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，该片段在基因筛选中鉴定为可抑制HIV-1复制的基因或基因片段。该片段定位于hnRNPU（N86-hnRNPU）的N-末端86个氨基酸，并介导野生型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在细胞核中的保留和细胞质中的伴随减少。由于HIV-1 mRNA的半衰期不受该片段的影响，这些观察结果表明N86-hnRNPU诱导HIV-1 mRNA输出的阻断。在我们的初步研究中，我们已经发现N86-hnRNPU片段通过免疫共沉淀与全长hnRNPU蛋白结合，并且通过共聚焦分析我们已经看到N86-hnRNPU和hnRNPU共定位于细胞核中。N86- hnRNPU片段的缺失分析表明，从N-末端去除68个氨基酸对片段的限制性能力没有影响，这表明19个氨基酸（hnRNPU中的68-86）可能足以进行限制。一种假设是N86-hnRNPU可能影响内源性hnRNPU在HIV-1 mRNA输出中的潜在积极作用。另一种假设是N86-hnRNPU影响参与mRNA输出的细胞因子，如TAP和SR蛋白9 G8。后者已被证明作为TAP介导的输出的输出适配器，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，导致观察到的HIV-1 mRNA输出阻断。我们将通过荧光原位杂交（FISH）可视化HIV-1 mRNA的分布，这可能会让我们深入了解基于细胞核中HIV-1 mRNA空间排列的阻断类型。最后，我们将测试N86-hnRNPU是否限制HIV-1感染的生物学目标，外周血单核细胞（PBMC）。总之，这些实验将使我们更接近于理解N86-hnRNPU介导的HIV-1 mRNA输出限制的机制。我们希望获得有价值的见解，以前未知的方面在HIV-1整合后的步骤，可能会导致新的治疗途径的识别。

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.