ABSTRACT
During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.
Subject(s)
HIV-1 , Nucleic Acid Conformation , RNA, Viral , rev Gene Products, Human Immunodeficiency Virus , HIV-1/metabolism , HIV-1/genetics , Binding Sites , rev Gene Products, Human Immunodeficiency Virus/metabolism , rev Gene Products, Human Immunodeficiency Virus/chemistry , rev Gene Products, Human Immunodeficiency Virus/genetics , RNA, Viral/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , Crystallography, X-Ray , Protein Binding , Models, Molecular , Humans , Response ElementsABSTRACT
Retroviruses must overcome cellular restrictions to the nucleocytoplasmic export of viral mRNAs that retain introns in order to complete their replication cycle. HIV accomplishes this using a system comprised of a trans-acting viral protein, Rev, and a cis-acting RNA secondary structure in the viral genome, the Rev-Response Element (RRE). HIV primary isolates differ with respect to the sequence and functional activity of the Rev-RRE system. Here, we describe a high throughput assay system for analyzing Rev-RRE functional activity using packageable viral vectors.
Subject(s)
RNA, Viral , Response Elements , rev Gene Products, Human Immunodeficiency Virus , Humans , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolism , Response Elements/genetics , RNA, Viral/genetics , HIV-1/genetics , HIV-1/physiology , Gene Expression Regulation, Viral , Virus Replication/genetics , Genetic Vectors/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The HIV-1 Rev protein expressed in the early stage of virus replication is involved in the nuclear export of some forms of virus RNA. Naturally occurring polymorphisms in the Rev protein could influence its activity. The association between the genetic features of different virus variants and HIV infection pathogenesis has been discussed for many years. In this study, Rev diversity among HIV-1 group M clades was analyzed to note the signatures that could influence Rev activity and, subsequently, clinical characteristics. From the Los Alamos HIV Sequence Database, 4962 Rev sequences were downloaded and 26 clades in HIV-1 group M were analyzed for amino acid changes, conservation in consensus sequences, and the presence of clade-specific amino acid substitutions (CSSs) and the Wu-Kabat protein variability coefficient (WK). Subtypes G, CRF 02_AG, B, and A1 showed the largest amino acid changes and diversity. The mean conservation of the Rev protein was 80.8%. In consensus sequences, signatures that could influence Rev activity were detected. In 15 out of 26 consensus sequences, an insertion associated with the reduced export activity of the Rev protein, 95QSQGTET96, was identified. A total of 32 CSSs were found in 16 clades, wherein A6 had the 41Q substitution in the functionally significant region of Rev. The high values of WK coefficient in sites 51 and 82, located on the Rev interaction surface, indicate the susceptibility of these positions to evolutionary replacements. Thus, the noted signatures require further investigation.
Subject(s)
Genetic Variation , HIV Infections , HIV-1 , rev Gene Products, Human Immunodeficiency Virus , HIV-1/genetics , HIV-1/classification , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolism , Humans , HIV Infections/virology , Phylogeny , Amino Acid Substitution , Amino Acid Sequence , Consensus SequenceABSTRACT
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.
Subject(s)
Anti-HIV Agents , HIV Infections , HIV-1 , Humans , HIV-1/genetics , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolism , RNA, Viral/genetics , Anti-HIV Agents/pharmacology , HIV Infections/drug therapy , Nucleic Acid ConformationABSTRACT
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.
Subject(s)
Active Transport, Cell Nucleus , HIV Infections , HIV-1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , tat Gene Products, Human Immunodeficiency Virus , Humans , Alternative Splicing , Cell Nucleus/metabolism , Gene Products, rev/genetics , HIV-1/physiology , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolism , RNA Splicing , RNA, Viral/genetics , RNA, Viral/metabolism , tat Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolismABSTRACT
HIV-1 Rev is an essential regulatory protein that transports unspliced and partially spliced viral mRNAs from the nucleus to the cytoplasm for the expression of viral structural proteins. During its nucleocytoplasmic shuttling, Rev interacts with several host proteins to use the cellular machinery for the advantage of the virus. Here, we report the 3.5 Å cryo-EM structure of a 4.8 MDa Rev-tubulin ring complex. Our structure shows that Rev's arginine-rich motif (ARM) binds to both the acidic surfaces and the C-terminal tails of α/ß-tubulin. The Rev-tubulin interaction is functionally homologous to that of kinesin-13, potently destabilizing microtubules at sub-stoichiometric levels. Expression of Rev in astrocytes and HeLa cells shows that it can modulate the microtubule cytoskeleton within the cellular environment. These results show a previously undefined regulatory role of Rev.
Subject(s)
HIV-1 , Humans , HeLa Cells , rev Gene Products, Human Immunodeficiency Virus/genetics , HIV-1/metabolism , Kinesins/genetics , Kinesins/metabolism , Tubulin/metabolismABSTRACT
The complex between the Rev protein of HIV-1 and the Rev Recognition Element (RRE) within the virus RNA promotes nuclear export of unspliced or incompletely spliced viral transcripts and is required for virus transmission. Here, we have screened a virtual collection of compounds using a pharmacophore based on the chemical similarity of previously characterized inhibitors to identify new chemical scaffolds blocking the RRE-Rev interaction. The best molecules discovered with this strategy inhibited the complex by binding to the RRE and exhibited substantial antiretroviral activity (between 0.582 and 11.3 µM EC50 values) likely associated to inhibitory actions on viral transcription and Rev function. These results have allowed us to identify structural features required for RRE-Rev inhibition as well as to add new compounds to the pool of possible candidates for developing antiretroviral agents based on blockage of HIV-1 RNA biogenesis.
Subject(s)
HIV-1 , HIV-1/physiology , rev Gene Products, Human Immunodeficiency Virus , Pharmacophore , RNA, Viral/metabolism , Active Transport, Cell NucleusABSTRACT
During HIV infection, intron-containing viral mRNAs are exported from the cell nucleus to the cytoplasm to complete the replication cycle. Cellular restrictions on the export of incompletely spliced transcripts are overcome by a viral protein, Rev, and an RNA structure found in all unspliced and incompletely spliced viral mRNAs, the Rev Response Element (RRE). Primary HIV isolates display substantial variation in the sequence and functional activity of Rev proteins. We analyzed Rev from two primary isolates with disparate activity that resulted in differences in in vitro fitness of replication-competent viral constructs. The results showed that amino acid differences within the oligomerization domain, but not the arginine-rich motif or the nuclear export signal, determined the level of Rev activity. Two specific amino acid substitutions were sufficient to alter the low-activity Rev to a high-activity phenotype. Other mutations in Rev sequences had unpredictable effects on activity that differed between the two Rev backbones. The sensitivity of Rev function level to small sequence changes likely permits modulation of Rev-RRE activity during HIV infection, which may play a role in pathogenesis. The functional consequences of Rev mutations differed between primary isolates, highlighting the challenge of generalizing studies of Rev conducted using laboratory HIV strains.
Subject(s)
HIV Infections , HIV Seropositivity , HIV-1 , Humans , HIV-1/genetics , rev Gene Products, Human Immunodeficiency Virus/genetics , HIV Infections/genetics , Gene Products, rev/genetics , Gene Products, rev/metabolism , Response Elements , HIV Seropositivity/genetics , RNA, Messenger/genetics , RNA, Viral/geneticsABSTRACT
Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction, whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing Rev-NLUC or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z') for the cell-free system was â¼0.85 when tested in 96-well format, and the anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.
Subject(s)
HIV Infections , HIV-1 , Humans , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolism , HIV-1/physiology , Luciferases, Firefly/genetics , Luciferases, Firefly/metabolism , Gene Products, rev/genetics , Gene Products, rev/metabolism , RNA/metabolism , Ribonucleases/metabolism , RNA, Viral/geneticsABSTRACT
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.
Subject(s)
HIV-1 , RNA, Viral , HIV-1/genetics , HIV-1/metabolism , Humans , Mutation , Peptides/chemistry , RNA, Viral/metabolism , Virus Replication , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Nucleocytoplasmic shuttling of viral elements, supported by several host factors, is essential for the replication of the human immunodeficiency virus (HIV). HIV-1 uses a nuclear RNA export pathway mediated by viral protein Rev to transport its Rev response element (RRE)-containing partially spliced and unspliced transcripts aided by the host nuclear RNA export protein CRM1. The factor(s) interacting with the CRM1-Rev complex are potential antiretroviral target(s) and could serve as a retroviral model system to study nuclear export machinery adapted by these viruses. We earlier reported that cellular Staufen-2 interacts with Rev, facilitating viral-RNA export. Here, we identified the formation of a complex between Staufen-2, CRM1 and Rev. Molecular docking and simulations mapped the interacting residues in the RNA-binding Domain 4 of Staufen-2 as R336 and R337, which were experimentally verified to be critical for interactions among Staufen-2, CRM1 and Rev by mutational analysis. Staufen-2 mutants defective in interaction with CRM1 or Rev failed to supplement the Rev-RNA export activity and viral production, demonstrating the importance of these interactions. Rev-dependent reporter assays and proviral DNA-construct transfection-based studies in Staufen-2 knockout cells in the presence of leptomycin-B (LMB) revealed a significant reduction in CRM1-mediated Rev-dependent RNA export with decreased virus production as compared to Staufen-2 knockout background or LMB treatment alone, suggesting the relevance of these interactions in augmenting RNA export activity of Rev. Our observations provide further insights into the mechanistic intricacies of unspliced viral-RNA export to the cytoplasm and support the notion that abrogating such interactions can reduce HIV-1 proliferation.
Subject(s)
HIV-1 , Humans , Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Genomics , HIV-1/physiology , Karyopherins/genetics , Karyopherins/metabolism , Molecular Docking Simulation , Nuclear Proteins/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolism , RNA, Nuclear/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Single-cell imaging has emerged as a powerful means to study viral replication dynamics and identify sites of virus−host interactions. Multivariate aspects of viral replication cycles yield challenges inherent to handling large, complex imaging datasets. Herein, we describe the design and implementation of an automated, imaging-based strategy, "Human Immunodeficiency Virus Red-Green-Blue" (HIV RGB), for deriving comprehensive single-cell measurements of HIV-1 unspliced (US) RNA nuclear export, translation, and bulk changes to viral RNA and protein (HIV-1 Rev and Gag) subcellular distribution over time. Differentially tagged fluorescent viral RNA and protein species are recorded using multicolor long-term (>24 h) time-lapse video microscopy, followed by image processing using a new open-source computational imaging workflow dubbed "Nuclear Ring Segmentation Analysis and Tracking" (NR-SAT) based on ImageJ plugins that have been integrated into the Konstanz Information Miner (KNIME) analytics platform. We describe a typical HIV RGB experimental setup, detail the image acquisition and NR-SAT workflow accompanied by a step-by-step tutorial, and demonstrate a use case wherein we test the effects of perturbing subcellular localization of the Rev protein, which is essential for viral US RNA nuclear export, on the kinetics of HIV-1 late-stage gene regulation. Collectively, HIV RGB represents a powerful platform for single-cell studies of HIV-1 post-transcriptional RNA regulation. Moreover, we discuss how similar NR-SAT-based design principles and open-source tools might be readily adapted to study a broad range of dynamic viral or cellular processes.
Subject(s)
HIV Infections , HIV Seropositivity , HIV-1 , Active Transport, Cell Nucleus , HIV-1/physiology , Humans , RNA, Viral/genetics , RNA, Viral/metabolism , Single-Cell Analysis , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
In silico-designed multiepitope conserved regions of human immunodeficiency virus 1 (HIV-1) proteins would be a beneficial strategy for antigen design which induces effective anti-HIV-1 T-cell responses. The conserved multiple HLA-DR-binding epitopes of Rev protein were identified using IEDB MHC-I prediction tools and SYFPEITHI webserver to screen potential T-cell epitopes. We analyzed toxicity, allergenicity, immunogenicity, hemolytic activity, cross-reactivity, cell-penetrating peptide (CPP) potency, and molecular docking of the candidate epitopes using several immune-informatics tools. Afterward, we designed a novel multiepitope construct based on non-toxic and non-allergenic Rev, Nef, Gp160 and P24-derived cytotoxic T cell (CTL) and T-helper cell (HTL) epitopes. Next, the designed construct (Nef-Rev-Gp160-P24) was subjected to three B-cell epitope prediction webservers, ProtParam and Protein-Sol to obtain the physicochemical features. Then, the recombinant multiepitope DNA and polypeptide constructs were complexed with different CPPs for nanoparticle formation and pass them via the cell membranes. Finally, the immunogenicity of multiepitope constructs in a variety of modalities was evaluated in mice. The results demonstrated that groups immunized with heterologous DNA+ MPG or HR9 CPP prime/rNef-Rev-Gp160-P24 polypeptide + LDP-NLS CPP boost regimens could significantly produce higher levels of IFN-γ and Granzyme B, and lower amounts of IL-10 than other groups. Moreover, higher levels of IgG2a and IgG2b were observed in all heterologous prime-boost regimens than homologous DNA or polypeptide regimens. Altogether, the present findings indicated that the Nef-Rev-Gp160-P24 polypeptide meets the criteria to be potentially useful as a multiepitope-based vaccine candidate against HIV-1 infection.
Subject(s)
AIDS Vaccines , Cell-Penetrating Peptides , rev Gene Products, Human Immunodeficiency Virus/immunology , Animals , Epitopes, T-Lymphocyte , HIV-1 , Mice , Molecular Docking SimulationABSTRACT
BACKGROUND: A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery. METHODS: A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels. FINDINGS: Virus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing. INTERPRETATION: These results provide "proof of concept' for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing. FUNDING: The work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.
Subject(s)
CRISPR-Cas Systems , Exons , Gene Editing , HIV Infections/therapy , HIV Infections/virology , HIV-1/genetics , Cell Line , Conserved Sequence , Fluorescent Antibody Technique , Gene Targeting , Genes, Reporter , Genetic Therapy , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Genome, Viral , Humans , Liposomes , Macrophages/metabolism , Macrophages/virology , Nanoparticles , Proviruses/genetics , RNA Interference , RNA, Messenger/administration & dosage , RNA, Messenger/genetics , rev Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/genetics , RNA, Guide, CRISPR-Cas SystemsABSTRACT
HIV-1 can incite activation of chemokine receptors, inflammatory mediators, and glutamate receptor-mediated excitotoxicity. The mechanisms associated with such immune activation can disrupt neuronal and glial functions. HIV-associated neurocognitive disorder (HAND) is being observed since the beginning of the AIDS epidemic due to a change in the functional integrity of cells from the central nervous system (CNS). Even with the presence of antiretroviral therapy, there is a decline in the functioning of the brain especially movement skills, noticeable swings in mood, and routine performance activities. Under the umbrella of HAND, various symptomatic and asymptomatic conditions are categorized and are on a rise despite the use of newer antiretroviral agents. Due to the use of long-lasting antiretroviral agents, this deadly disease is becoming a manageable chronic condition with the occurrence of asymptomatic neurocognitive impairment (ANI), symptomatic mild neurocognitive disorder, or HIV-associated dementia. In-depth research in the pathogenesis of HIV has focused on various mechanisms involved in neuronal dysfunction and associated toxicities ultimately showcasing the involvement of various pathways. Increasing evidence-based studies have emphasized a need to focus and explore the specific pathways in inflammation-associated neurodegenerative disorders. In the current review, we have highlighted the association of various HIV proteins and neuronal cells with their involvement in various pathways responsible for the development of neurotoxicity.
Subject(s)
AIDS Dementia Complex/immunology , AIDS Dementia Complex/virology , Central Nervous System/virology , HIV-1/metabolism , Viral Proteins/metabolism , AIDS Dementia Complex/physiopathology , Anti-Retroviral Agents/therapeutic use , Astrocytes/virology , Central Nervous System/physiopathology , Genome , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp41/metabolism , HIV Infections/complications , HIV Infections/metabolism , Human Immunodeficiency Virus Proteins/metabolism , Humans , Inflammation , Kynurenine/metabolism , Macrophages/virology , Microglia/virology , Neurons/virology , Oligodendroglia/virology , Receptors, N-Methyl-D-Aspartate/metabolism , Viral Load , Viral Regulatory and Accessory Proteins/metabolism , Viroporin Proteins/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , rev Gene Products, Human Immunodeficiency Virus/metabolism , tat Gene Products, Human Immunodeficiency Virus/metabolism , vpr Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
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.
Subject(s)
HIV Long Terminal Repeat , HIV-1/genetics , Promoter Regions, Genetic , Binding Sites , Gene Expression Regulation, Viral , Genes, Reporter , Genetic Variation , HIV Infections/virology , HIV-1/classification , HIV-1/isolation & purification , HIV-1/physiology , Humans , NF-kappa B , Transcription, Genetic , Virus Replication , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolism , tat Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4+ T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size.
Subject(s)
HIV Infections/diagnosis , HIV Infections/virology , HIV-1/physiology , Nucleic Acid Amplification Techniques , Viral Load , Virus Latency , rev Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/genetics , Cell Line , Conserved Sequence , Genetic Variation , HIV Infections/drug therapy , Humans , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Polymerase Chain Reaction , RNA, Viral , Reproducibility of Results , Sensitivity and Specificity , rev Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
Human immunodeficiency virus-1 (HIV-1) infection remains to be one of the major threats throughout the world. Many researchers are working in this area to find a cure for HIV-1. The group of the FDA approved drugs which are currently used against HIV-1 in the clinical practice include nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), integrase inhibitors (InIs), and protease inhibitors (PIs). Fixed dose combinations (FDCs) of these drugs are available and are used as per the anti-retroviral therapy (ART) guidelines. Despite these, unfortunately, there is no cure for HIV1 infection to date. The present review is focused upon describing the importance of a post-transcriptional regulatory protein "Rev", responsible for latent HIV-1 infection as a possible, and promising therapeutic target against HIV-1.
Subject(s)
Anti-HIV Agents/pharmacology , Drug Delivery Systems , HIV Infections/drug therapy , HIV-1 , rev Gene Products, Human Immunodeficiency Virus/metabolism , Gene Expression Regulation, Viral/drug effects , Humans , rev Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
The transcription of the HIV-1 provirus results in only one type of transcript-full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.
Subject(s)
HIV-1/genetics , RNA Splicing , RNA, Messenger/metabolism , RNA, Viral/metabolism , Alternative Splicing , Exons , Gene Expression Regulation, Viral , Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Regulatory Sequences, Nucleic Acid , Virus Latency/genetics , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern.IMPORTANCE To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.