RESUMO
BACKGROUND: The genome of human immunodeficiency virus type 1 (HIV-1) is encapsulated in a core consisting of viral capsid proteins (CA). After viral entry, the HIV-1 core dissociates and releases the viral genome into the target cell, this process is called uncoating. Uncoating of HIV-1 core is one of the critical events in viral replication and several studies show that host proteins positively or negatively regulate this process by interacting directly with the HIV-1 CA. RESULTS: Here, we show that arginyl-tRNA-protein transferase 1 (ATE1) plays an important role in the uncoating process by governing the optimal core stability. Yeast two-hybrid screening of a human cDNA library identified ATE1 as an HIV-1-CA-interacting protein and direct interaction of ATE1 with Pr55gag and p160gag - pol via HIV-1 CA was observed by cell-based pull-down assay. ATE1 knockdown in HIV-1 producer cells resulted in the production of less infectious viruses, which have normal amounts of the early products of the reverse transcription reaction but reduced amounts of the late products of the reverse transcription. Interestingly, ATE1 overexpression in HIV-1 producer cells also resulted in the production of poor infectious viruses. Cell-based fate-of-capsid assay, a commonly used method for evaluating uncoating by measuring core stability, showed that the amounts of pelletable cores in cells infected with the virus produced from ATE1-knockdown cells increased compared with those detected in the cells infected with the control virus. In contrast, the amounts of pelletable cores in cells infected with the virus produced from ATE1-overexpressing cells decreased compared with those detected in the cells infected with the control virus. CONCLUSIONS: These results indicate that ATE1 expression levels in HIV-1 producer cells contribute to the adequate formation of a stable HIV-1 core. These findings provide insights into a novel mechanism of HIV-1 uncoating and revealed ATE1 as a new host factor regulating HIV-1 replication.
Assuntos
Aminoaciltransferases/metabolismo , Capsídeo/química , Infecções por HIV/enzimologia , HIV-1/metabolismo , Aminoaciltransferases/genética , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Infecções por HIV/genética , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Estabilidade Proteica , Replicação Viral , Desenvelopamento do VírusRESUMO
BACKGROUND & AIMS: While certain nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection, their effects are yet to be optimized and the emergence of NRTI-resistant HBV variants is an issue because of the requirement for lifelong treatment. The development of agents that more profoundly suppress wild-type and drug-resistant HBVs, and that have a long-acting effect, are crucial to improve patient outcomes. METHODS: Herein, we synthesized a novel long-acting 4'-modified NRTI termed E-CFCP. We tested its anti-HBV activity in vitro, before evaluating its anti-HBV activity in HBV-infected human-liver-chimeric mice (PXB-mice). E-CFCP's long-acting features and E-CFCP-triphosphate's interactions with the HBV reverse transcriptase (HBV-RT) were examined. RESULTS: E-CFCP potently blocked HBVWTD1 production (IC50qPCR_cell=1.8 nM) in HepG2.2.15 cells and HBVWTC2 (IC50SB_cell=0.7 nM), entecavir (ETV)-resistant HBVETV-RL180M/S202G/M204V (IC50SB_cell=77.5 nM), and adefovir-resistant HBVADV-RA181T/N236T production (IC50SB_cell=14.1 nM) in Huh7 cells. E-CFCP profoundly inhibited intracellular HBV DNA production to below the detection limit, but ETV and tenofovir alafenamide (TAF) failed to do so. E-CFCP also showed less toxicity than ETV and TAF. E-CFCP better penetrated hepatocytes and was better tri-phosphorylated; E-CFCP-triphosphate persisted intracellularly for longer than ETV-triphosphate. Once-daily peroral E-CFCP administration over 2 weeks (0.02~0.2 mg/kg/day) reduced HBVWTC2-viremia by 2-3 logs in PXB-mice without significant toxicities and the reduction persisted over 1-3 weeks following treatment cessation, suggesting once-weekly dosing capabilities. E-CFCP also reduced HBVETV-RL180M/S202G/M204V-viremia by 2 logs over 2 weeks, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V-viremia. E-CFCP's 4'-cyano and fluorine interact with both HBVWT-RT and HBVETV-RL180M/S202G-M204 -RT via Van der Waals and polar forces, being important for E-CFCP-triphosphate's interactions and anti-HBV potency. CONCLUSION: E-CFCP represents the first reported potential long-acting NRTI with potent activity against wild-type and treatment-resistant HBV. LAY SUMMARY: Although there are currently effective treatment options for HBV, treatment-resistant variants and the need for lifelong therapy pose a significant challenge. Therefore, the development of new treatment options is crucial to improve outcomes and quality of life. Herein, we report preclinical evidence showing that the anti-HBV agent, E-CFCP, has potent activity against wild-type and treatment-resistant variants. In addition, once-weekly oral dosing may be possible, which is preferrable to the current daily dosing regimens.
Assuntos
Desenvolvimento de Medicamentos/métodos , Farmacorresistência Viral/efeitos dos fármacos , Vírus da Hepatite B , Hepatite B , Inibidores da Transcriptase Reversa/farmacologia , Animais , Preparações de Ação Retardada/farmacologia , Modelos Animais de Doenças , Vias de Administração de Medicamentos , Esquema de Medicação , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Humanos , Camundongos , DNA Polimerase Dirigida por RNA/metabolismo , TempoRESUMO
The human immunodeficiency virus type 1 (HIV-1) capsid (CA) is an essential viral component of HIV-1 infection and an attractive therapeutic target for antivirals. Here, we report that a small molecule, ACAi-028, inhibits HIV-1 replication by targeting a hydrophobic pocket in the N-terminal domain of CA (CA-NTD). ACAi-028 is 1 of more than 40 candidate anti-HIV-1 compounds identified by in silico screening and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Our binding model showed that ACAi-028 interacts with the Q13, S16, and T19 amino acid residues, via hydrogen bonds, in the targeting pocket of CA-NTD. Using recombinant fusion methods, TZM-bl, time-of-addition, and colorimetric reverse transcriptase (RT) assays, the compound was found to exert anti-HIV-1 activity in the early stage between reverse transcription and proviral DNA integration, without any effect on RT activity in vitro, suggesting that this compound may affect HIV-1 core disassembly (uncoating) as well as a CA inhibitor, PF74. Moreover, electrospray ionization mass spectrometry (ESI-MS) also showed that the compound binds directly and noncovalently to the CA monomer. CA multimerization and thermal stability assays showed that ACAi-028 decreased CA multimerization and thermal stability via S16 or T19 residues. These results indicate that ACAi-028 is a new CA inhibitor by binding to the novel hydrophobic pocket in CA-NTD. This study demonstrates that a compound, ACAi-028, targeting the hydrophobic pocket should be a promising anti-HIV-1 inhibitor.
Assuntos
Fármacos Anti-HIV , HIV-1 , Fármacos Anti-HIV/farmacologia , Capsídeo , Proteínas do Capsídeo/genética , Humanos , Fenilalanina/farmacologia , Replicação ViralRESUMO
The cellular environment affects optimal viral replication because viruses cannot replicate without their host cells. In particular, metabolic resources such as carbohydrates, lipids, and ATP are crucial for viral replication, which is sensitive to cellular metabolism. Intriguingly, recent studies have demonstrated that human immunodeficiency virus type 1 (HIV-1) infection induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis in CD4+ T cells to produce the virus efficiently. However, the importance of aerobic glycolysis in maintaining the quality of viral components and viral infectivity has not yet been fully investigated. Here, we show that aerobic glycolysis is necessary not only to override the inhibitory effect of virion-incorporated glycolytic enzymes, but also to maintain the enzymatic activity of reverse transcriptase and the adequate packaging of envelope proteins into HIV-1 particles. To investigate the effect of metabolic remodeling on the phenotypic properties of HIV-1 produced by infected cells, we replaced glucose with galactose in the culture medium because the cells grown in galactose-containing medium are forced to carry out oxidative metabolism instead of aerobic glycolysis. We found that the packaging levels of glyceraldehyde 3-phosphate dehydrogenase, alpha-enolase and pyruvate kinase muscle type 2, which decrease HIV-1 infectivity by packaging into viral particles, are increased in progeny viruses produced by the cells grown in galactose-containing medium. Furthermore, we found that the entry and reverse transcription efficiency of the progeny viruses were reduced, which was caused by a decrease in the enzymatic activity of reverse transcriptase in the viral particles and a decrease in the packaging levels of envelope proteins and reverse transcriptase. These results indicate that the aerobic glycolysis environment in HIV-1-infected cells may contribute to the quality control of viruses.
Assuntos
Glucose/metabolismo , Glicólise , HIV-1/patogenicidade , Vírion/metabolismo , Aerobiose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Proteínas de Ligação a DNA/metabolismo , Galactose/farmacologia , Produtos do Gene env/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Glicólise/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Proteínas de Membrana/metabolismo , Fosfopiruvato Hidratase/metabolismo , Transcrição Reversa/efeitos dos fármacos , Transcrição Reversa/genética , Hormônios Tireóideos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Empacotamento do Genoma Viral/efeitos dos fármacos , Proteínas de Ligação a Hormônio da TireoideRESUMO
BACKGROUND: A protein exhibiting more than one biochemical function is termed a moonlighting protein. Glycolytic enzymes are typical moonlighting proteins, and these enzymes control the infection of various viruses. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and alpha-enolase (ENO1) are incorporated into human immunodeficiency virus type 1 (HIV-1) particles from viral producer cells and suppress viral reverse transcription independently each other. However, it remains unclear whether these proteins expressed in viral target cells affect the early phase of HIV-1 replication. RESULTS: Here we show that the GAPDH expression level in viral target cells does not affect the early phase of HIV-1 replication, but ENO1 has a capacity to suppress viral integration in viral target cells. In contrast to GAPDH, suppression of ENO1 expression by RNA interference in the target cells increased viral infectivity, but had no effect on the expression levels of the HIV-1 receptors CD4, CCR5 and CXCR4 and on the level of HIV-1 entry. Quantitative analysis of HIV-1 reverse transcription products showed that the number of copies of the late products (R/gag) and two-long-terminal-repeat circular forms of viral cDNAs did not change but that of the integrated (Alu-gag) form increased. In contrast, overexpression of ENO1 in viral target cells decreased viral infectivity owing to the low viral integration efficiency. Results of subcellular fractionation experiments suggest that the HIV integration at the nucleus was negatively regulated by ENO1 localized in the nucleus. In addition, the overexpression of ENO1 in both viral producer cells and target cells most markedly suppressed the viral replication. CONCLUSIONS: These results indicate that ENO1 in the viral target cells prevents HIV-1 integration. Importantly, ENO1, but not GAPDH, has the bifunctional inhibitory activity against HIV-1 replication. The results provide and new insights into the function of ENO1 as a moonlighting protein in HIV-1 infection.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , HIV-1/fisiologia , Fosfopiruvato Hidratase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Integração Viral/fisiologia , Biomarcadores Tumorais/genética , Linhagem Celular , Núcleo Celular/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Infecções por HIV/virologia , Humanos , Fosfopiruvato Hidratase/genética , Transcrição Reversa , Proteínas Supressoras de Tumor/genética , Replicação ViralRESUMO
The effective antigen (Ag) uptake by microfold cells (M-cells) is important for the induction of an efficient mucosal immune responses. Here, we show that 10-hydroxydecanoic acid (10-HDAA) from royal jelly (RJ) potentially supports M-cell differentiation and induces effective antigen-specific mucosal immune responses in cynomolgus macaques. 10-HDAA increases the expression level of receptor activator of nuclear factor-kappaB (NF-κB) (RANK) in Caco-2 cells, which suggests that 10-HDAA potentially prompts the differentiation of Caco-2 cells into M-cells and increased transcytosis efficiency. This idea is supported by the following observations. Intranasal administration of 10-HDAA increased the number of M-cells in the epithelium overlying nasopharynx-associated lymphoid tissue (NALT) in macaques. Oral administration of 10-HDAA increased the number of M-cells in the follicle-associated epithelium (FAE) covering Peyer's patches (PPs) and significantly increased the antigen-specific immunoglobulin A (IgA) level in macaques. These findings suggest that the exogenous honeybee-derived medium-chain fatty acid 10-HDAA may effectively enhance antigen-specific immune responses.
Assuntos
Ácidos Decanoicos/farmacologia , Imunidade nas Mucosas/efeitos dos fármacos , Imunoglobulina A/biossíntese , Animais , Antígenos/imunologia , Células CACO-2 , Diferenciação Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Mucosa Intestinal/imunologia , Macaca fascicularis , Masculino , Ligante RANK/genéticaRESUMO
Our previous study showed that the phosphorylation of a highly conserved serine residue, Ser16 in the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein is promoted by virion-incorporated extracellular signal-regulated kinase 2 (ERK2) and required for proper peptidyl-prolyl isomerase (Pin1)-mediated uncoating. Interestingly, western blot analysis demonstrated that phosphorylated/activated mitogen-activated protein kinase kinase 1/2 (MEK1/2), the upstream activator of ERK2, as well as ERK2 are incorporated into virions. Here, we show that the MEK1/2 selective allosteric inhibitor Trametinib reduces HIV-1 infectivity via the decrease in virion-incorporated ERK2 phosphorylation. The treatment of chronic HIV-1-infected T-cell line, CEM/LAV-1 cells with Trametinib results in a decrease in ERK2 phosphorylation in the virions. The viruses have relatively low infectivity and impaired reverse transcription. Cell-based fate-of-capsid uncoating assay showed that the reduction in infectivity was caused by a functional impairment of the uncoating process. Furthermore, the viruses from Trametinib-treated CEM/LAV-1 cells also showed decreased reverse transcription efficiency and attenuated multiple rounds of replication in human peripheral blood mononuclear cells (PBMCs). Taken together, these findings suggest that Trametinib suppresses HIV-1 replication by abrogating the proper disassembly of CA core.
Assuntos
Capsídeo/fisiologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia , Capsídeo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação/efeitos dos fármacosRESUMO
Human immunodeficiency virus type 1 (HIV-1) recruits diverse cellular factors into viral particles during its morphogenesis, which apparently play roles in modulating its infectivity. In our study, proteomic techniques demonstrated that a key glycolytic protein, pyruvate kinase muscle type 2 (PKM2), is incorporated into viral particles. Here, we show that virion-packaged PKM2 significantly reduces viral infectivity by affecting the incorporation level of a cellular tRNALys3 into virions. Enhanced expression of PKM2 in HIV-1-producing cells led to a higher incorporation level of PKM2 into progeny virions without affecting the viral maturation process. Compared with the control virus, the high-level-PKM2-packaging virus showed decreased levels of both reverse transcription products and cellular tRNALys3 packaging, suggesting that the shortage of intravirion tRNALys3 suppresses reverse transcription efficiency in target cells. Interestingly, the enhanced expression of PKM2 also suppressed the virion recruitment of other nonpriming cellular tRNAs such as tRNALys1,2 and tRNAAsn, which are known to be selectively packaged into virions, without affecting the steady level of the cytoplasmic pool of those tRNAs in producer cells, suggesting that PKM2 specifically impedes the selective incorporation of tRNAs into virions. Taken together, our findings indicate that PKM2 is a vital host factor that negatively affects HIV-1 infectivity by targeting the tRNALys3-mediated initiation of reverse transcription in target cells.
Assuntos
HIV-1/fisiologia , Piruvato Quinase/fisiologia , Células HEK293 , Humanos , Piruvato Quinase/genética , RNA de Transferência , Transcrição Reversa , Vírion/fisiologia , Montagem de Vírus , Internalização do VírusRESUMO
Human immunodeficiency virus type-1 (HIV-1) particles contain not only viral-encoded but also host-encoded proteins. Interestingly, several studies showed that host proteins play a critical role in viral infectivity, replication and/or immunoreactivity in the next target cells. Here, we show that alpha-enolase (ENO1) is incorporated into HIV-1 virions and the virion-incorporated ENO1 prevents the early stage of HIV-1 reverse transcription. We found that viral particles contain two isoforms of ENO1 with different isoelectric points by two-dimensional electrophoresis. Suppression of ENO1 expression by RNA interference in the HIV-1 producer cells decreased ENO1 incorporation into virions without altering the packaging of viral structural proteins and viral production but increased viral infectivity. Although the low-level-ENO1-packaging virus maintained comparable levels of reverse transcriptase activity, viral genomic RNA and tRNALys3 packaging to the control virus, its levels of early cDNA products of reverse transcription were higher than those of the control virus. In contrast, the high-level-ENO1-packaging virus, which was produced from ENO1-overexpressing cells, showed decreased infectivity and the levels of early cDNA products. Taken together, these findings reveal a novel function of ENO1 as a negative regulation factor targeting HIV-1 reverse transcription.
Assuntos
HIV-1/fisiologia , Fosfopiruvato Hidratase/metabolismo , Transcrição Reversa , Vírion/fisiologia , Linhagem Celular , HIV-1/enzimologia , HIV-1/patogenicidade , Humanos , Fosfopiruvato Hidratase/genética , Interferência de RNA , Vírion/enzimologia , Montagem de VírusRESUMO
Dimerization of HIV-1 protease (PR) subunits is an essential process for PR's acquisition of proteolytic activity, which plays a critical role in the maturation of HIV-1. Recombinant wild-type PR (PR(WT)) proved to dimerize, as examined with electrospray ionization mass spectrometry; however, two active site interface PR mutants (PR(T26A) and PR(R87K)) remained monomeric. On the other hand, two termini interface PR mutants (PR(1-C95A) and PR(97/99)) took both monomeric and dimeric forms. Differential scanning fluorimetry indicated that PR(1-C95A) and PR(97/99) dimers were substantially less stable than PR(WT) dimers. These data indicate that intermolecular interactions of two monomers occur first at the active site interface, generating unstable or transient dimers, and interactions at the termini interface subsequently occur, generating stable dimers. Darunavir (DRV), an HIV-1 protease inhibitor, inhibits not only proteolytic activity but also PR dimerization. DRV bound to protease monomers in a one-to-one molar ratio, inhibiting the first step of PR dimerization, whereas conventional protease inhibitors (such as saquinavir) that inhibit enzymatic activity but not dimerization failed to bind to monomers. DRV also bound to mutant PRs containing the transframe region-added PR (TFR-PR(D25N) and TFR-PR(D25N-7AA)), whereas saquinavir did not bind to TFR-PR(D25N) or TFR-PR(D25N-7AA). Notably, DRV failed to bind to mutant PR containing four amino acid substitutions (V32I, L33F, I54M, and I84V) that confer resistance to DRV on HIV-1. To our knowledge, the present report represents the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV, which should help design further, more potent novel PIs.
Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , Sulfonamidas/farmacologia , Domínio Catalítico , Darunavir , Dimerização , Modelos Moleculares , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) must take advantage of its own proteins with two or more functions to successfully replicate. Although many attempts have been made to determine the function of viral proteins encoded in the HIV-1 genome, the role of the p2 peptide, a spacer between the capsid and the nucleocapsid in HIV-1 Gag in early-phase HIV infection still remains unclarified. RESULTS: In this study, we show that the p2 peptide enhances HIV-1 acute infection by increasing intracellular ATP production via the activation of mitochondrial cytochrome c oxidase (MT-CO) involved in the respiratory chain. We found that cell-permeable p2-peptide-treated cells were more effectively infected by HIV-1 than control cells. To characterize the effect of the p2 peptide on HIV-1 replication in MAGIC-5 cells, various HIV-1 cDNA products were measured by quantitative real-time PCR. The levels of the late (R/gag), 2-LTR circular (2-LTR), and integrated (Alu) forms of viral cDNAs increased in the presence of the p2 peptide. Interestingly, yeast two-hybrid analysis revealed a novel interaction between the p2 peptide and the mitochondrial intermembrane space domain (N(214)-F(235)) of MT-CO subunit I (MT-CO1). Mutational analysis indicated that Gln(6) in the p2 peptide is important for the interaction with MT-CO1. The p2 peptide activated MT-CO1 in vitro in a concentration-dependent manner, and fluorescence-microscopy analysis demonstrated that the p2 peptide had a significant effect on mitochondrial targeting. Furthermore, the analysis of HIV-1 lacking a functional p2 peptide demonstrated the inhibition of intracellular ATP production in MT-4 cells and monocyte-derived macrophages (MDMs) and a decrease in reverse transcription efficiency following infection of MT-4 cells and MDMs. CONCLUSIONS: These findings provide evidence that the p2 peptide is a viral positive allosteric modulator of MT-CO and the increased intracellular ATP production after HIV infection in a p2-peptide-dependent manner is essential for efficient reverse transcription in early-phase HIV-1 infection.
Assuntos
Trifosfato de Adenosina/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Macrófagos/virologia , Mitocôndrias/enzimologia , Fragmentos de Peptídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Reversa , Técnicas do Sistema de Duplo-Híbrido , Regulação para Cima , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
N-myristoyltransferase (NMT) catalyzes protein N-myristoylation. It has been suggested that the isozyme NMT1 enhances the replication of human immunodeficiency virus type-1 (HIV-1). However, the details of the mechanism by which NMT1 does so remain unclear. In this study, we investigated NMT1-binding proteins by co-immunoprecipitation and mass spectrometry. As a result, several RNA-binding proteins including ribosomal proteins, NMT isozymes, and hnRNP A2/B1 were observed to bind to NMT1, as mediated mainly by RNA. Interestingly, only hRNP A2/B1 was found to associate with NMT1 without mediation by RNA. It was also suggested that hnRNP A2/B1 contributes to the formation of complexes of high molecular weights involving NMT1. Knockdown of hnRNP A2/B1 resulted in the enhancement of viral replication with an increase in the expression level of viral RNA in HIV-1-producing cells. On the other hand, knockdown of NMT1 resulted in the attenuation of viral replication with the decrease in the expression level of viral RNA in HIV-1-producing cells. Additionally, overexpression of NMT1 induced the enhancement of viral replication with the increase in the expression level of the viral RNA. These findings suggest that both NMT1 and hnRNP A2/B1 take part in the regulation of HIV-1 RNA expression through their mutual opposite effects on the viral RNA expression in HIV-1-producing cells.
Assuntos
Aciltransferases/fisiologia , Regulação Viral da Expressão Gênica , HIV-1/fisiologia , RNA Viral/genética , Replicação Viral/fisiologia , Aciltransferases/metabolismo , Células HEK293 , HIV-1/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Ligação ProteicaRESUMO
We reported previously that Pin1 facilitates human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid core through the phosphorylated Ser(16)-Pro(17) motif. However, the specific kinase responsible for Ser(16) phosphorylation has remained unknown. Here, we showed that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser(16). The characterization of immature virions produced by exposing chronically HIV-1LAV-1-infected CEM/LAV-1 cells to 10 µM saquinavir indicated that Ser(16) is phosphorylated after the initiation of Pr55(Gag) processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser(16) residue in the Ser(16)-Pro(17) motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser(16) phosphorylation level inside virions, and virus partially defective in Ser(16) phosphorylation showed impaired reverse transcription and attenuated replication owing to attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser(16) phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed impaired reverse transcription and attenuated HIV-1 replication. Taken together, these findings provide insights into the as-yet-obscure processes in Pin1-dependent HIV-1 uncoating.
Assuntos
Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Peptidilprolil Isomerase/metabolismo , Processamento de Proteína Pós-Traducional , Desenvelopamento do Vírus , Linhagem Celular , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Replicação ViralRESUMO
The presence of anti-CCR5 and anti-HIV-1 envelope glycoprotein (ENV) gp41 antibodies (Abs) at sites of HIV-1 exposure was effective in preventing its transmission to HIV-1-exposed seronegative (ESN) subjects. Here, we design an immunogen that can induce Abs against CCR5 and SIVmac239 ENV simultaneously and show that bovine alpha-2-HS-glycoprotein (bAHSG) functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and ENV. Initially, we generated a rhesus CCR5-derived cyclopeptide (cDDR5) conjugated with a recombinant trimeric SIVmac239 Env. When inguinally administered to rhesus macaques, the immunogen simultaneously induced both anti-CCR5 and anti-ENV Abs in sera, and the purified serum IgG fraction exerted an inhibitory effect on SIVmac239 infection in vitro. When further boosted with bAHSG, the responses of both Abs were significantly enhanced. To examine the cross-reactivity of bAHSG, it was administered to naïve cynomolgus macaques. The results showed a statistically significant increase in IgG response against cynomolgus CCR5 and SIVmac239 ENV, and the induction of neutralizing activity against SIVmac239. These findings suggest that bAHSG is useful for immune strategies aimed at generating Abs against CCR5 and ENV simultaneously to confer HIV-protective immunity.
Assuntos
Vacinas contra a AIDS/imunologia , Produtos do Gene env/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Imunização Secundária/métodos , Receptores CCR5/imunologia , Vírus da Imunodeficiência Símia/imunologia , alfa-2-Glicoproteína-HS/imunologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Antígenos/imunologia , Bovinos , Proteína gp120 do Envelope de HIV , Proteína gp41 do Envelope de HIV/imunologia , Imunidade Humoral , Macaca fascicularis , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/imunologiaRESUMO
HIV-1 subtype B replication in the CNS can occur in CD4+ T cells or macrophages/microglia in adults. However, little is known about CNS infection in children or the ability of subtype C HIV-1 to evolve macrophage-tropic variants. In this study, we examined HIV-1 variants in ART-naïve children aged three years or younger to determine viral genotypes and phenotypes associated with HIV-1 subtype C pediatric CNS infection. We examined HIV-1 subtype C populations in blood and CSF of 43 Malawian children with neurodevelopmental delay or acute neurological symptoms. Using single genome amplification (SGA) and phylogenetic analysis of the full-length env gene, we defined four states: equilibrated virus in blood and CSF (nâ=â20, 47%), intermediate compartmentalization (nâ=â11, 25%), and two distinct types of compartmentalized CSF virus (nâ=â12, 28%). Older age and a higher CSF/blood viral load ratio were associated with compartmentalization, consistent with independent replication in the CNS. Cell tropism was assessed using pseudotyped reporter viruses to enter a cell line on which CD4 and CCR5 receptor expression can be differentially induced. In a subset of compartmentalized cases (nâ=â2, 17%), the CNS virus was able to infect cells with low CD4 surface expression, a hallmark of macrophage-tropic viruses, and intermediate compartmentalization early was associated with an intermediate CD4 entry phenotype. Transmission of multiple variants was observed for 5 children; in several cases, one variant was sequestered within the CNS, consistent with early stochastic colonization of the CNS by virus. Thus we hypothesize two pathways to compartmentalization: early stochastic sequestration in the CNS of one of multiple variants transmitted from mother to child, and emergence of compartmentalized variants later in infection, on average at age 13.5 months, and becoming fully apparent in the CSF by age 18 months. Overall, compartmentalized viral replication in the CNS occurred in half of children by year three.
Assuntos
Sistema Nervoso Central/virologia , Infecções por HIV/sangue , Infecções por HIV/líquido cefalorraquidiano , HIV-1/metabolismo , Antígenos CD4/biossíntese , Antígenos CD4/metabolismo , Linhagem Celular , Pré-Escolar , Genótipo , Células HEK293 , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Lactente , Recém-Nascido , Macrófagos/virologia , Dados de Sequência Molecular , Fenótipo , Filogenia , Receptores CCR5/biossíntese , Receptores CCR5/metabolismo , Carga Viral , Replicação Viral/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: One of the major functions of Nef is in the enhancement of the infectivity of the human and simian immunodeficiency viruses (HIV and SIV, respectively). However, the detailed mechanism of the enhancement of viral infectivity by Nef remains unclear. Additionally, studies of mechanisms by which Nef enhances the infectivity of SIV are not as intensive as those of HIV-1. METHODS: We generated short-lived Nef constructed by fusing Nef to a proteasome-mediated protein degradation sequence to characterize the Nef role in viral infectivity. RESULTS: The apparent expression level of the short-lived Nef was found to be extremely lower than that of the wild-type Nef. Moreover, the expression level of the short-lived Nef increased with the treatment with a proteasome inhibitor. The infectivity of HIV-1 with the short-lived Nef was significantly lower than that with the wild-type Nef. On the other hand, the short-lived Nef enhanced the infectivity of SIVmac239, an ability observed to be interestingly equivalent to that of the wild-type Nef. The short-lived Nef was not detected in SIVmac239, but the wild-type Nef was, suggesting that the incorporation of Nef into SIVmac239 is not important for the enhancement of SIVmac239 infectivity. CONCLUSIONS: Altogether, the findings suggest that the mechanisms of infectivity enhancement by Nef are different between HIV-1 and SIVmac239. Lastly, we propose the following hypothesis: even when the expression level of a protein is extremely low, the protein may still be sufficiently functional.
Assuntos
HIV-1/fisiologia , Vírus da Imunodeficiência Símia/fisiologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Humanos , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Vírus da Imunodeficiência Símia/patogenicidade , VirulênciaRESUMO
Raltegravir (RAL), a human immunodeficiency virus (HIV)-1 integrase inhibitor, has been administered as part of antiretroviral therapy. Studies in patients with HIV-1 have shown high variability in the pharmacokinetics of RAL, and in healthy volunteers, coadministration of proton-pump inhibitors has been shown to increase the plasma RAL concentrations. Here, we found that RAL containing a 1,3,4-oxadiazole ring is converted to a hydrolysis product (H-RAL) with a cleaved 1,3,4-oxadiazole ring at pH 1.0 and 13.0 conditions in vitro, thereby reducing the anti-HIV activity of the drug. The inclusion of cyclodextrins (beta-cyclodextrin [ßCD], random methyl-ßCD [RAM-ßCD], and hydroxypropyl-ßCD [HP-ßCD]) can protect RAL from pH-induced changes. The conversion of RAL to H-RAL was detected by using various mass spectrometry analyses. The chromatogram of H-RAL increased in a time-dependent manner similar to another 1,3,4-oxadiazole-containing drug, zibotentan, using high-performance liquid chromatography. Oral bioavailability and target protein interactions of H-RAL were predicted to be lower than those of RAL. Moreover, H-RAL exhibited significantly reduced anti-HIV-1 activity, whereas combinations with ßCD, RAM-ßCD, and HP-ßCD attenuated this effect in cell-based assays. These findings suggest that ßCDs can potentially protect against the conversion of RAL to H-RAL under acidic conditions in the stomach, thereby preserving the anti-HIV-1 effect of RAL. Although clinical trials are needed for evaluation, we anticipate that protective devices such as ßCDs may improve the pharmacokinetics of RAL, leading to better treatment outcomes, including reduced dosing, long-term anti-HIV-1 activity, and deeper HIV-1 suppression.
RESUMO
Nuclear localization signal (NLS) of HIV-1 integrase (IN) is implicated in nuclear import of HIV-1 preintegration complex (PIC). Here, we established a multiclass drug-resistant HIV-1 variant (HIVKGD) by consecutively exposing an HIV-1 variant to various antiretroviral agents including IN strand transfer inhibitors (INSTIs). HIVKGD was extremely susceptible to a previously reported HIV-1 protease inhibitor, GRL-142, with IC50 of 130 femtomolar. When cells were exposed to HIVKGD IN-containing recombinant HIV in the presence of GRL-142, significant decrease of unintegrated 2-LTR circular cDNA was observed, suggesting that nuclear import of PIC was severely compromised by GRL-142. X-ray crystallographic analyses revealed that GRL-142 interacts with NLS's putative sequence (DQAEHLK) and sterically blocks the nuclear transport of GRL-142-bound HIVKGD's PIC. Highly INSTI-resistant HIV-1 variants isolated from heavily INSTI-experienced patients proved to be susceptible to GRL-142, suggesting that NLS-targeting agents would serve as salvage therapy agents for highly INSTI-resistant variant-harboring individuals. The data should offer a new modality to block HIV-1 infectivity and replication and shed light on developing NLS inhibitors for AIDS therapy.
Assuntos
Integrase de HIV , HIV-1 , Humanos , Sinais de Localização Nuclear/genética , HIV-1/genética , Integrase de HIV/genética , AntiviraisRESUMO
COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.
Assuntos
Antivirais , COVID-19 , Proteases 3C de Coronavírus , Inibidores de Proteases , SARS-CoV-2 , Animais , Humanos , Camundongos , Antivirais/farmacologia , Benzotiazóis , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/química , Proteases 3C de Coronavírus/antagonistas & inibidoresRESUMO
BACKGROUND: Host proteins are incorporated inside human immunodeficiency virus type 1 (HIV-1) virions during assembly and can either positively or negatively regulate HIV-1 infection. Although the identification efficiency of host proteins is improved by mass spectrometry, how those host proteins affect HIV-1 replication has not yet been fully clarified. RESULTS: In this study, we show that virion-associated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) does not allosterically inactivate HIV-1 reverse transcriptase (RT) but decreases the efficiency of reverse transcription reactions by decreasing the packaging efficiency of lysyl-tRNA synthetase (LysRS) and tRNA(Lys3) into HIV-1 virions. Two-dimensional (2D) gel electrophoresis demonstrated that some isozymes of GAPDH with different isoelectric points were expressed in HIV-1-producing CEM/LAV-1 cells, and a proportion of GAPDH was selectively incorporated into the virions. Suppression of GAPDH expression by RNA interference in CEM/LAV-1 cells resulted in decreased GAPDH packaging inside the virions, and the GAPDH-packaging-defective virus maintained at least control levels of viral production but increased the infectivity. Quantitative analysis of reverse transcription products indicated that the levels of early cDNA products of the GAPDH-packaging-defective virus were higher than those of the control virus owing to the higher packaging efficiencies of LysRS and tRNA(Lys3) into the virions rather than the GAPDH-dependent negative allosteric modulation for RT. Furthermore, immunoprecipitation assay using an anti-GAPDH antibody showed that GAPDH directly interacted with Pr55(gag) and p160(gag)-pol and the overexpression of LysRS in HIV-1-producing cells resulted in a decrease in the efficiency of GAPDH packaging in HIV particles. In contrast, the viruses produced from cells expressing a high level of GAPDH showed decreased infectivity in TZM-bl cells and reverse transcription efficiency in TZM-bl cells and peripheral blood mononuclear cells (PBMCs). CONCLUSIONS: These findings indicate that GAPDH negatively regulates HIV-1 infection and provide insights into a novel function of GAPDH in the HIV-1 life cycle and a new host defense mechanism against HIV-1 infection.