REGULATION OF HIV-1 RNA SPLICING

关于人类免疫缺陷病毒1型（HIV-1）的结构和复制的信息为开发一些抗病毒药物提供了理论基础。 谨慎的做法是继续探索病毒生命周期所有阶段的调控，以开发抑制抗药性HIV-1变异体复制的新方法。 为此，将研究HIV-1选择性剪接的调节，这是病毒生命周期中的一个步骤，才刚刚开始被理解。病毒mRNA在HIV-1感染细胞中以非常不同的丰度存在，这意味着剪接位点的利用受到调节。 将研究调节达特、vpr、rev和env/nef mRNA剪接的顺式元件和细胞因子。 达特外显子2和3内的几个顺式元件（外显子剪接沉默子）已被表征为用于抑制达特3 '剪接位点处的剪接。 将使用体外剪接测定来鉴定与这些元件结合并介导抑制的细胞因子。 将在达特外显子剪接沉默子保守的原型M组HIV-1毒株和缺乏这些元件的原型O组或离群毒株中研究这些元件突变的影响。 在vpr 3'剪接位点剪接的RNA也以低丰度存在于感染的细胞中。 将使用体外剪接测定和体内转染分析来鉴定在该剪接位点抑制剪接的元件。 最丰富的病毒mRNA在rev和env/nef 3'剪接位点剪接。 已经表明，其中一个rev剪接位点与env/nef剪接分支点重叠。 因此，剪接因子可以竞争病毒RNA上的相同位点，这可以调节rev和env/nef mRNA剪接之间的平衡。 将对该模型进行测试，并确定竞争细胞因子。 对HIV-1剪接调控和细胞因子作用的研究将有助于我们理解病毒在不同细胞类型中的复制。

Information about the structure and replication of human immunodeficiency virus type 1 (HIV-1) has provided a rationale for the development of a number of antiviral drugs. It is prudent to continue exploring regulation of all phases of the virus life cycle in order to develop new approaches for inhibiting replication of drug-resistant HIV-1 variants. To this end, the regulation of HIV-1 alternative splicing, a step in the virus life cycle that is only beginning to be understood will be studied. Viral mRNAs are present at very different abundances in the HIV-1 infected cell which implies that splice site utilization is regulated. Cis elements and cellular factors regulating splicing of tat, vpr, rev and env/nef mRNAs will be studied. Several cis elements (exon splicing silencers) within tat exon 2 and 3 have been characterized that act to inhibit splicing at the tat 3'splice sites. In vitro splicing assays will be used to identify cellular factors that bind to these elements and mediate the inhibition. The effect of mutations in these elements will be studies in the context of number prototype group M HIV-1 strains in which the tat exon splicing silencers are conserved and in a prototype group O or outlier strain lacking these elements. RNA spliced at the vpr 3' splice site is also present at low abundance in infected cells. In vitro splicing assays and in vivo transfection analysis will be used to identify elements inhibiting splicing at this splice site. The most abundant viral mRNAs are spliced at the rev and env/nef 3' splice sites. It has been shown that one of the rev splice sites overlaps an env/nef splicing branchpoint. Thus, splicing factors may compete for the same site on the viral RNA and this may regulate the balance between rev and env/nef mRNA splicing. This model will be tested and the competing cellular factors will be identified. Studies on the splicing regulation of HIV-1 and the role of cellular factors should contribute to our understanding of viral replication in diverse cell types.