Mode of Action of a Fragment of hnRNPU in Blocking HIV-1 mRNA Export

描述（由申请人提供）：我们建议研究异质核核糖核蛋白U （hnRNPU）片段的作用模式，该片段是在基因筛选中发现的可以抑制HIV-1复制的基因或基因片段。该片段定位于hnRNPU的n端86个氨基酸（N86-hnRNPU），介导了野生型HIV-1复制的有效阻断。对作用机制的进一步分析表明，N86-hnRNPU诱导HIV-1 mrna在细胞核中保留，并伴随细胞质中的减少。由于HIV-1 mRNA的半衰期不受片段的影响，这些观察结果表明N86-hnRNPU诱导了HIV-1 mRNA输出的阻断。在前期研究中，我们通过共免疫沉淀发现N86-hnRNPU片段与全长hnRNPU蛋白结合，并通过共聚焦分析发现N86-hnRNPU与hnRNPU在细胞核内共定位。N86- hnRNPU片段的缺失分析表明，从n端去除68个氨基酸对片段的限制能力没有影响，这表明19个氨基酸（hnRNPU中68-86个）可能足以进行限制。一种假设是N86-hnRNPU可能会影响内源性hnRNPU在HIV-1 mRNA输出中的潜在积极作用。另一种假设是N86-hnRNPU影响mRNA输出的细胞因子，如TAP和SR蛋白9G8。后者已被证明作为tap介导的输出适配器并与HIV-1 mrna结合。我们将研究上述（或其他）细胞蛋白是否被N86-hnRNPU抑制，从而导致观察到的HIV-1 mRNA输出受阻。我们将通过荧光原位杂交（FISH）可视化HIV-1 mrna的分布，这可能使我们深入了解基于HIV-1 mrna在细胞核中的空间排列的块的类型。最后，我们将测试N86-hnRNPU是否在生物靶点外周血单核细胞（PBMCs）中限制HIV-1感染。总之，这些实验将使我们进一步了解N86-hnRNPU介导的限制HIV-1 mRNA输出的机制。我们希望对HIV-1整合后步骤中以前未知的方面获得有价值的见解，这可能导致确定新的治疗途径。

DESCRIPTION (provided by applicant): We propose to investigate the mode of action of a fragment of heterogeneous nuclear ribonuclear protein U (hnRNPU) that was identified in a genetic screen for genes or gene fragments that could inhibit HIV-1 replication. This fragment maps to the N-terminal 86 amino acids of hnRNPU (N86-hnRNPU) and mediates a potent block in wild type HIV-1 replication. Further analysis into the mechanism of action revealed that N86-hnRNPU induced retention of HIV-1 mRNAs in the nucleus and a concomitant reduction in the cytoplasm. Since the half-life of HIV-1 mRNAs is unaffected by the fragment, these observations suggest that N86-hnRNPU induces a block in HIV-1 mRNA export. In our preliminary studies, we've found that the N86-hnRNPU fragment associates with the full-length hnRNPU protein via co-immunoprecipitation, and through confocal analysis we have seen that N86-hnRNPU and hnRNPU co-localize in the nucleus. Deletion analysis of the N86- hnRNPU fragment has shown that removal of 68 amino acids from the N-terminus has no effect on the restrictive capacity of the fragment suggesting that the 19 amino acids (68-86 in hnRNPU) may be sufficient for restriction. One hypothesis is that N86-hnRNPU may be affecting a potentially positive role of endogenous hnRNPU in HIV-1 mRNA export. An alternative hypothesis is that N86-hnRNPU affects cellular factors involved in mRNA export such as TAP and the SR protein 9G8. The later has been shown to act as an export adaptor for TAP-mediated export and bind to HIV-1 mRNAs. We will study whether the above mentioned (or other) cellular proteins are inhibited by N86-hnRNPU resulting in the observed block in HIV-1 mRNA export. We will visualize the distribution of HIV-1 mRNAs via fluorescence in situ hybridization (FISH), which may give us insights into the type of block based of the spatial arrangement of HIV-1 mRNAs in the nucleus. Finally, we will test whether N86-hnRNPU restricts HIV-1 infection in the biological target, peripheral blood mononuclear cells (PBMCs). Together, these experiments will lead us closer to understanding the mechanism of N86-hnRNPU mediated restriction of HIV-1 mRNA export. We hope to gain valuable insights into previously unknown aspects in HIV-1 post-integrations steps that might lead to the identification of novel therapeutic avenues. PUBLIC HEALTH RELEVANCE: We propose to identify the mechanism by which a fragment of hnRNPU blocks HIV-1 mRNA cytoplasmic export. We will identify cellular partners that might be necessary for restriction, study the distribution of HIV-1 mRNAs in the cell, and determine the ability of the fragment to restrict HIV-1 replication in peripheral blood mononuclear cells. These efforts may provide previously unknown insights into HIV-1 replication and mRNA export.