ANALYSIS OF HIV-1 CELL ENTRY USING CHEMICALLY INDUCED T CELL MUTANTS

我们先前已经描述了EMS诱导的CEM突变体的分离， 表达中等水平的表面CD 4（野生型的50- 75 表达），但与野生型CEM细胞以及与 其他具有类似CD 4表达的突变体对HIV 1的易感性 感染这些突变体产生的合胞体数量减少， 用gp 160-牛痘载体（VSC 25）感染。此外，传染性 使用HIV 1（MN株）的检测显示， 感染性，通过病毒滴定、长期RT测定和 定量PCR（感染后72小时）。其中两个变种人 对PKC激活剂TPA的诱导反应异常。类似于 在野生型CEM中，它们在感染后1小时内将其表面CD 4下调50%。 TPA治疗。相比之下，TPA处理24小时诱导了 CD 4和CD 25（IL 2 R）在亲本系以及其他突变体，但不是 这两个变种人TPA诱导的膜受体的磷酸化是 发现CD 4和CD 5正常，但CD 7不正常。使用的CAT测定 HIV 1 LTR（139）-CAT质粒+ TPA诱导完全阴性， 这两个变种人 用对照质粒转染，Actin-CAT和RSV-CAT导致 CAT活动有趣的是，用LTR-CAT + pCV 1（达特）共转染， 不导致CAT基因通过TAR元件的反式激活。凝胶 使用NF-k结合寡核苷酸和核提取物的阻滞试验 TPA和TNF诱导的细胞，显示出NF-k结合蛋白的诱导， 突变体，但在一个显着的，降低的水平。进一步的生化 对这些突变体的分析将允许鉴定细胞基因， 这对最佳的HIV I感染率很重要， PKC磷酸化途径。

We have previously described the isolation of EMS-induced CEM mutants which express intermediate levels of surface CD4 (50-75% of wild type expression), yet differ markedly from wild type CEM cells as well as from other mutants with similar CD4 expression in their susceptibility to HIV 1 infection. These mutants produced reduced number of syncytia following infection with the gpl6O-vaccinia vector (VSC25). Furthermore,,infectivity assays using HIV 1 (MN strain), showed a significant reduction in their infectibility, as judged by virus titrations, longterm RT assays, and quantitative PCR (ar 72 hr post infection). Two of these mutants were found to respond abnormally to induction with the PKC activator TPA. Similar to wild type CEM, they down regulated their surface CD4 by 50% within 1 hr of TPA treatment. In contrast, TPA treatment for 24 hr. induces expression of CD4 and CD25 (IL2R) on the parental line as well as other mutants, but not on these two mutants. TPA induced phosphorylation of membrane receptors was found to be normal for CD4 and CD5, but not for CD7. CAT assays using the HIV 1 LTR (139)-CAT plasmid + TPA induction were completely negative for these two mutants. Transfection with control plasmids, e.g., Actin-CAT and RSV-CAT resulted in CAT activity. Interestingly, cotransfection with LTR-CAT + pCV1 (tat) did not result in transactivation of the CAT gene via the TAR element. Gel retardation assay using the NF-k binding oligo and nuclear extracts from TPA and TNF-induced cells, showed induction of NF-k binding protein in the mutants, but at a significantly ,reduced levels. Further biochemical analyses of these mutants would allow identification of cellular gene(s) which are important for optimal HIV I infection land are dependent on the PKC phosphorylation pathway.