EFFECTS OF STRESS/STRESS RESPONSE GENES ON REGULATION OF HIV-1 GENE EXPRESSION

用DNA损伤剂如紫外线辐射处理人类细胞， 可导致潜伏病毒的激活和病毒的增加 生产这可能对疾病进展有重要意义 在治疗HIV-1感染者期间， 使用细胞毒性药物的患者。我们在遗传毒性应激方面的专业知识 反应已被应用于HIV-1反应的研究。 这 该项目由NIH校内艾滋病靶向抗病毒项目资助 91和92财政年度，93财政年度仅为一名研究员提供部分工资支持： 从那时起，它一直得到DTP资金的支持。 最初我们 重点关注细胞毒性剂对稳定整合的HIV的诱导- 启动子-CAT构建体。我们已经发现，一个或多个细胞因子中， 介质可以影响DNA损伤反应;此外，我们还 发现由紫外线照射的细胞产生的可溶性因子可以 激活稳定整合的HIV-CAT。由于紫外线照射可以 诱导细胞Tat样因子，我们寻找细胞因子 参与HIV-1调节的研究主要集中在具有达特- 我们用西北印迹法 方法在整个核中的各种TAR RNA结合蛋白（TBP） 萃取物 发现TBP的水平在不同的细胞系中不同， 并且在许多情况下是DNA损伤诱导的。 通过这种方法，我们 已知的TAR结合蛋白的数量增加了一倍多， 这是TBP对压力反应的第一次展示。 我们计划进一步 表征这些TBP及其对遗传毒性应激的反应，包括 细胞因子在其激活中的作用。将制定方法， 纯化这些DNA损伤诱导的RNA结合蛋白， 基于部分蛋白质序列测定分离cDNA克隆。 这些蛋白质的鉴定可能会提供重要的见解 HIV-1的调节和HIV-1感染细胞对 细胞毒性剂。

Treatment of human cells with DNA-damaging agents, such as UV radiation, can lead to the activation of latent virus and to increased virus production. This may have important implications in disease progression both from environmental sources and during treatment of HIV-1-infected patients with cytotoxic agents. Our expertise in genotoxic stress responses has been applied to the study of responses in HIV-1. This project was funded by the NIH Intramural AIDS Targeted Antiviral Program for FY91 and FY92 with only partial salary support for a fellow in FY93: since that time, it has been supported by DTP funding. Initially, we focused on the induction by cytotoxic agents of stably-integrated HIV- promoter-CAT constructs. We have found that one or more cytokines in the medium can affect DNA-damage responsiveness; in addition, we have also found that a soluble factor(s) produced by UV-irradiated cells can activate stably-integrated HIV-CAT in trans. Since UV-irradiation can induce a cellular Tat-like factor, our search for cellular factors involved in HIV-1 regulation has focused primarily on proteins with Tat- like properties, and we have identified by a Northwestern blotting approach a variety of TAR RNA-binding proteins (TBP) in whole nuclear extracts. The levels of TBP were found to vary in different cell lines, and were DNA-damage inducible in many cases. With this approach, we have more than doubled the number of known TAR-binding proteins and have shown the first demonstration of a TBP response to stress. We plan to further characterize these TBP and their responses to genotoxic stress including the role of cytokines in their activation. Approaches will be developed to purify these DNA-damage-inducible RNA-binding proteins and to ultimately isolate cDNA clones based on partial protein sequence determinations. Identification of these proteins will probably provide important insights into the regulation of HIV-1 and the responses of HIV-1-infected cells to cytotoxic agents.