Human immunodeficiency virus Rev facilitates the cytoplasmic accumulation of viral RNAs that contain aRev binding site. A human Rev-interacting protein (hRIP) was originally identified based on its ability to interact with the Rev nuclear export signal (NES) in yeast two-hybrid assays. To date, however, the function of hRIP and a role for hRIP in Rev-directed RNA export have remained elusive. Here we ablate hRIP activity with a dominant-negative mutant or RNA interference and analyze Rev function by RNA in situ hybridization. We find, unexpectedly, that in the absence of functional hRIP, Rev-directed RNAs mislocalize and aberrantly accumulate at the nuclear periphery, where hRIP is localized. In contrast, in the absence of Rev or the Rev cofactor CRM1, Rev-directed RNAs remain nuclear. We further show that the RNA mislocalization pattern resulting from loss of hRIP activity is highly specific to Rev function: the intracellular distribution of cellular poly(A) + mRNA, nuclear proteins, and, most important, NES-containing proteins, are unaffected. Thus, hRIP is an essential cellular Rev cofactor, which acts at a previously unanticipated step in HIV-1 RNA export: movement of RNAs from the nuclear periphery to the cytoplasm. Animal viruses often encode regulatory proteins, which facilitate expression of their viral genes in the host cell. One such example is the human immunodeficiency virus type 1 (HIV-1) Rev protein, which uses a novel mechanism to regulate viral messenger RNA (mRNA) export from the nucleus. Rev is a sequence-specific RNA binding protein that facilitates the cytoplasmic accumulation of the intron-containing HIV-1 gag-pol and env mRNAs (Cullen 2002). Rev interacts with a cis-acting viral RNA element in these mRNAs designated the Rev responsive element (RRE; Pollard and Malim 1998; Hope 1999). Rev contains two distinct functional domains: an arginine-rich RNA binding motif (ARM) and a leucinerich "effector" domain. Rev's ARM domain confers sequence-specific RNA binding, protein oligomerization, and nuclear targeting (Frankel and Young 1998). Rev's effector domain contains a leucine-rich nuclear export signal (NES), which binds the nuclear export receptor CRM1 (Weis 2002). Other cellular proteins have been shown to interact directly or indirectly with the Rev NES, including the kinesin-like Rev/Rex effector binding protein (REBP) and the eukaryotic initiation factor-5 alpha (eIF-5␣; Bevec et al. 1996;Dayton 1996;Kjems and Askjaer 2000;Venkatesh et al. 2003).The human Rev-interacting protein (hRIP), also known as hRab or Hrb, was identified using a yeast twohybrid screen with Rev as the bait (Bogerd et al. 1995;Fritz et al. 1995). Additional studies demonstrated a strong correlation between the ability of Rev NES mutants to support Rev function in yeast and their ability to interact with hRIP in the two-hybrid assay (Stutz et al. 1996). However, subsequent genetic analyses indicate the Rev-hRIP interaction is indirect, and is likely bridged by CRM1 (Fritz and Green 1996;Henderson and Percipalle 1997;Ne...

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...