HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.     

Real-time kinetics of HIV-1 Rev-Rev response element interactions. Definition of minimal binding sites on RNA and protein and stoichiometric analysis.

The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA target, Rev response element (RRE) RNA was determined in vitro using a biosensor technique. Our results showed that the primary Rev binding site is a core stem-loop RNA molecule of 30 nucleotides that bound Rev at a 1:1 ratio, whereas the 244-nucleotide full-length RRE bound four Rev monomers. At high Rev concentrations, additional binding of Rev to RRE was observed with ratios of more than 10:1. Because RRE mutants that lacked the core binding site and were inactive in vivo bound Rev nonspecifically at these concentrations, the real stoichiometric ratio of Rev-RRE is probably closer to 4:1. Binding affinity of Rev for RRE was approximately 10(-10) M, whereas the affinity for the core RNA was about 10(-11) M, the difference being due to the contribution of low affinity binding sites on the RRE. Mathematical analysis suggested cooperativity of Rev binding, probably mediated by the Rev oligomerization domains. C-terminal deletions of Rev had no effect on RRE binding, but truncation of the N terminus by as few as 11 residues significantly reduced binding specificity. This method was also useful to rapidly evaluate the potential of aminoglycoside antibiotics, to inhibit the Rev-RRE interaction.     

Analysis of the EIAV Rev-responsive element (RRE) reveals a conserved RNA motif required for high affinity Rev binding in both HIV-1 and EIAV

A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.     

Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.     

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K_d value of 410?nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.     

Cooperative dimerization of a stably folded protein directed by a flexible RNA in the assembly of the HIV Rev dimer–RRE stem II complex

The binding of the HIV‐1 Rev protein as an oligomer to a viral RNA element, the Rev‐response element (RRE), mediates nuclear export of genomic RNA. Assembly of the Rev–RRE ribonucleoprotein (RNP) complex is nucleated by the binding of the first Rev molecule to stem IIB of the RRE. This is followed by stepwise addition of a total of ~six Rev molecules along the RRE through a combination of RNA–protein and protein–protein interactions. RRE stem II, which forms a three‐way junction consisting of stems IIA, IIB and IIC, has been shown to bind to two Rev molecules in a cooperative manner, with the second Rev molecule binding to the junction region of stem II. The results of base substitutions at the stem II junction, and characterization of stem II junction variants selected from a randomized library showed that an “open” flexible structure is preferred for binding of the second Rev molecule, and that binding of the second Rev molecule to the junction region is not sequence‐specific. Alanine substitutions of a number of Rev amino acid residues implicated to be important for Rev folding in previous structural studies were found to result in a dramatic decrease in the binding of the second Rev molecule. These results support the model that proper folding of Rev is critical in ensuring that the flexible RRE is able to correctly position Rev molecules for specific RNP assembly, and suggests that targeting Rev folding may be effective in the inhibition of Rev function. Copyright © 2015 John Wiley & Sons, Ltd.     

Bipartite 3'-cis-acting signal for replication in yeast 23 S RNA virus and its repair

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome is small (2.9 kb) and only encodes its RNA-dependent RNA polymerase. Recently, we have succeeded in generating 23 S RNA virus from an expression vector containing the entire viral cDNA sequence. Using this in vivo launching system, we analyzed the 3'-cis-acting signals for replication. The 3'-non-coding region of 23 S RNA contains two cis-elements. One is a stretch of 4 Cs at the 3' end, and the other is a mismatched pair in a stem-loop structure that partially overlaps the terminal 4 Cs. In the latter element, the loop or stem sequence is not important but the stem structure with the mismatch pair is essential. The mismatched bases should be purines. Any combination of purines at the mismatch pair bestowed capability of replication on the RNA, whereas converting it to a single bulge at either side of the stem abolished the activity. The terminal and penultimate Cs at the 3' end could be eliminated or modified to other nucleotides in the launching plasmid without affecting virus generation. However, the viruses generated regained or restored these Cs at the 3' terminus. Considering the importance of the viral 3' ends in RNA replication, these results suggest that this 3' end repair may contribute to the persistence of 23 S RNA virus in yeast by maintaining the genomic RNA termini intact. We discuss possible mechanisms for this 3' end repair in vivo.     

The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE ‘conformers’, we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.     

Construction of HIV Rev peptides containing peptide nucleic acid that bind HIV RRE IIB RNA

Peptides containing peptide nucleic acid (PNA) have been designed and synthesized to construct molecules recognizing a bulge or a loop structure of RNA. Such peptides were here designed from the HIV Rev protein that can bind the stem-loop IIB of the Rev responsive element (RRE) RNA. Variations of PNA modulated the binding affinities of the peptides to RRE IIB RNA.     

Identification and characterization of a nuclear factor that specifically binds to the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1)

The Rev protein of human immunodeficiency virus type 1 (HIV-1) differentially transactivates the expression of viral structural proteins by allowing the accumulation of unspliced and singly spliced viral mRNA in the cytoplasm. The cis-acting RNA target sequence for the Rev protein, termed the Rev response element (RRE), is present in the env gene and is predicted to form a highly ordered RNA secondary structure. Recent data indicate that Rev directly binds to RRE and, further, that this binding can be mapped to a 90-nucleotide subfragment at the 5' end of RRE. We now report that RRE also binds specifically and predominantly to a nuclear factor of approximately 56 kD. Mapping of the binding site reveals that the same subfragment that binds Rev also binds this nuclear factor. We designate this protein as NFRRE for nuclear factor, RRE binding. Rev and NFRRE appear to bind simultaneously to RRE. NFRRE is widely distributed in various mammalian cells. We speculate that this factor plays an important role in Rev-mediated transactivation and is likely to be involved in the processing or transport of cellular mRNA.     

Binding sites for Rev and ASF/SF2 map to a 55-nucleotide purine-rich exonic element in equine infectious anemia virus RNA

The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.     

Evaluation of mismatch-binding ligands as inhibitors for Rev-RRE interaction

Drugs targeting the stem-loop IIB of Rev responsible element (RRE) of HIV-1 mRNA are potential therapeutic agents for HIV-1 infection. The stem loop is characterized by an internal loop consist of consecutive G-G and G-A mismatches, which is the single binding site for Rev protein for nuclear export of viral mRNA. We report here that ligands binding to G-G and G-A mismatches in duplex DNA also bind to the internal loop in competition with Rev peptide and lead to the dissociation of pre-formed Rev-RRE complex in a model system.     

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.     

Single-nucleotide changes in the HIV Rev-response element mediate resistance to compounds that inhibit Rev function

Previously we described the identification of two compounds (3-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide [103833] and 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile [104366]) that interfered with HIV replication through the inhibition of Rev function. We now describe resistant viral variants that arose after drug selection, using virus derived from two different HIV proviral clones, NL4-3 and R7/3. With HIV(NL4-3), each compound selected a different single point mutation in the Rev response element (RRE) at the bottom of stem-loop IIC. Either mutation led to the lengthening of the stem-loop IIC stem by an additional base pair, creating an RRE that was more responsive to lower concentrations of Rev than the wild type. Surprisingly, wild-type HIV(R7/3) was also found to be inhibited when tested with these compounds, in spite of the fact this virus already has an RNA stem-loop IIC similar to the one in the resistant NL4-3 variant. When drug resistance was selected in HIV(R7/3), a virus arose with two nucleotide changes that mapped to the envelope region outside the RRE. One of these nucleotide changes was synonymous with respect to env, and one was not. The combination of both nucleotide changes appeared to be necessary for the resistance phenotype as the individual point mutations by themselves did not convey resistance. Thus, although drug-resistant variants can be generated with both viral strains, the underlying mechanism is clearly different. These results highlight that minor nucleotide changes in HIV RNA, outside the primary Rev binding site, can significantly alter the efficiency of the Rev/RRE pathway.     

Dynamics of RNA-protein interactions in the HIV-1 Rev-RRE complex visualized by 6-thioguanosine-mediated photocrosslinking.

Expression of the structural proteins of human immunodeficiency virus type 1 (HIV-1) requires the direct interaction of multiple copies of the viral protein Rev with its target RNA, the Rev response element (RRE). RRE is a complex 351-nt RNA that is highly structured and located within the viral env gene. During initial Rev-RRE recognition, Rev binds with high affinity to a bubble structure located within the RRE RNA stem-loop II. We have used a site-specific photocrosslinking method based on 6-thioguanosine (6-thioG) photochemistry to probe the conformation of the high-affinity binding site of RRE RNA and its interactions with Rev protein under physiological conditions. A minimal duplex RNA containing the bubble region of RRE and 12 flanking base pairs was synthesized chemically. Two different RRE constructs with a single photoactive nucleoside (6-thio-dG or 6-thioG) at position 47 or 48 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent crosslinks in RRE RNA. Mapping of crosslink sites by RNA sequencing revealed that 6-thioG at position 47 or 48 crosslinked to A73. In the presence of Rev, both RNA-RNA and RNA-protein crosslinks were observed, however, the RNA-RNA crosslink site was unchanged. Our results provide direct evidence that, during RNA-protein recognition, Rev is in close proximity to O6 of G47 and G48 in the major groove of RRE RNA. Our results also show that the bubble region of RRE RNA has a biologically relevant structure where G47 and G48 are in close proximity to A73 and this RNA structure is not changed significantly upon Rev binding. We propose that Rev protein recognizes and binds to specific structural elements of RRE RNA containing non-Watson-Crick base pairs and such structures could be a determinant for recognition by other RNA-binding proteins. Our site-specific crosslinking methods provide a general approach to capture dynamic states of biologically relevant RNA structures that are otherwise missed by NMR and X-ray crystallographic studies.     

Portable encapsidation signal of the L-A double-stranded RNA virus of S. cerevisiae

The (+) single-stranded RNA (ssRNA) of the L-A virus is the species packaged to form new viral particles. Empty L-A viral particles specifically bind viral (+) ssRNA, and a sequence 400 bases from the 3' end is necessary for this activity. We show that its stem-loop structure, the A residue protruding from the stem, and the loop sequence are all important for the binding, and that this 34 base region is sufficient for the binding. M1, a satellite virus of L-A, has a similar structure on its (+) strand that is likewise sufficient for the binding. Heterologous RNA with the binding sequence from L-A or M1, when expressed in vivo, was packaged in L-A viral particles. Thus, the sites necessary to bind to empty particles are encapsidation signals for the L-A virus. Since the pol domain of the 180 kd minor coat protein appears to be responsible for the binding, this result suggests that the RNA polymerase molecule recognizes the viral genome for packaging.     

Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies.

The naturally uncapped genomic and mRNAs of poliovirus initiate translation by an internal ribosome-binding mechanism. The mRNA 5' untranslated region (UTR) of poliovirus is approximately 750 nucleotides in length and has seven to eight (depending on the serotype) AUG codons upstream of the initiator AUG. The sequence required for internal ribosome binding has been termed the ribosome landing pad (RLP). To better understand the mechanisms of internal initiation, we have determined the boundaries and critical elements of the RLP of poliovirus type 2 (Lansing strain) in vivo. By using deletion analysis, we demonstrate the existence of a core RLP in the poliovirus mRNA 5' UTR whose boundaries are between nucleotides 134 and 155 at the 5' end and nucleotides 556 and 585 at the 3' end. Sequences flanking the core RLP affect translational activity. The importance of several stem-loop structures in the RLP for internal initiation has been determined. Mutation of the phylogenetically conserved loop sequences in the proximal stem-loop structure of the RLP (stem-loop structure III; nucleotides 127 to 165) abolished internal translation. However, deletion of the second stem-loop in the RLP (stem-loop structure IV; nucleotides 189 to 223) reduced internal translation by only 50%. Internal deletions encompassing nucleotides 240 to 300, 350 to 380, or 450 to 480, predicted to disrupt stem-loop structure V and possibly VI, also abrogated internal initiation. Small point mutations within a short polypyrimidine sequence, highly conserved among all picornaviruses, abolished translation. A conservation of distance between the conserved polypyrimidine tract and a downstream AUG could play an important role in the mechanism of internal initiation.