determine whether the association of nef with the cellular kinase is essential for nef function in vivo, a kinase-negative point mutation was introduced into the pathogenic molecular clone SIVMAC39. In the mutant, arg137arg138 have been changed to leu137leu138. This mutant nef also does not downregulate cell surface CD4 antigen. Juvenile macaques were inoculated with the mutant virus to analyze the significance of the kinase association function in vivo. During the first four weeks of infection, relatively low virus loads were observed, but increased after eight. Virus recovered at 4 and 8 weeks after inoculation was composed of variants in which the nef gene sequence reverted to the prototype sequence, i.E., codons for leu137leu138 had mutated back to arg137arg138. The revertant nefs were able to associate with the cellular serine kinase and downregulated cell surface cd4 antigen. These data demonstrate a strong selective pressure for the reversion of nef from a kinase-negative phenotype to a kinase-positive phenotype in vivo. This reversion is directly linked to increased viral burden in the infected host.