Examination of Unspliced HIV-1 mRNA Translation

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. Sam68C, 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 Sam68C, and determined the minimal mutant required for inhibition of Rev-dependent expression is Sam6814(45-54)-300. Sam68C 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 Sam68C co-localized with the Rev-exported mRNAs in perinuclear bundles. Further investigation has shown that Sam68C inhibition of incompletely spliced HIV-1 mRNAs is independent of the perinuclear bundling of the viral mRNA. We go on to show that Sam68C specifically inhibits the translation of the incompletely spliced HIV-1 mRNAs. Translational inhibition by Sam68C 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 Sam68C 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 Sam68C 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.Ph