Functional Segregation of Overlapping Genes in HIV.

Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a "segregated" organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.

An unusual topological structure of the HIV-1 Rev response element.

Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.

HIV-1 Tat commandeers nuclear export of Rev-viral RNA complex by controlling hnRNPA2-mediated splicing.

IMPORTANCE: HIV-infected host cells impose varied degrees of regulation on viral replication, from very high to abortive. Proliferation of HIV in astrocytes is limited when compared to immune cells, such as CD4+ T lymphocytes. Understanding such differential regulation is one of the key questions in the field as these cells permit HIV persistence and rebound viremia, challenging HIV treatment and clinical cure. This study focuses on understanding the molecular mechanism behind such cell-specific disparities. We show that one of the key mechanisms is the regulation of heterogenous nuclear ribonucleoprotein A2, a host factor involved in alternative splicing and RNA processing, by HIV-1 Tat in CD4+ T lymphocytes, not observed in astrocytes. This regulation causes an increase in the levels of unspliced/partially spliced viral RNA and nuclear export of Rev-RNA complexes which results in high viral propagation in CD4+ T lymphocytes. The study reveals a new mechanism imposed by HIV on host cells that determines the fate of infection.

Exploring the HIV-1 Rev Recognition Element (RRE)-Rev Inhibitory Capacity and Antiretroviral Action of Benfluron Analogs.

Human immunodeficiency virus-type 1 (HIV-1) remains one of the leading contributors to the global burden of disease, and novel antiretroviral agents with alternative mechanisms are needed to cure this infection. Here, we describe an exploratory attempt to optimize the antiretroviral properties of benfluron, a cytostatic agent previously reported to exhibit strong anti-HIV activity likely based on inhibitory actions on virus transcription and Rev-mediated viral RNA export. After obtaining six analogs designed to modify the benzo[c]fluorenone system of the parent molecule, we examined their antiretroviral and toxicity properties together with their capacity to recognize the Rev Recognition Element (RRE) of the virus RNA and inhibit the RRE-Rev interaction. The results indicated that both the benzo[c] and cyclopentanone components of benfluron are required for strong RRE-Rev target engagement and antiretroviral activity and revealed the relative impact of these moieties on RRE affinity, RRE-Rev inhibition, antiviral action and cellular toxicity. These data provide insights into the biological properties of the benzo[c]fluorenone scaffold and contribute to facilitating the design of new anti-HIV agents based on the inhibition of Rev function.

Role of Mutations in Differential Recognition of Viral RNA Molecules by Peptides.

The conserved noncoding RNA elements in viral genomes interact with proteins to regulate various events during viral replication. We report studies on the recognition mechanisms of two helical peptides, namely, a native (Rev) peptide and a lab-evolved (RSG1.2) peptide, by a highly conserved viral RNA element from the human immunodeficiency virus 1 genome. Specifically, we investigated the physical interactions between the viral RNA molecule and helical peptides by computing free energy changes on mutating key amino acid residues involved in recognition of an internal loop in the viral RNA molecule.

An emerging and variant viral promoter of HIV-1 subtype C exhibits low-level gene expression noise.

BACKGROUND: We observe the emergence of several promoter-variant viral strains in India during recent years. The variant viral promoters contain additional copies of transcription factor binding sites present in the viral modulatory region or enhancer, including RBEIII, LEF-1, Ap-1 and/or NF-κB. These sites are crucial for governing viral gene expression and latency. Here, we infer that one variant viral promoter R2N3-LTR containing two copies of RBF-2 binding sites (an RBEIII site duplication) and three copies of NF-κB motifs may demonstrate low levels of gene expression noise as compared to the canonical RN3-LTR or a different variant R2N4-LTR (a duplication of an RBEIII site and an NF-κB motif). To demonstrate this, we constructed a panel of sub-genomic viral vectors of promoter-variant LTRs co-expressing two reporter proteins (mScarlet and Gaussia luciferase) under the dual-control of Tat and Rev. We established stable pools of CEM.NKR-CCR5 cells (CEM-CCR5RL reporter cells) and evaluated reporter gene expression under different conditions of cell activation. RESULTS: The R2N3-LTR established stringent latency that was highly resistant to reversal by potent cell activators such as TNF-α or PMA, or even to a cocktail of activators, compared to the canonical RN3- or the variant R2N4-LTR. The R2N3-LTR exhibited low-level basal gene expression in the absence of cell activation that enhanced marginally but significantly when activated. In the presence of Tat and Rev, trans-complemented in the form of an infectious virus, the R2N3-LTR demonstrated gene expression at levels comparable to the wild-type viral promoter. The R2N3-LTR is responsive to Tat and Rev factors derived from viral strains representing diverse genetic subtypes. CONCLUSION: With extremely low-level transcriptional noise, the R2N3-LTR can serve as an excellent model to examine the establishment, maintenance, and reversal of HIV-1 latency. The R2N3-LTR would also be an ideal viral promoter to develop high-throughput screening assays to identify potent latency-reversing agents since the LTR is not affected by the usual background noise of the cell.

Modulation of HIV-1 gene expression by binding of a ULM motif in the Rev protein to UHM-containing splicing factors.

The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.

ANP32A and ANP32B are key factors in the Rev-dependent CRM1 pathway for nuclear export of HIV-1 unspliced mRNA.

The nuclear export receptor CRM1 is an important regulator involved in the shuttling of various cellular and viral RNAs between the nucleus and the cytoplasm. HIV-1 Rev interacts with CRM1 in the late phase of HIV-1 replication to promote nuclear export of unspliced and single spliced HIV-1 transcripts. However, other cellular factors involved in the CRM1-dependent viral RNA nuclear export remain largely unknown. Here, we demonstrate that ANP32A and ANP32B mediate the export of unspliced or partially spliced viral mRNA via interactions with Rev and CRM1. We found that a double, but not single, knockout of ANP32A and ANP32B significantly decreased the expression of gag protein. Reconstitution of either ANP32A or ANP32B restored the viral production equally. Disruption of both ANP32A and ANP32B expression led to a dramatic accumulation of unspliced viral mRNA in the nucleus. We further identified that ANP32A and ANP32B interact with both Rev and CRM1 to promote RNA transport. Our data strongly suggest that ANP32A and ANP32B play an important role in the Rev-CRM1 pathway, which is essential for HIV-1 replication, and our findings provide a candidate therapeutic target for host defense against retroviral infection.

Altered-specificity mutants of the HIV Rev arginine-rich motif-RRE IIB interaction.

Arginine-rich motifs (ARMs) bind RNA structures with high affinity and specificity, and the human immunodeficiency virus (HIV) exploits ARM-RNA interactions to regulate its lifecycle. The expression of HIV structural genes relies on recognition between the ARM of its Rev protein and its primary binding site, an internal loop in the viral RNA, the Rev-response element region IIB (IIB). Many functional variants of the Rev ARM-IIB interaction have been discovered, yet how easily it can evolve new specificities is poorly explored. A double mutant of Rev ARM, R35G-N40 V, uses an unknown strategy to recognize IIB. Here, isothermal titration calorimetry and gel shift assays show that the R35G-N40V-IIB interaction has high affinity and specificity in vitro and a larger unfavorable entropy change upon binding than that of wild-type Rev ARM-IIB. In stark contrast with the critical dependence of wild-type Rev on Arg35, Arg39, Asn40, and Arg44, mutational profiling shows R35G-N40V is highly mutable at positions 40 and 44 and dependent on Gly35, Arg38, Arg39, Arg42, and Arg43. Affinity measurements in vitro and reporter assay measurements in vivo are consistent with the wild-type Rev ARM and R35G-N40V maintaining their recognition strategies when binding IIB mutants specific to wild-type Rev ARM and R35G-N40V, respectively. Some single amino acid mutants of wild-type Rev ARM and R35G-N40V have enhanced specificity, recognizing mutant IIBs yet not wild-type IIB. These results provide another example of viral ARM-RNA interactions evolving new specificities with few mutations, consistent with neutral theories of evolution.

A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA.

Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.

Highly efficient cellular uptake of a cell-penetrating peptide (CPP) derived from the capsid protein of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is one of the smallest, nonenveloped, single-stranded DNA viruses. The PCV2 capsid protein (Cap) is the sole viral structural protein and main antigenic determinant. Previous sequence analysis has revealed that the N terminus of the PCV2 Cap contains a nuclear localization signal (NLS) enriched in positively charged residues. Here, we report that PCV2's NLS can function as a cell-penetrating peptide (CPP). We observed that this NLS can carry macromolecules, e.g. enhanced GFP (EGFP), into cells when they are fused to the NLS, indicating that it can function as a CPP, similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT). We also found that the first 17 residues of the NLS (NLS-A) have a key role in cellular uptake. In addition to entering cells via multiple endocytic processes, NLS-A was also rapidly internalized via direct translocation enabled by increased membrane permeability and was evenly distributed throughout cells when its concentration in cell cultures was ≥10 µm Of note, cellular NLS-A uptake was ∼10 times more efficient than that of HIV TAT. We inferred that the externalized NLS of the PCV2 Cap may accumulate to a high concentration (≥10 µm) at a local membrane area, increasing membrane permeability to facilitate viral entry into the cell to release its genome into a viral DNA reproduction center. We conclude that NLS-A has potential as a versatile vehicle for shuttling foreign molecules into cells, including pharmaceuticals for therapeutic interventions.

HIV-1 Rev interacts with HERV-K RcREs present in the human genome and promotes export of unspliced HERV-K proviral RNA.

BACKGROUND: The HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. They are present as several almost complete proviral copies and numerous fragments in the human genome. Many HERV-K proviruses express a regulatory protein Rec, which binds to an element present in HERV-K mRNAs called the RcRE. This interaction is necessary for the nucleo-cytoplasmic export and expression of HERV-K mRNAs that retain introns and plays a role analogous to that of Rev and the RRE in HIV replication. There are over 900 HERV-K RcREs distributed throughout the human genome. Thus, it was of interest to determine if Rev could functionally interact with selected RcRE elements that map either to HERV-K proviruses or human gene regions. This interaction would have the potential to alter the expression of both HERV-K mRNAs and cellular mRNAs during HIV-1 infection. RESULTS: In this study we employed a combination of RNAseq, bioinformatics and cell-based functional assays. Potential RcREs were identified through a number of bioinformatic approaches. They were then tested for their ability to promote export and translation of a reporter mRNA with a retained intron in conjunction with Rev or Rec. Some of the selected elements functioned well with either Rev, Rec or both, whereas some showed little or no function. Rev function on individual RcREs varied and was also dependent on the Rev sequence. We also performed RNAseq on total and cytoplasmic RNA isolated from SupT1 cells expressing HIV Rev, with or without Tat, or HERV-K Rec. Proviral mRNA from three HERV-K loci (4p16.1b, 22q11.23 and most significantly 3q12.3) accumulated in the cytoplasm in the presence of Rev or Tat and Rev, but not Rec. Consistent with this, the 3' RcRE from 3q12.3 functioned well with HIV-Rev in our reporter assay. In contrast, this RcRE showed little or no function with Rec. CONCLUSIONS: The HIV Rev protein can functionally interact with many RcREs present in the human genome, depending on the RcRE sequence, as well as the Rev sequence. This leads to export of some of the HERV-K proviral mRNAs and also has the potential to change the expression of non-viral genes.

Impact of pre-amplification conditions on sensitivity of the tat/rev induced limiting dilution assay.

Antiretroviral therapy (ART) can lower a patient's HIV plasma viral load to an undetectable level, but following cessation of ART viremia rapidly rebounds. It has been shown that ART does not eliminate latent viruses sequestered into anatomical and cellular reservoirs. Therefore, in patients that have ceased ART, the following rebound in HIV viremia is caused by the activation of latent HIV reservoirs. A major issue in HIV cure research is the quantification of these latent HIV reservoirs. Various reservoir measurement methods exist, but the gold standard technique remains the culture-based quantitative viral outgrowth assay (QVOA). Recently, a new PCR-based assay, named the tat/rev induced limiting dilution assay (TILDA) was described which measures the frequency of inducible latently infected CD4+ T cells that actively produce multiply-spliced RNA coding for the Tat/Rev proteins. The objective of this study was to further optimize the assay by examining the influence of varied factors, such as the amount of products transferred from the pre-amplification step to the PCR reaction, storage of pre-amplification products prior to PCR runs, and the number of cells used, on the assay's sensitivity and reproducibility. We also investigated whether the assay could be used to quantify HIV reservoirs in monocytes/macrophages.

Binding and thermodynamics of REV peptide-ctDNA interaction.

The thermodynamics of DNA-ligand binding is important as it provides useful information to understand the details of binding processes. HIV-1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV-1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV-1 were investigated using spectroscopic (UV-visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a Tm of 75.5 °C, whereas the ctDNA-REV peptide complex exhibited an incremental shift in the Tm by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive Bâ†’Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr-bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA-REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K-308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low ΔH values indicating that the ctDNA-REV peptide interaction is driven largely by entropy. The heat capacity change (ΔCp ) was insignificant, an unusual finding in the area of DNA-peptide interaction studies. The variation in the values obtained for ΔH, ΔS, and ΔG with temperature further suggests that ctDNA-REV peptide interaction is entropically driven. ITC based analysis of salt dependence of binding constant gave a charge value (Z) = +4.01, as determined for the δlnK/δln[Na+ ] parameter, suggesting the participation of only 3-4 Arg out of 11 Arg charge from REV peptide. The stoichiometry observed for the complex was three molar charge of REV peptide binding per molar charge of ctDNA. ITC based analysis further confirmed that the binding between ctDNA and REV peptide is governed by electrostatic interaction. Molecular interactions including H-bonding, van der Waals forces, and solvent molecules rearrangement, underlie the binding of REV peptide to ctDNA.

The human immunodeficiency virus (HIV) Rev-binding protein (HRB) is a co-factor for HIV-1 Nef-mediated CD4 downregulation.

Human immunodeficiency virus type 1 (HIV-1)-mediated CD4 downregulation is an important determinant of viral replication in vivo. Research on cellular co-factors involved in this process could lead to the identification of potential therapeutic targets. We found that CD4 surface levels were significantly higher in HIV-1-infected cells knocked-down for the HIV Rev-binding protein (HRB) compared with control cells. HRB knock-down affected CD4 downregulation induced by Nef but not by HIV-1 Vpu. Interestingly, the knock-down of the related protein HRBL (HRB-like), but not of the HRB interaction partner EPS15 (epidermal growth factor receptor pathway substrate 15), increased CD4 levels in Vpu-expressing cells significantly. Both of these proteins are known to be involved in HIV-1-mediated CD4 downregulation as co-factors of HIV-1 Nef. These results identify HRB as a previously unknown co-factor for HIV-1 Nef-mediated CD4 downregulation and highlight differences with the related protein HRBL, which affects the CD4 downregulation in a dual role as co-factor of both HIV-1 Nef and Vpu.

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.

Pokeweed antiviral protein alters splicing of HIV-1 RNAs, resulting in reduced virus production.

Processing of HIV-1 transcripts results in three populations in the cytoplasm of infected cells: full-length RNA, singly spliced, and multiply spliced RNAs. Rev, regulator of virion expression, is an essential regulatory protein of HIV-1 required for transporting unspliced and singly spliced viral transcripts from the nucleus to the cytoplasm. Export allows these RNAs to be translated and the full-length RNA to be packaged into virus particles. In our study, we investigate the activity of pokeweed antiviral protein (PAP), a glycosidase isolated from the pokeweed plant Phytolacca americana, on the processing of viral RNAs. We show that coexpression of PAP with a proviral clone alters the splicing ratio of HIV-1 RNAs. Specifically, PAP causes the accumulation of multiply spliced 2-kb RNAs at the expense of full-length 9-kb and singly spliced 4-kb RNAs. The change in splicing ratio is due to a decrease in activity of Rev. We show that PAP depurinates the rev open reading frame and that this damage to the viral RNA inhibits its translation. By decreasing Rev expression, PAP indirectly reduces the availability of full-length 9-kb RNA for packaging and translation of the encoded structural proteins required for synthesis of viral particles. The decline we observe in virus protein expression is not due to cellular toxicity as PAP did not diminish translation rate. Our results describing the reduced activity of a regulatory protein of HIV-1, with resulting change in virus mRNA ratios, provides new insight into the antiviral mechanism of PAP.

Thermodynamics of Rev-RNA interactions in HIV-1 Rev-RRE assembly.

The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function.

A cell-penetrating antibody fragment against HIV-1 Rev has high antiviral activity: characterization of the paratope.

The HIV-1 protein Rev oligomerizes on viral transcripts and directs their nuclear export. Previously, a Fab against Rev generated by phage display was used to crystallize and solve the structure of the Rev oligomerization domain. Here we have investigated the capability of this Fab to block Rev oligomerization and inhibit HIV-1 replication. The Fab itself did not have antiviral activity, but when a Tat-derived cell-penetrating peptide was appended, the resulting molecule (FabRev1-Tat) was strongly inhibitory of three different CCR5-tropic HIV-1 isolates (IC50 = 0.09-0.44 µg/ml), as assessed by suppression of reverse transcriptase activity in infected peripheral blood mononuclear cells, and had low cell toxicity (TC50 > 100 µg/ml). FabRev1-Tat was taken up by both peripheral blood mononuclear and HEK293T cells, appearing in both the cytoplasm and nucleus, as shown by immunofluorescence confocal laser scanning microscopy. Computational alanine scanning was used to identify key residues in the complementarity-determining regions to guide mutagenesis experiments. Residues in the light chain CDR3 (LCDR3) were assessed to be important. Residues in LCDR3 were mutated, and LCDR3-Tyr(92) was found to be critical for binding to Rev, as judged by surface plasmon resonance and electron microscopy. Peptides corresponding to all six CDR regions were synthesized and tested for Rev binding. None of the linear peptides had significant affinity for Rev, but four of the amide-cyclic forms did. Especially cyclic-LCDR3 (LGGYPAASYRTA) had high affinity for Rev and was able to effectively depolymerize Rev filaments, as shown by both surface plasmon resonance and electron microscopy.

Cooperativity among Rev-associated nuclear export signals regulates HIV-1 gene expression and is a determinant of virus species tropism.

UNLABELLED: Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCE: Cells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression.