Replication of HIV-1 requires nuclear export and translation of the incompletely spliced 4 and 9 kb classes of HIV-1 mRNA, which encode the structural and enzymatic proteins of the virus. HIV-1 Rev binds to the Rev-responsive element (RRE) contained in the introns of incompletely spliced HIV-1 mRNAs and mediates their nuclear export via the Crm1 pathway. Sam68C, is a C-terminal deletion mutant of the endogenous human protein Sam68, and has been shown to be a potent inhibitor of Rev-dependent reporters. In this study we have performed deletion analysis of Sam68C, and determined the minimal mutant required for inhibition of Rev-dependent expression is Sam6814(45-54)-300. Sam68C inhibition is specific to RRE/Rev/Crm1 transported mRNAs: the Rev/Crm1 exported reporter construct GagRRE is inhibited while the Tap/p15 transported GagCTE reporter construct is not. Previous work from our lab showed that Sam68C co-localized with the Rev-exported mRNAs in perinuclear bundles. Further investigation has shown that Sam68C inhibition of incompletely spliced HIV-1 mRNAs is independent of the perinuclear bundling of the viral mRNA. We go on to show that Sam68C specifically inhibits the translation of the incompletely spliced HIV-1 mRNAs. Translational inhibition by Sam68C is correlated with a loss of PABP1 binding with no attendant change in abundance, polyadenylation or polyadenosine tail length of the affected mRNAs.
The selective inhibition of Crm1 exported HIV-1 mRNAs by Sam68C suggests that it is able to recognize unique characteristics of these viral mRNPs. We show that Rev and the RRE are required, but individually neither is sufficient for complete Sam68C inhibition. Study of the incompletely spliced HIV-1 mRNP revealed that the nuclear cap binding complex, CBP20/80, is not exchanged for eIF4E. Additionally, in cells expressing the HIV-1 provirus, CBP80 relocalizes to the cytoplasm and co-sediments with polysomes. This supports the hypothesis that incompletely spliced HIV-1 mRNAs are translated in an eIF4E-independent, CBP20/80-dependent fashion. This property of the 9kb and 4kb HIV-1 mRNAs could be utilized to develop new therapeutic approaches to controlling HIV-1 infection.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/16787 |
| Date | 20 January 2009 |
| Creators | Marsh, Kimberley Anne |
| Contributors | Cochrane, Alan |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
| Format | 10950046 bytes, application/pdf |
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