The cellular HIV-1 Rev cofactor hRIP is required for viral replication

Abstract

An important goal of contemporary HIV type 1 (HIV-1) research is to identify cellular cofactors required for viral replication. The HIV-1 Rev protein facilitates the cytoplasmic accumulation of the intron-containing viral gag-pol and env mRNAs and is required for viral replication. We have previously shown that a cellular protein, human Rev-interacting protein (hRIP), is an essential Rev cofactor that promotes the release of incompletely spliced HIV-1 RNAs from the perinuclear region. Here, we use complementary genetic approaches to ablate hRIP activity and analyze HIV-1 replication and viral RNA localization. We find that ablation of hRIP activity by a dominant-negative mutant or RNA interference inhibits virus production by mislocalizing Rev-directed RNAs to the nuclear periphery. We further show that depletion of endogenous hRIP by RNA interference results in the loss of viral replication in human cell lines and primary macrophages; virus production was restored to wild-type levels after reintroduction of hRIP protein. Taken together, our results indicate that hRIP is an essential cellular cofactor for Rev function and HIV-1 replication. Because hRIP is not required for cell viability, it may be an attractive target for the development of new antiviral strategies.Rev-directed RNA export ͉ RNA localization R eplication of lentiviruses, such as HIV type 1 (HIV-1), entails a tightly regulated complex life cycle that depends upon specific interactions between viral RNAs and viral and cellular proteins. In principle, every step in the viral life cycle provides an opportunity for therapeutic intervention (1-3). Anti-HIV drugs that are currently available or in clinical development inhibit critical steps in viral replication such as reverse transcription, viral protein maturation, virus entry, and integration of the proviral DNA (4-7). However, therapeutic strategies that inactivate other critical steps in the viral life cycle remain unrealized (8-11).An essential and characteristic step in HIV-1 replication is the export of the intron-containing gag-pol and env mRNAs from the nucleus to the cytoplasm. The viral regulatory protein Rev mediates this event, in conjunction with the cellular nuclear export machinery and several protein cofactors (12-15). Rev contains two functional domains: an arginine-rich RNA-binding motif, which specifically interacts with a cis-acting element within intron-containing viral RNAs designated the Rev-responsive element (RRE) (16,17), and a leucine-rich ''effector'' domain that constitutes a nuclear export signal (14,15,(17)(18)(19).We have recently shown that the human Rev-interacting protein (hRIP) is a cellular cofactor required for Rev function (20). In the absence of functional hRIP, Rev-directed RNAs mislocalize and aberrantly accumulate at the nuclear periphery, where hRIP is localized. Importantly, the requirement for hRIP is highly specific, because comparable ablation of hRIP activity did not affect the intracellular distribution of cellular poly(A) ϩ mRNA, nuclear proteins, or...

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