Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA

The leader RNA sequence of human immunodeficiency virus type 1 (HIV-1) consists of a complex series of stem loop structures that are critical for viral replication. Three-dimensional structural analysis by NMR of one of these structures, the SL1 stem loop of the packaging signal region, revealed a highly conserved purine rich loop with a structure nearly identical to the Rev-binding loop of the Rev response element. Using band-shift assays, surface plasmon resonance, and further NMR analysis, we demonstrate that this loop binds Rev. HIV-1 appears to have a second Rev-binding site close to the major splice donor site that may have an additional role in the viral life cycle.     

HIV-2 genome dimerization is required for the correct processing of Gag: a second-site reversion in matrix can restore both processes in dimerization-impaired mutant viruses

A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5' ends. In vitro, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich ((392)-GGAG-(395)) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the (392)-GGAG-(395) motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein.     

HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET

The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Psi) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Psi+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Psi- RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Psi does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Psi+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Psi thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.     

The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein

The major packaging signal of human immunodeficiency virus type 1 (HIV-1) RNA has been localised to the region 3' to the major splice donor within the leader sequence. Secondary structural studies for this region of the HIV-1 genome have shown the existence of a stem-loop structure capped by a purine-rich tetraloop. Extensive mapping data presented here lead to the complete characterisation of the structure of the stem-loop, including a new purine-rich internal loop in the lower part of the structure and the previously established GGAG tetraloop at its tip. Biochemical analysis reveals that both internal loop and tetraloop are primary sites for interaction with Gag polyprotein, and that binding of Gag protein leads to a conformational change which alters the RNA structure. NMR spectroscopy has been used to determine the three-dimensional structure of this complete stem-loop structure. The structural analysis reveals a significant difference between the apical part of the stem-loop structure, which adopts a well-defined conformation, and the purine-rich internal loop, which is instead very flexible. In contrast to what is generally observed for internal loop structures in RNA, this region of the encapsidation signal adopts a structure lacking stable interstrand interactions capable of stabilising a unique conformation. We suggest that the stem-loop structure represents a nucleation site for Gag protein binding, and that the protein exploits the flexibility of the internal loop to initiate the unwinding of the structure with successive addition of Gag molecules interacting with the RNA and each other through conserved I (interaction) domains.     

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.     

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a long distance interaction (LDI) and a branched multiple-hairpin (BMH) structure. The BMH structure exposes the dimer initiation site (DIS) hairpin, whereas this motif is occluded in the LDI structure. Consequently, these structures differ in their capacity to form RNA dimers in vitro. The BMH structure is dimerization-competent, due to DIS hairpin formation, but also presents the splice donor (SD) and RNA packaging (Psi) hairpins. In the LDI structure, an extended RNA packaging (Psi(E)) hairpin is folded, which includes the splice donor site and gag coding sequences. The gag initiation codon is engaged in a long distance base pairing interaction with sequences in the upstream U5 region in the BMH structure, thus forming the evolutionarily conserved U5-AUG duplex. Therefore, the LDI-BMH equilibrium may affect not only the process of RNA dimer formation but also translation initiation. In this study, we designed mutations in the 3'-terminal region of the leader RNA that alter the equilibrium of the LDI-BMH structures. The mutant leader RNAs are affected in RNA dimer formation, but not in their translation efficiency. These results indicate that the LDI-BMH status does not regulate HIV-1 RNA translation, despite the differential presentation of the gag initiation codon in both leader RNA structures.     

Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions.

Encapsidation of HIV-1 genomic RNA is mediated by specific interactions between the RNA packaging signal and the Gag protein. During maturation of the virion, the Gag protein is processed into smaller fragments, including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathione- S -transferase (GST) Gag and NC fusion proteins from HIV-1, to the entire HIV-1 and -2 leader RNAencompassing the packaging signal. We have mapped the binding sites at conditions where only about two complexes are formed and find that GST-Gag and GST-NC fusion proteins bind specifically to discrete sites within the leader. Analysis of the HIV-1 leader indicated that GST-Gag strongly associates with the PSI stem-loop and to a lesser extent with regions near the primer binding site. GST-NC binds the same regions but with reversed preferences. The HIV-1 proteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context.     

Insight into the intrinsic flexibility of the SL1 stem-loop from genomic RNA of HIV-1 as probed by molecular dynamics simulation

The SL1 stem-loop is the dimerization initiation site for linking the two copies of the RNA forming the HIV-1 genome. The 26 nucleotides stem contains a defect consisting on a highly conserved G-rich 1-3 asymmetrical internal loop, which is a major site for nucleocapsid protein binding. Several NMR attempts were undertaken to determine the internal loop structure in the SL1 monomer. However, the RNA constructs used in the different studies were largely mutated, in particular with replacement of the nine nucleotides apical loop by a tetraloop, and divergent results were obtained ranging from a rigid structure to a particularly large flexibility. To investigate the reasons for such discrepancies, we used molecular dynamics simulation of the SL1 monomer to probe the effect of mutations on the conformational stability of the internal loop and of the whole stem. It is found that in the wild-type sequence, the internal loop displays conformational variability originating mainly from the nine nucleotides apical loop flexibility that causes large conformational fluctuations (without changing the average structure) in the 7 bp duplex linking the apical and internal loops. The large amplitude atomic motions in the duplex are transmitted to the internal loop in which they induce conformational changes characterized by a labile hydrogen bond network such as G5 successively H-bonded to A29 and G30. On the contrary, with a four nucleotides apical loop, conformational fluctuations in the duplex are reduced by a factor of 2 and are not sufficiently energizing for promoting changes in the internal loop structure at the time scale of the simulations.     

Evidence that a Kissing Loop Structure Facilitates Genomic RNA Dimerisation in HIV-1

Genomic RNA isolated from retroviral particles is a dimer composed of two identical strands. A region called the dimer linkage signal close to the 5′ end of the RNA may be involved in forming the dimer. Several models for the formation of the HIV-1 RNA dimer have been proposed. In the kissing loop model, dimerisation results from base-pairing between homologous sequences in an RNA stem – loop. In the guanine tetrad model interstrand guanine contacts form the dimer. We have made mutations preventing the dimerisation of subgenomic RNAsin vitroby these mechanisms. To prevent the kissing loop dimer forming we changed the complementary loop sequence from 711GCGCGC716 to 711AAACGC716. To prevent the guanine tetrad dimer forming we changed G819 to U. These mutations were introduced into a clone of HIV-1_NL4-3separately and collectively. All three clones produced infectious virions. Dimeric RNA with similar thermal stabilities was isolated from viruses containing either the single or the double mutations. The results suggest that sequences involved in forming a guanine tetrad are not important for HIV-1 RNA dimerisation. In contrast sequences involved in forming a kissing loop complex are not absolutely required, but are important in forming a stable HIV-1 RNA dimer.     

Association of potent human antiviral cytidine deaminases with 7SL RNA and viral RNP in HIV-1 virions

7SL RNA promotes the formation of the signal recognition particle that targets secretory and membrane proteins to the endoplasmic reticulum. 7SL RNA is also selectively packaged by many retroviruses, including HIV-1. Here, we demonstrate that 7SL RNA is an integral component of the viral ribonucleoprotein (RNP) complex containing Gag, viral genomic RNA, and tRNA(3)(Lys). Only the potent anti-HIV-1 cytidine deaminases can bind to 7SL RNA and target to HIV-1 RNP. A conserved motif in the amino-terminal region of A3G is important for 7SL RNA interaction. The weak anti-HIV-1 A3C did not interact with 7SL RNA and failed to target to viral RNPs, despite efficient virion packaging. However, a chimeric construct of A3C plus the 7SL-binding amino terminus of A3G did target to viral RNPs and showed enhanced anti-HIV-1 activity. 7SL RNA binding is a conserved feature of human anti-HIV-1 cytidine deaminases. Thus, potent anti-HIV-1 cytidine deaminases have evolved to possess a unique RNA-binding ability for precise HIV-1 targeting and viral inhibition.     

Steric Block High Affinity Oligonucleotide Analogues: A New Tool for Mapping RNA-Protein Binding Sites

Steric-block ON analogues are efficient inhibitors of RNA-protein interaction and therefore have potential to probe RNA sequences for putative protein binding sites and to investigate mechanisms of protein binding. The packaging process of HIV-1 is highly specific involving an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. Using oligonucleotide probes we have confirmed that the terminal purine loop is the major Gag binding site on SL3 and that a secondary Gag binding site exists at an internal purine bulge. We also demonstrate direct binding of oligonucleotide to their binding sites and confirm this interaction does not alter global RNA conformation, making them highly specific, nondisruptive probes of RNA protein interactions.     

Steric block high affinity oligonucleotide analogues: a new tool for mapping RNA-protein binding sites

Steric-block ON analogues are efficient inhibitors of RNA-protein interaction and therefore have potential to probe RNA sequences for putative protein binding sites and to investigate mechanisms of protein binding. The packaging process of HIV-1 is highly specific involving an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. Using oligonucleotide probes we have confirmed that the terminal purine loop is the major Gag binding site on SL3 and that a secondary Gag binding site exists at an internal purine bulge. We also demonstrate direct binding of oligonucleotide to their binding sites and confirm this interaction does not alter global RNA conformation, making them highly specific, nondisruptive probes of RNA protein interactions.     

Molecular dynamics simulation for probing the flexibility of the 35 nucleotide SL1 sequence kissing complex from HIV-1Lai genomic RNA

The SL1 stem-loop located in the encapsidation domain is responsible for initiating the dimerisation of HIV-1 genomic RNA by means of a loop-loop interaction known as Kissing Complex (KC). The SL1 secondary structure has been predicted as a 35 nucleotides [K. G. Murti, M. Bondurant, and A. Tereba. J Virol 37, 411-419 (1981)] stem-loop composed of a 4 base pairs (bp) terminal duplex, a 4 nt asymmetrical internal loop, a 7 bp internal duplex, and a 9 nt apical loop. Several high resolution structures of the monomer and of KC of a 23 nt sequence containing only the internal duplex and the apical loop of SL1 are available in the literature. No experimental high resolution structure of the complete native SL1 sequence has been reported so far, either for the monomer or for KC. The asymmetrical internal loop has been described from NMR studies of different monomeric hairpin sequences, leading to divergent results, which suggests its high flexibility. In this work, we built a SL1(35) KC model which was submitted to a 31 ns molecular dynamics simulation (MD). Our results allows to describe the internal dynamics of SL1(35) KC and the differences of behavior of the different parts of the dimer. Thus, we could show the stability of the interactions between the two apical loops and of the terminal duplexes, the destabilization of the internal duplexes and the high flexibility of the asymmetrical internal loops.     

YB-1 stabilizes HIV-1 genomic RNA and enhances viral production

HIV-1 utilizes cellular factors for efficient replication. The viral RNA is different from cellular mRNAs in many aspects, and is prone to attacks by cellular RNA quality control systems. To establish effective infection, the virus has evolved multiple mechanisms to protect its RNA. Here, we show that expression of the Y-box binding protein 1 (YB-1) enhanced the production of HIV-1. Downregulation of endogenous YB-1 in producer cells decreased viral production. YB-1 increased viral protein expression by stabilizing HIV-1 RNAs. The stem loop 2 in the HIV-1 RNA packaging signal was mapped to be the YB-1-responsive element. Taken together, these results indicate that YB-1 stabilizes HIV-1 genomic RNA and thereby enhances HIV-1 gene expression and viral production.