Nuclear Hiv 1 Gag Specifically Associates With Unspliced Viral Rna During Transcription

Author : Rebecca Maldonado
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (951 download)

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Download or read book Nuclear Interactions of the Rous Sarcoma Virus Gag Polyprotein with the Unspliced Viral RNA. written by Rebecca Maldonado and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Retroviruses are single-stranded RNA viruses that encode viral enzymes for the reverse transcription of the RNA genome into a DNA provirus, which integrates into the host chromosome. The integrated proviral DNA is transcribed into viral RNA (vRNA) by the host RNA polymerase II. The unspliced vRNA has three fates. First, it can be spliced into sub-genomic mRNAs that encode for viral proteins. Second, it can remain unspliced to serve as the mRNA for the translation of the Gag and Gag-Pol structural proteins. Third, it can be packaged as genomic RNA (gRNA) in new virions. Gag is thought to select the unspliced gRNA for packaging. Although it was previously thought that Gag functioned solely in the cytoplasm, the Gag proteins of multiple retroviruses have recently been observed to traffic through the nucleus. Data from our laboratory suggest that Gag nuclear trafficking for the avian oncoretrovirus, Rous sarcoma virus (RSV), is linked to efficient gRNA packaging. For RSV Gag mutants that have reduced nuclear trafficking, packaging is decreased. Restoration of Gag nuclear trafficking rescues RNA packaging. RSV Gag is imported into the nucleus by the host karyopherins Importin-[alpha]/[beta], Importin 11, and TNPO3. RSV Gag exits via the host nuclear export protein Crm1. Our biochemical evidence shows that Gag interaction with Crm1 is enhanced by Gag binding to nucleic acid. Together these data suggest that Gag is interacting with the vRNA in the nucleus. The goal of the work presented in this dissertation is to visualize Gag: RNA complexes in the nucleus. Under steady-state conditions RSV Gag is primarily located in the cytoplasm and at the plasma membrane with a small portion of the Gag present in the nucleus. To visualize Gag: vRNA complexes in the nucleus, we created an infectious proviral construct containing 24 copies of MS2 stem loops (RC.V8-24x) labeled with MS2-YFP. Using confocal microscopy, we observed co-localization between RSV Gag and the RC.V8-24x in the nucleus. An RSV Gag nuclear export mutant that accumulates in distinct nuclear foci (Gag.L219A) co-localizes with vRNA in the nucleus at high levels compared to non-vRNAs. Interestingly, deletion of cis-acting sequences in the vRNA, known to be required for Gag: RNA interactions, did not greatly decrease co-localization of the vRNA with Gag. Gag.L291A preferentially co-localizes with the vRNA in the nucleus. However, a low but consistent level of co-localization exists with non-viral and nascent RNAs, suggesting a localization mechanism tied to transcription. Gag.L219A co-localizes with splicing factors SF2 and SC35, which are known to bind to RNAs co-transcriptionally. Further, overexpression of Clk1, a kinase known to phosphorylate these splicing factors causing them to travel to sites of active transcription, promotes increased co-localization between Gag.L219A and these splicing factors. Additionally, Gag.L219A nuclear foci formation is prevented by the transcription inhibitor actinomcyin D. Together, these data suggest that RSV Gag is localizing to sites of active transcription. We hypothesize that RSV Gag is traveling to sites of RNA synthesis to co-transcriptionally select the unspliced vRNA for packaging into new virions. To determine whether Gag: RNA nuclear interactions are conserved among retroviruses, we utilized a cell line that contains a stably integrated, doxycycline-inducible HIV-1 provirus that expresses HIV-1 Gag fused to GFP. The unspliced HIV-1 RNA was visualized via fluorescence in situ hybridization using probes directed against the gag coding sequence. Interestingly, HIV-1 Gag also co-localizes with its unspliced vRNA in the nucleus. These data allow us to speculate a purpose for Gag nuclear trafficking in retroviral replication that includes localizing to sites with high levels of vRNA, as a mechanism to pair the Gag structural protein with the gRNA for subsequent movement to the plasma membrane for packaging.