RAF-1 ACTIVATES TRANSCRIPTION FROM THE HIV-LONG TERMINAL REPEAT

细胞因子或佛波醇治疗潜伏有HIV-前病毒的细胞 酯类导致转录因子核因子-kappaB的激活，以及 HIV-LTR转录水平的增加和向生产性的转变 感染。这些药物还激活丝氨酸/苏氨酸激酶Raf-1。 由于Raf-1是有丝分裂信号级联的必要组成部分， 至少部分通过激活AP-1驱动的基因表达而起作用 和ETS结合基序，我们测试了Raf-1的激活能力 从HIV-LTR转录而来。使用瞬时共转染实验， 我们发现Raf-1可以激活HIV-LTR的表达。突变者 这增加了Raf-1、Raf-pm17和Raf-BXB的转化活性 表现出更高的激活活性，而一个突变体没有 Transform，Raf-301，不反式激活HIV-LTR驱动的表达。 对一系列5‘端缺失突变体的分析揭示了 由Raf-BXB激活的NT-120和NT-91之间的序列。点 该区域的突变将核因子-kappaB基序鉴定为Raf- HIV-LTR中的反应元件。激活的Raf-1的能力 模拟细胞因子激活HIV-LTR的表达表明 Raf-1的激活状态可能是HIV潜伏期的重要决定因素。

Treatment of cells harboring latent HIV-provirus with cytokines or phorbol esters results in the activation of transcription factor NF-kappaB, an increase in transcription from the HIV-LTR and a switch to productive infection. These agents also activate the serine/threonine kinase, Raf-1. Since Raf-1 is a necessary component of the mitogenic signaling cascade, acting at least in part by activating expression of genes driven by AP-1 and Ets binding motifs, we tested the ability of Raf-1 to activate transcription from the HIV-LTR. Using a transient cotransfection assay, we have found that Raf-1 activates expression from the HIV-LTR. Mutants that increased the transforming activity of Raf-1, Raf-pm17, and Raf-BXB displayed increased transactivation activity, and a mutant that did not transform, Raf-301, did not transactivate HIV-LTR-driven expression. Analysis of a series of 5' deletion mutants revealed the importance of sequences between nt-120 and nt-91 for activation by Raf-BXB. Point mutations in this region identified the NF-kappaB motif as the Raf- responsive element in the HIV-LTR. The ability of activated Raf-1 to mimic cytokine activation of HIV-LTR expression suggests that the activation state of Raf-1 may be an important determinant of HIV latency.