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.
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.
