RESUMEN
IMPORTANCE: Twenty-five years after the first report that HIV-2 infection can reduce HIV-1-associated pathogenesis in dual-infected patients, the mechanisms are still not well understood. We explored these mechanisms in cell culture and showed first that these viruses can co-infect individual cells. Under specific conditions, HIV-2 inhibits HIV-1 through two distinct mechanisms, a broad-spectrum interferon response and an HIV-1-specific inhibition conferred by the HIV-2 TAR. The former could play a prominent role in dually infected individuals, whereas the latter targets HIV-1 promoter activity through competition for HIV-1 Tat binding when the same target cell is dually infected. That mechanism suppresses HIV-1 transcription by stalling RNA polymerase II complexes at the promoter through a minimal inhibitory region within the HIV-2 TAR. This work delineates the sequence of appearance and the modus operandi of each mechanism.
Asunto(s)
Coinfección , Regulación Viral de la Expresión Génica , Duplicado del Terminal Largo de VIH , VIH-1 , VIH-2 , Interferones , ARN Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Coinfección/inmunología , Coinfección/virología , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , VIH-1/inmunología , VIH-2/genética , VIH-2/inmunología , VIH-2/metabolismo , ARN Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Interferones/inmunología , Regiones Promotoras Genéticas/genética , Unión Competitiva , ARN Polimerasa II/metabolismo , Transcripción GenéticaRESUMEN
The AIDS pandemic is still of importance. HIV-1 and HIV-2 are the causative agents of this pandemic, and in the absence of a viable vaccine, drugs are continually required to provide quality of life for infected patients. The HIV capsid (CA) protein performs critical functions in the life cycle of HIV-1 and HIV-2, is broadly conserved across major strains and subtypes, and is underexploited. Therefore, it has become a therapeutic target of interest. Here, we report a novel series of 2-pyridone-bearing phenylalanine derivatives as HIV capsid modulators. Compound FTC-2 is the most potent anti-HIV-1 compound in the new series of compounds, with acceptable cytotoxicity in MT-4 cells (selectivity index HIV-1 > 49.57; HIV-2 > 17.08). However, compound TD-1a has the lowest EC50 in the anti-HIV-2 assays (EC50 = 4.86 ± 1.71 µM; CC50= 86.54 ± 29.24 µM). A water solubility test found that TD-1a showed a moderately increased water solubility compared with PF74, while the water solubility of FTC-2 was improved hundreds of times. Furthermore, we use molecular simulation studies to provide insight into the molecular contacts between the new compounds and HIV CA. We also computationally predict drug-like properties and metabolic stability for FTC-2 and TD-1a. Based on this analysis, TD-1a is predicted to have improved drug-like properties and metabolic stability over PF74. This study increases the repertoire of CA modulators and has important implications for developing anti-HIV agents with novel mechanisms, especially those that inhibit the often overlooked HIV-2.
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Fármacos Anti-VIH , VIH-1 , Humanos , Cápside , Fenilalanina , Calidad de Vida , Replicación Viral , VIH-1/metabolismo , Proteínas de la Cápside/metabolismo , VIH-2/metabolismo , Agua/metabolismo , Relación Estructura-ActividadRESUMEN
HIV-2, compared to HIV-1, elicits potent and broadly neutralizing antibodies, and uses a broad range of co-receptors. However, both sensitivity to neutralization and breadth of co-receptor use varies between HIV-2 isolates, and the molecular background is still not fully understood. Thus, in the current study, we have deciphered relationships between HIV-2 neutralization sensitivity, co-receptor use and viral envelope glycoprotein (Env) molecular motifs. A panel of primary HIV-2 isolates, with predefined use of co-receptors, was assessed for neutralization sensitivity using a set of HIV-2 Env-directed monoclonal antibodies and co-receptor indicator cell lines. Neutralization sensitivity of the isolates was analysed in relation target cell co-receptor expression, in addition to amino acid motifs and predicted structures of Env regions. Results showed that HIV-2 isolates were more resistant to neutralizing antibodies when entering target cells via the alternative co-receptor GPR15, as compared to CCR5. A similar pattern was noted for isolates using the alternative co-receptor CXCR6. Sensitivity to neutralizing antibodies appeared also to be linked to specific Env motifs in V1/V2 and C3 regions. Our findings suggest that HIV-2 sensitivity to neutralization depends both on which co-receptor is used for cell entry and on specific Env motifs. This study highlights the multifactorial mechanisms behind HIV-2 neutralization sensitivity.
Asunto(s)
Infecciones por VIH , VIH-1 , Anticuerpos Neutralizantes , Anticuerpos ampliamente neutralizantes , Anticuerpos Anti-VIH , Proteína gp120 de Envoltorio del VIH , VIH-1/metabolismo , VIH-2/metabolismo , Humanos , Receptores Acoplados a Proteínas G , Receptores de Péptidos , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
The discovery of the G-protein coupled-receptor (GPCR) CXCR4 as a major coreceptor of HIV-1 entry about three decades ago explained why the chemokine SDF-1/CXCL12 inhibits specific viral strains. The knowledge that RANTES, MlP-1α, and MlP-1ß specifically inhibit other primary HIV-1 strains allowed the rapid discovery of CCR5 as second major viral coreceptor and explained why individuals with deletions in CCR5 are protected against sexual HIV-1 transmission. Here, we provide an update on endogenous ligands of GPCRs that act as endogenous inhibitors of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) entry. In addition, we summarize the development of optimized derivatives of endogenous GPCR ligands and their perspectives as antiviral agents and beyond. Finally, we provide examples for other endogenous peptides that may contribute to our innate immune defense against HIV-1 and other viral pathogens and offer prospects for preventive or therapeutic development.
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Inhibidores de Fusión de VIH , Infecciones por VIH , VIH-1 , Animales , Inhibidores de Fusión de VIH/farmacología , Inhibidores de Fusión de VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/metabolismo , VIH-1/fisiología , VIH-2/metabolismo , VIH-2/fisiología , Humanos , Ligandos , Péptidos/uso terapéutico , Receptores CCR5 , Receptores Acoplados a Proteínas G/uso terapéutico , Transducción de Señal , Virus de la Inmunodeficiencia de los SimiosRESUMEN
The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin recruits the histone methyl-transferase SETDB1 to spread H3K9me3 repressive marks across genes and transgenes in an integration site-dependent manner. The deposition of these repressive marks leads to heterochromatin formation and inhibits gene expression, but the underlying mechanism is not fully understood. Here, we show that TASOR silencing or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identify new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 synergistically repress HIV expression from its LTR. Similar to the RNA-induced transcriptional silencing complex found in fission yeast, we show that TASOR interacts with the RNA exosome and RNA Polymerase II, predominantly under its elongating state. Finally, we show that TASOR facilitates the association of RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral expression.
Asunto(s)
Represión Epigenética , VIH-2/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estabilidad del ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ensamble y Desensamble de Cromatina , Expresión Génica , Silenciador del Gen , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , Células HeLa , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Fosfoproteínas , Provirus/genética , ARN Polimerasa II/metabolismo , SchizosaccharomycesRESUMEN
Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 (A3) cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2, but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions, whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partners in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif-encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here, we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.
Asunto(s)
Citidina Desaminasa/metabolismo , Productos del Gen vif/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Citidina Desaminasa/genética , Células HEK293 , Infecciones por VIH , Humanos , Antígenos de Histocompatibilidad Menor/genética , Receptor EphB2RESUMEN
The HIV-1 accessory proteins Vif, Vpu, and Nef can promote infection by overcoming the inhibitory effects of the host cell restriction factors APOBEC3G, Tetherin, and SERINC5, respectively. However, how the HIV-1 accessory protein Vpr enhances infection in macrophages but not in CD4+ T cells remains elusive. Here, we report that Vpr counteracts lysosomal-associated transmembrane protein 5 (LAPTM5), a potent inhibitor of HIV-1 particle infectivity, to enhance HIV-1 infection in macrophages. LAPTM5 transports HIV-1 envelope glycoproteins to lysosomes for degradation, thereby inhibiting virion infectivity. Vpr counteracts the restrictive effects of LAPTM5 by triggering its degradation via DCAF1. In the absence of Vpr, the silencing of LAPTM5 precisely phenocopied the effect of Vpr on HIV-1 infection. In contrast, Vpr did not enhance HIV-1 infection in the absence of LAPTM5. Moreover, LAPTM5 was highly expressed in macrophages but not in CD4+ T lymphocytes. Re-expressing LAPTM5 reconstituted the Vpr-dependent promotion of HIV-1 infection in primary CD4+ T cells, as observed in macrophages. Herein, we demonstrate the molecular mechanism used by Vpr to overcome LAPTM5 restriction in macrophages, providing a potential strategy for anti-HIV/AIDS therapeutics.
Asunto(s)
Infecciones por VIH/metabolismo , VIH-1/metabolismo , Interacciones Microbiota-Huesped , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Silenciador del Gen , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , VIH-2/metabolismo , VIH-2/patogenicidad , Interacciones Microbiota-Huesped/genética , Humanos , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Virus de la Inmunodeficiencia de los Simios/metabolismo , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia Arriba , Virión/metabolismoRESUMEN
The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein-DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein-DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency.
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Infecciones por VIH/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Regulación Viral de la Expresión Génica , Redes Reguladoras de Genes , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-2/genética , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Factores de Transcripción/genética , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
HIV-2 infection is frequently neglected in HIV/AIDS campaigns. However, a special emphasis must be given to HIV-2 as an untreated infection that also leads to AIDS and death, and for which the efficacy of most available drugs is limited against HIV-2. HIV envelope glycoproteins mediate binding to the receptor CD4 and co-receptors at the surface of the target cell, enabling fusion with the cell membrane and viral entry. Here, we developed and optimized a computer-assisted drug design approach of an important HIV-2 glycoprotein that allows us to explore and gain further insights at the molecular level into protein structures and interactions crucial for the inhibition of HIV-2 cell entry. The 3D structure of a key HIV-2ROD gp125 region was generated by a homology modeling campaign. To disclose the importance of the main structural features and compare them with experimental results, 3D-models of six mutants were also generated. These mutations revealed the selective impact on the behavior of the protein. Furthermore, molecular dynamics simulations were performed to optimize the models, and the dynamic behavior was tackled to account for structure flexibility and interactions network formation. Structurally, the mutations studied lead to a loss of aromatic features, which is very important for the establishment of π-π interactions and could induce a structural preference by a specific coreceptor. These new insights into the structure-function relationship of HIV-2 gp125 V3 and surrounding regions will help in the design of better models and the design of new small molecules capable to inhibit the attachment and binding of HIV with host cells.
Asunto(s)
Diseño de Fármacos , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/metabolismo , VIH-2/metabolismo , Dominios Proteicos , Secuencia de Aminoácidos , Antígenos CD4/química , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/metabolismo , Humanos , Simulación de Dinámica Molecular , Relación Estructura-Actividad , Internalización del Virus/efectos de los fármacosRESUMEN
INTRODUCTION: Most of the typical chemokine receptors (CKRs) have been identified as coreceptors for a variety of human and simian immunodeficiency viruses (HIVs and SIVs). This study evaluated CCRL2 to examine if it was an HIV/SIV coreceptor. METHODS: The Human glioma cell line, NP-2, is normally resistant to infection by HIV and SIV. The cell was transduced with amplified cluster of differentiation 4 (CD4) as a receptor and CCR5, CXCR4 and CCRL2 as coreceptor candidates to produce NP-2/CD4/coreceptor cells (). The cells were infected with multiplicity of infection (MOI) 1.0. Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa staining. Proviral DNA was detected by polymerase chain reaction (PCR), and reverse transcriptase (RT) activity was measured. RESULTS: IFA detected viral antigens of the primary isolates, HIV-1HAN2 and HIV-2MIR in infected NP-2/CD4/CCRL2 cells, indicated CCRL2 as a functional coreceptor. IFA results were confirmed by the detection of proviral DNA and measurement of RT-activity in the spent cell supernatants. Additionally, MGC was detected in HIV-2MIR-infected NP-2/CD4/CCCRL2 cells. HIV-2MIR were found more potent users of CCRL2 than HIV-1HAN2. Moreover, GWAS studies, gene ontology and cell signaling pathways of the HIV-associated genes show interaction of CCRL2 with HIV/SIV envelope protein. CONCLUSIONS: In vitro experiments showed CCRL2 to function as a newly identified coreceptor for primary HIV-2 isolates conveniently. The findings contribute additional insights into HIV/SIV transmission and pathogenesis. However, its in vivo relevance still needs to be evaluated. Confirming in vivo relevance, ligands of CCRL2 can be investigated as potential targets for HIV entry-inhibitor drugs.
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Infecciones por VIH/metabolismo , VIH-2/metabolismo , Receptores CCR/metabolismo , Infecciones por VIH/genética , VIH-1/genética , VIH-1/metabolismo , VIH-2/genética , Humanos , Células Jurkat , Receptores CCR/genéticaRESUMEN
The family of human APOBEC3 (A3) restriction factors is formed by seven different proteins, A3A-D and A3F-H. Among these A3s, A3B harbors strong restriction activity against several retroviruses, such as SIV, and MLV. How lentiviruses and other retroviruses, prevalent in many primate species, counteract A3B is poorly understood. In this study, we found that A3B strongly inhibited SIVmac and HIV-2 infectivity, which was antagonized by their Vif proteins. Both SIVmac and HIV-2 Vifs diminished the protein level of A3B in viral producer cells, and hindered A3B incorporation into viral particles. We observed that HIV-2 Vif binds A3B and induces its degradation by assembly of an A3-Vif-CUL5-ElonginB/C E3-ligase complex. A3B and HIV-2 Vif localize and interact in the nucleus. In addition, we also found that the accessory protein Bet of prototype foamy virus (PFV) significantly antagonized the anti-SIVmac activity of A3B. Like Vif, Bet prevented the incorporation of A3B into viral particles. However, in contrast to Vif Bet did not induce the degradation of A3B. Rather, Bet binds A3B to block formation of high molecular weight A3B complexes and induces A3B cytoplasmic trapping. In summary, these findings indicate that A3B is recognized by diverse retroviruses and counteracted by virus-specific pathways that could be targeted to inhibit A3B mutating activity in cancers.
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Citidina Desaminasa/antagonistas & inhibidores , Citidina Desaminasa/metabolismo , VIH-2/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas de los Retroviridae/metabolismo , Spumavirus/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Elonguina/genética , Elonguina/metabolismo , Productos del Gen vif/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Virus de la Inmunodeficiencia de los Simios/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Virión/metabolismoRESUMEN
During the progression of HIV-1 infection, macrophage tropic HIV-1 that use the CCR5 co-receptor undergoes a change in co-receptor use to CXCR4 that is predominately T cell tropic. This change in co-receptor preference makes the virus able to infect T cells. HIV-2 is known to infect MDMs and T cells and is dual tropic. The aim of this study was to elucidate the differential expression profiles of host miRNAs and their role in cells infected with HIV-1/HIV-2. To achieve this goal, a comparative global miRNA expression profile was determined in human PBMCs and MDMs infected with HIV-1/HIV-2. Differentially expressed miRNAs were identified in HIV-1/HIV-2 infected PBMCs and MDMs using the next-generation sequencing (NGS) technique. A comparative global miRNA expression profile in infected MDMs and PBMCs with HIV-1 and HIV-2 identified differential expression of several host miRNAs. These differentially expressed miRNAs are likely to be involved in many signaling pathways, like the p53 signaling pathway, PI3K-Akt signaling pathways, MAPK signaling pathways, FoxO signaling pathway, and viral carcinogenesis. Thus, a comparative study of the differential expression of host miRNAs in MDMs and T cell in response to HIV-1 and HIV-2 infection will help us to identify unique biomarkers that can differentiate HIV-1 and HIV-2 infection.
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Infecciones por VIH/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , MicroARNs/biosíntesis , Monocitos/metabolismo , Transcriptoma , Infecciones por VIH/patología , Humanos , Macrófagos/patología , Macrófagos/virología , Monocitos/patología , Monocitos/virologíaRESUMEN
Simian immunodeficiency virus (SIV) and human immunodeficiency virus 2 (HIV-2) display unique ability to infect nondividing target cells. Viral protein X (Vpx) of HIV-2/SIV is known to be involved in the nuclear import of viral genome in nondividing cells, but the mechanism remains poorly understood. In the present investigation for the first time we provide evidence that Vpx of SIVsmPBj1.9 physically interacts with human nucleoporin 153 (Nup153), which is known to provide a docking site for protein-cargo complexes at the nuclear pore complex (NPC). Results from superresolution-structured illumination microscopy studies reveal that Vpx interaction with NPC-associated Nup153 is critical for its efficient nuclear translocation. Virion-associated MAPK/ERK-2-mediated phosphorylation of Vpx plays a critical role in its interaction with human Nup153 and this interaction was found to be evolutionarily conserved in various SIV isolates and HIV-2. Interestingly, MAPK/ERK-2 packaging defective SIV failed to promote the efficient nuclear import of viral genome and suggests that MAPK/ERK-2-mediated Vpx phosphorylation is important for its interaction with Nup153, which is critical for lentiviruses to establish infection in nondividing target cells. Together, our data elucidate the mechanism by which Vpx orchestrates the challenging task of nuclear translocation of HIV-2/SIV genome in nondividing target cells.
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Genoma Viral , VIH-2/genética , Proteínas de Complejo Poro Nuclear/genética , Virus de la Inmunodeficiencia de los Simios/genética , Proteínas Reguladoras y Accesorias Virales/genética , Transporte Activo de Núcleo Celular/genética , Animales , Regulación de la Expresión Génica , Células HEK293 , VIH-2/metabolismo , Haplorrinos , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Fosforilación , Transducción de Señal , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Virión/genética , Virión/metabolismoRESUMEN
Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain- and HD domain-containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase-mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for â¼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.
Asunto(s)
Infecciones por VIH/metabolismo , VIH/metabolismo , Macrófagos/virología , Transcripción Reversa , Ribonucleótidos/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Células Cultivadas , Desoxirribonucleótidos/genética , Desoxirribonucleótidos/metabolismo , VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , VIH-2/genética , VIH-2/metabolismo , Humanos , Células Jurkat , Macrófagos/metabolismo , Mutagénesis , Ribonucleótidos/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismoRESUMEN
HIV protease inhibitors (PIs) approved by the FDA (US Food and Drug Administration) are a major class of antiretroviral. HIV-2 protease (PR2) is naturally resistant to most of them as PIs were designed for HIV-1 protease (PR1). In this study, we explored the impact of amino-acid substitutions between PR1 and PR2 on the structure of protease (PR) by comparing the structural variability of 13 regions using 24 PR1 and PR2 structures complexed with diverse ligands. Our analyses confirmed structural rigidity of the catalytic region and highlighted the important role of three regions in the conservation of the catalytic region conformation. Surprisingly, we showed that the flap region, corresponding to a flexible region, exhibits similar conformations in PR1 and PR2. Furthermore, we identified regions exhibiting different conformations in PR1 and PR2, which could be explained by the intrinsic flexibility of these regions, by crystal packing, or by PR1 and PR2 substitutions. Some substitutions induce structural changes in the R2 and R4 regions that could have an impact on the properties of PI-binding site and could thus modify PI binding mode. Substitutions involved in structural changes in the elbow region could alter the flexibility of the PR2 flap regions relative to PR1, and thus play a role in the transition from the semi-open form to the closed form, and have an impact on ligand binding. These results improve the understanding of the impact of sequence variations between PR1 and PR2 on the natural resistance of HIV-2 to commercially available PIs.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Inhibidores de la Proteasa del VIH , VIH-1 , Proteasa del VIH/genética , Proteasa del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , VIH-2/genética , VIH-2/metabolismo , Unión ProteicaRESUMEN
Innate immunity is the first line of host defence against pathogens. Suppression of innate immune responses is essential for the survival of all viruses. However, the interplay between innate immunity and HIV/SIV is only poorly characterized. We have discovered Vpx as a novel inhibitor of innate immune activation that associates with STING signalosomes and interferes with the nuclear translocation of NF-κB and the induction of innate immune genes. This new function of Vpx could be separated from its role in mediating degradation of the antiviral factor SAMHD1, and is conserved among diverse HIV-2/SIV Vpx. Vpx selectively suppressed cGAS-STING-mediated nuclear factor-κB signalling. Furthermore, Vpx and Vpr had complementary activities against cGAS-STING activity. Since SIVMAC lacking both Vpx and Vpr was less pathogenic than SIV deficient for Vpr or Vpx alone, suppression of innate immunity by HIV/SIV is probably a key pathogenic determinant, making it a promising target for intervention.
Asunto(s)
VIH-2/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-2/genética , Células HeLa , Humanos , Inmunidad Innata/genética , Masculino , Ratones Endogámicos BALB C , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/inmunología , Adulto Joven , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The HIV-1 capsid (CA) utilizes CPSF6 for nuclear entry and integration site targeting. Previous studies demonstrated that the HIV-1 CA C-terminal domain (CTD) contains a highly conserved K182 residue involved in interaction with CPSF6. In contrast, certain HIV-2 strains possess a substitution at this residue (K182R). To assess whether CA-CPSF6 interaction via the CA CTD is conserved among primate lentiviruses, we examined resistance of several HIV-1- and HIV-2-lineage viruses to a truncated form of CPSF6, CPSF6-358. The results demonstrated that viruses belonging to the HIV-2-lineage maintain interaction with CPSF6 regardless of the presence of the K182R substitution, in contrast to the case with HIV-1-lineage viruses. Our structure-guided mutagenesis indicated that the differential requirement for CA-CPSF6 interaction is regulated in part by residues near the 182nd amino acid of CA. These results demonstrate a previously unrecognized distinction between HIV-1 and HIV-2, which may reflect differences in their evolutionary histories.
Asunto(s)
Cápside/metabolismo , VIH-1/genética , VIH-2/genética , Factores de Escisión y Poliadenilación de ARNm/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Cápside/ultraestructura , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , VIH-2/crecimiento & desarrollo , VIH-2/metabolismo , Interacciones Huésped-Patógeno , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Replicación Viral , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
BACKGROUND: HIV-self-testing (HIVST) could be a strategy to get more people tested for HIV in resource limited settings. One of the prerequisites of a successful HIVST programme is the availability of an easy to use, valid HIV-test which is robust against field conditions and procedural errors by untrained lay users. METHODS AND FINDINGS: The primary objective of this study was to evaluate the ability of untrained persons to correctly interpret the OraQuick HIV Self-Test results with oral fluid compared with results obtained by trained users using the matched lot OraQuick Rapid HIV-1/2 Antibody Test and blinded to the results of the Self-Test. Sensitivity of the OraQuick HIV Self-Test in untrained users was 101 in 102 (99.02%; 95%CI = 93.88-99.95%)-and specificity- 1,241 in 1,241 (100.0%; 95%CI = 99.62-100.0%). Forty-eight Self-Tests were excluded in the accuracy analysis (due to a result read as invalid, not sure or ambiguous) resulting in a test system failure rate of 3.45% (95% CI 2.56%-4.55%). At least one observation of difficulty or error with one or more of the test steps were seen in 1,193 (84.6%) participants. Age, education and health literacy were independently associated with the sum score of procedural errors and difficulties. Four tests did not provide a valid result as determined by the trained user's interpretation of the Self-Test. CONCLUSIONS: The OraQuick HIV Self-Test provides reliable and repeatable results in a rural field environment in spite of procedural errors.
Asunto(s)
Serodiagnóstico del SIDA/métodos , Anticuerpos Anti-VIH/metabolismo , Infecciones por VIH , VIH-1/metabolismo , VIH-2/metabolismo , Saliva/metabolismo , Autocuidado , Adolescente , Adulto , Femenino , Infecciones por VIH/diagnóstico , Infecciones por VIH/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Población Rural , SudáfricaRESUMEN
The rapid confirmatory Bio-Rad Geenius HIV 1/2 assay was evaluated as an alternative to the HIV-1 Western blot (WB) confirmatory assay. A total of 370 retrospective samples collected from 356 patients were tested. Sensitivity of the Geenius assay to detect HIV-1 and HIV-2 infections was 100% and 97%, respectively, and that of the WB assay was 86% and 39%, respectively. Geenius reduced the number of indeterminate results by 85% and exhibited a differentiation capacity for HIV-1 and HIV-2 of 100% and 89%, respectively. Three of 10 patients presenting with an early HIV infection (1 to 2 weeks before seroconversion by WB) were positive using Geenius. None of the HIV-negative samples were positive using Geenius or WB. However, 7% and 10% of them were indeterminate with Geenius and WB, respectively, leading to a specificity rate of 93% for Geenius and 90% for WB. Ninety cadaveric samples (54 negative, 23 HIV-1 positive, and 3 HIV-1 indeterminate) were tested with Geenius, leading to a sensitivity of 100%, a specificity of 96%, and an indeterminate rate of 4%. Our results indicate that the Bio-Rad Geenius HIV 1/2 rapid test exhibits better sensitivity to detect HIV-1 infections and better performance than WB to confirm and differentiate between HIV-1 and HIV-2 infections. The performance of this new confirmatory assay to detect early infections, to reduce the rate of indeterminate status, and to confirm HIV-1 infection in cadaveric blood samples makes Geenius a potent reliable alternative to the WB.