Oncogene Science, Inc., 106 Charles Lindbergh Blvd., Uniondale, NY 11553

Received 6/2/97; Accepted 6/4/97

3. REV STRUCTURE AND FUNCTION

Rev is a 116 amino acid RNA binding phosphoprotein that binds a cis-acting RNA regulatory element contained within the env mRNA, termed the Rev response element (RRE) (8, 9, 10, 11). Mutational analyses of Rev have revealed several discrete domains: i) an amino terminal domain that determines RRE binding and nuclear localization, ii) an oligomerization domain, flanking the RRE binding domain, and iii) a carboxy terminal domain that acts as a nuclear export signal (NES) and binding site for cellular proteins, known as the activation domain (Figure 2).

Figure 2. Rev domains. The RRE binding domain (hatched box) is located between amino acid 35-50 in HIV-1 Rev, and is rich in arginine residues. This domain is flanked by sequences important for oligomerization (shaded). The black box represents the activation domain or nuclear export signal (NES). The sequences of other NES in related lentiviruses are shown in the insert.

The arginine-rich motif, located between amino acids 35 and 50 in the Rev protein, is responsible for nuclear localization as well as for the sequence-specific interaction with the RRE (10, 12, 13, 14, 15). A 17 amino acid peptide from this highly basic domain has been shown by circular dichroism to form an a-helix that binds the RRE with the same affinity as the full-length protein (15). The sequences immediately adjacent to this basic domain are critical for Rev oligomerization, which is required for full activity in vivo (13, 16, 17, 18). Subsequent to binding, Rev monomers multimerize on the RRE, in a process mediated by both protein-protein interactions, and protein-RNA interaction (13, 19, 20). Cellular cofactors binding to the activation domain facilitate multimerization (21, 18).

In addition to the nuclear localization and RNA binding domain, a protein activation domain which is required to mediate Rev effector functions in vivo is located at the carboxy terminus (22-26). A leucine-rich region has been identified as the critical part of this domain, which is required for interaction with cellular protein(s) involved in the transport of HIV mRNAs (24, 25). This domain also acts as a nuclear export signal (NES) (27, 28, 29, 30). NES have been identified in Rev proteins from non-primate lentiviruses (30), as well as in several cellular proteins: the inhibitor of cAMP-dependent protein kinase (PKI) (28), the fragile X mental retardation protein (FMRP) (31), and the amphibian transcription factor IIIA (32). Unlike the better known nuclear localization sequences (NLS), this domain contains critical hydrophobic residues (28), and like the NLS, all these peptide domains are functionally interchangeable (28, 30, 31, 32) and capable of directing the export of unrelated proteins (27, 28).

Rev represents a paradigm for the arginine-rich family of RNA binding proteins, and one of the best studied. The target for Rev binding, the RRE, is a highly structured 234 nucleotide RNA that forms an array of stem-loops (33, 34, 35). It has been demonstrated that the Rev binding site is located in a 13-nucleotide bulge structure in stem-loop IIB, shown in Figure 3 (11, 13, 14, 19, 20, 36, 37) (Figure 3). The secondary and tertiary structure of the RRE has been deduced from the in vitro selection of randomized RREs (38,39) and variation of these sequences (40). Two purine-purine pairs within the internal bulge of stem-loop IIB have been identified (37, 39- 42). These non-canonical base pairs open the major groove of the A-form RNA double helix, making the bases more accessible to the arginine-rich, positively- charged Rev peptide (39, 41).

In addition to these in vitro studies, a genetic strategy has been used to isolate Rev "suppressor" mutations that alleviated the deleterious effects of mutations in stem-loop IIB of the RRE (43). Taken together, these studies suggested that the arginine-rich a-helix of Rev docked into the major groove of the RNA double helix in the bulge of stem-loop IIB. The three dimensional structure of the high affinity RRE site (stem-loop IIB) complexed with the arginine-rich Rev peptide has recently been determined by nuclear magnetic resonance (NMR) techniques (44, 45). These studies confirm the purine-purine base pairs, separated by a non-conserved residue in the bulge, that cause the backbone to twist in an S-shaped fold. As predicted, the major groove is doubled in width, allowing the arginine-rich a-helix to fit, making contacts with the phosphate backbone and with purine residues (46).

Figure 3. Structure of the Rev Responsive element (RRE). The stem-loop structure of the HIV-1 RRE is depicted here. Stem-loop IIB (SLIIB) (shaded) contains the Rev binding site (RBE), shown within the box. The two non-canonical purine-purine base pairs are indicated.

Rev is functional in a wide variety of eukaryotic cells, including yeast, Drosophila, Xenopus oocytes, and mammals (25, 47, 48, 49). Thus, the Rev cellular cofactor(s) may be evolutionary conserved proteins, essential for the function of normal cells. Several cellular factors have been described to interact with Rev. The murine protein YL2 and its human homologue p32 were shown to interact with the basic domain of Rev, the same domain that interacts with the RRE and contributes to the oligomerization process (50, 51, 52). The p32 protein associates with ASF/SF2, an essential splicing factor (53),

and is thought to function as a link to the cellular splicing machinery. Rev has been shown to recruit ASF/SF2 itself to the Rev-RRE complex in vitro, thus causing inhibition of splicing (52). However, ASF/SF2 is not specific for Rev, since it also binds to the basic RNA binding domain of Tat (54), and it does not bind to the activation or effector domain of Rev, which has been shown to be essential for Rev function (22-26).

The Rev leucine-rich effector domain was considered a more likely candidate for interaction with cellular factors specific for Rev function, since mutations in this domain (Figure 2) abrogate Rev function whether fused to its own RRE-binding domain or to heterologous RNA binding sequences (55). In further support of this model, non-functional Rev mutants in the activation domain which contain an intact RNA binding domain, exhibit a potent dominant-negative effect (12, 23, 25). At least two cellular proteins have been shown to bind to the activation domain of Rev: the eukaryotic initiation factor 5A (eIF-5A) (56), and a novel class of nuclear pore-associated proteins (57, 58, 59, 60). Although the role of eIF5A in mediating Rev function is not completely understood, it has been shown that non-functional mutants of this protein that still retain their ability to bind Rev inhibit Rev-mediated nuclear export, in yeast and in human T cells (61). A novel yeast cellular protein that is part of the nuclear pore complex, called Rip1p (59), and its mammalian homologue , hRIP/Rab (57, 58) were found to bind to the activation domain of Rev, as well as to that of HTLV-I Rex (58), as required for a true cofactor of HIV-1 Rev function, since Rev and Rex, together with the Rev proteins from other lentiviruses have functionally equivalent activation domains (58). The RIP/Rab protein contains a series of repeats containing the amino acids phenylalanine and glycine, known as FG repeats. These repeats are characteristic of a class of nuclear pore proteins called FG nucleoporins (62). Rev has also recently been shown to interact with multiple FG nucleoporins in yeast and in mammalian cells (60), and the ability of Rev mutants to interact with these proteins correlates with their ability to promote nuclear export of RNA (60). These cellular proteins are important in the nuclear export process, and they have been shown to bind other NES in cellular proteins, such as PKI (29, see Section 3.1).

Two main hypotheses have been proposed for the mechanism by which Rev causes the relocalization of unspliced or partially spliced viral mRNAs in the cytoplasm: 1. inhibition of some aspect of pre-mRNA splicing by Rev, leading to increased mRNA transport to the cytoplasm, and 2. direct effect of Rev to increase the nuclear export of pre-mRNA species.

Most of the evidence in favor of the role of Rev in inhibition of splicing was originated in in vitro experimental systems. These studies showed that inefficient splicing is a pre-requisite for Rev function and that Rev inhibits the splicing of RRE-containing introns (63, 64). An arginine-rich peptide from the NLS/RNA binding domain of Rev has been shown to block the entry of the essential U4/U6.U5 small ribonuclear protein complex in the spliceosome assembly in vitro (65). However, this block does not require the presence of the Rev activation domain, that has been shown to be essential for Rev function in vivo. In addition, it has not been demostrated that this in vitro-observed inhibition of splicing is required in vivo for Rev function.

Although both models are plausible and not necessarily mutually exclusive, a recent large body of data points to RNA export rather than splicing as the mechanism of action of Rev, and a direct effect of Rev on the cellular nuclear transport machinery has now been demonstrated (49, 57-60). Earlier evidence in support of a role of Rev in nuclear export stemmed from the fact that no incompletely spliced viral mRNAs are exported to the cytoplasm in the absence of Rev, in human T cells containing stably integrated proviruses (66). Moreover, a sequence from an unrelated retrovirus, the Mason-Pfizer monkey virus, was shown to enable Rev-independent HIV replication, possibly by interacting with a cellular factor that plays a role in mRNA transport analogous to that of the Rev protein (67). More recently, the simultaneous discovery of the nucleoporin RIP/Rab by three independent laboratories (57-59) as a cellular cofactor for Rev function confirmed that Rev plays a direct role in the nuclear export of pre-mRNAs. As described in Section 3.1, several cellular proteins have been shown to contain nuclear export signals (NES) functionally homologous to that of Rev (28, 31, 32). Taken together, this evidence indicates that Rev acts as an adaptor to allow RRE-containing viral mRNAs to access a pre-existing cellular export pathway (29).