REGULATION OF HIV-1 RNA SPLICING

有关人类免疫缺陷病毒1型(HIV-1)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对病毒生命周期所有阶段的管理，以便开发新的方法来抑制具有抗药性的艾滋病毒-1变种的复制。为此，将研究HIV-1选择性剪接的调节，这是病毒生命周期中刚刚开始了解的一个步骤。病毒mRNAs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控。将研究调控TAT、VPR、REV和env/nef mRNAs剪接的顺式元件和细胞因子。TAT外显子2和3中的几个顺式元件(外显子剪接沉默因子)已被鉴定为抑制TAT 3‘剪接位点的剪接。体外剪接试验将被用来识别与这些元件结合的细胞因子，并介导抑制作用。这些元素的突变的影响将在Tat外显子剪接消音器保守的M型HIV-1原型毒株和缺乏这些元素的O型原型毒株或异常毒株的背景下进行研究。在VPR 3‘剪接点剪接的RNA在感染细胞中也存在低丰度。体外剪接试验和体内转染分析将被用来确定在这个剪接位置抑制剪接的元件。最丰富的病毒mRNAs在rev和env/nef 3‘剪接点剪接。已经证明，其中一个rev剪接位点与env/nef剪接分支点重叠。因此，剪接因子可能竞争病毒RNA上的同一位置，这可能调节REV和env/nef mRNA剪接之间的平衡。这一模型将被测试，并将确定相互竞争的细胞因素。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.