RESUMO
Tigliane diterpenoids possess exceptionally complex structures comprising common 5/7/6/3-membered ABCD-rings and disparate oxygen functionalities. While tiglianes display a wide range of biological activities, compounds with HIV latency-reversing activity can eliminate viral reservoirs, thereby serving as promising leads for new anti-HIV agents. Herein, we report collective total syntheses of phorbol (13) and 11 tiglianes 14-24 with various acylation patterns and oxidation states, and their evaluation as HIV latency-reversing agents. The syntheses were strategically divided into five stages to increase the structural complexity. First, our previously established sequence enabled the expeditious preparation of ABC-tricycle 9 in 15 steps. Second, hydroxylation of 9 and ring-contractive D-ring formation furnished phorbol (13). Third, site-selective attachment of two acyl groups to 13 produced four phorbol diesters 14-17. Fourth, the oxygen functionalities were regio- and stereoselectively installed to yield five tiglianes 18-22. Fifth, further oxidation to the most densely oxygenated acerifolin A (23) and tigilanol tiglate (24) was realized through organizing a 3D shape of the B-ring. Assessment of the HIV latency-reversing activities of the 12 tiglianes revealed seven tiglianes (14-17 and 22-24) with 20- to 300-fold improved efficacy compared with prostratin (12), a representative latency-reversing agent. Therefore, the robust synthetic routes to a variety of tiglianes with promising activities devised in this study provide opportunities for advancing HIV eradication strategies.
Assuntos
Diterpenos , Infecções por HIV , Forbóis , Humanos , Latência Viral , OxigênioRESUMO
Viruses require host cells to replicate and proliferate, which indicates that viruses hijack the cellular machinery. Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4-positive T cells, and efficiently uses cellular proteins to replicate. Cells already have proteins that inhibit the replication of the foreign HIV-1, but their function is suppressed by viral proteins. Intriguingly, HIV-1 infection also changes the cellular metabolism to aerobic glycolysis. This phenomenon has been interpreted as a cellular response to maintain homeostasis during viral infection, yet HIV-1 efficiently replicates even in this environment. In this review, we discuss the regulatory role of glycolytic enzymes in viral replication and the impact of aerobic glycolysis on viral infection by introducing various host proteins involved in viral replication. Furthermore, we would like to propose a "glyceraldehyde-3-phosphate dehydrogenase-induced shock (G-shock) and kill strategy" that maximizes the antiviral effect of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to eliminate latently HIV-1-infected cells.
Assuntos
Glicólise , Infecções por HIV , HIV-1 , Replicação Viral , Humanos , HIV-1/fisiologia , Glicólise/fisiologia , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Infecções por HIV/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismoRESUMO
Structurally simplified analogues of ansellone A, in which the decalin skeleton is replaced with a lipophilic chain, were prepared and their HIV latency-reversing activities biologically evaluated. In particular, two analogues bearing ether and alkenyl side chains, respectively, showed comparable activities to that of ansellone A. Each of the simplified compounds was easily synthesized using Prins cyclisation chemistry.
Assuntos
Infecções por HIV , Humanos , Relação Estrutura-AtividadeRESUMO
The first total synthesis of marine sesterterpenoid ansellone G (2) was accomplished. This strategy utilizes the Prins cyclization reaction of a chloro-substituted homoallyl alcohol to synthesize the hydrobenzopyran skeleton. The preintroduction of the chloro groups facilitated the functional group transformation for 2 after constructing the carbon framework. Furthermore, we also successfully synthesized phorbadione (3) by dehydrating the tertiary alcohol. The HIV latency-reversing activity of the synthesized 2, 3, and deacetylated 2 was also evaluated.
Assuntos
Álcoois , CiclizaçãoRESUMO
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 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
One compound sometimes shows two biological functions, becoming important aspect of recent drug discovery. This study began with an attempt to confirm the previously reported molecular mechanism of the anti-human immunodeficiency virus (HIV) heterocyclic compound BMMP [2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine], i.e., induction of abnormal uncoating of the viral core at the post-entry step. Our mechanistic study gave results consistent with this mechanism. We further attempted to find out the molecular target of BMMP by a pulldown approach using previously synthesized biotinylated BMMP (Biotin-BMMP) and successfully identified heterogenous nuclear ribonucleoprotein M (hnRNP M) as a BMMP-binding protein. This protein was found not to be accountable for the anti-HIV activity of BMMP. As hnRNP M has been reported to promote cancer metastasis, we tested this mechanism and found that BMMP suppressed migration of the human lung carcinoma cell line A549 stimulated with transforming growth factor-ß (TGF-ß). Mechanistic study showed that BMMP suppressed the expression of CD44 mRNA via the regulation of hnRNP M. Furthermore, six new derivatives of BMMP were synthesized, and the patterns of their activities against HIV-1 and cell migration were not uniform, suggesting that the anti-HIV mechanism and the anti-cell migration mechanism of BMMP are independent. Taken together, the anti-cell migration activity of the anti-HIV heterocyclic compound BMMP was newly discovered by identification of its binding protein hnRNP M using a chemical biology approach.
Assuntos
Fármacos Anti-HIV/química , Compostos Heterocíclicos/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Compostos Heterocíclicos/metabolismo , Compostos Heterocíclicos/farmacologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Ligação Proteica , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismoRESUMO
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
BACKGROUND: Lifestyle-related diseases such as diabetes are steadily increasing worldwide. In Sudan, there are a variety of plant species used traditionally for the treatment of diabetes, obesity and other symptoms which need to be validated through scientific studies for their claimed traditional uses. Therefore, in the current study, the free radical scavenging activity, α-glucosidase inhibitory and pancreatic lipase inhibitory activities of 70% ethanol and water extracts of eighteen Sudanese medicinal plants were investigated using various in vitro assays. Moreover, the cytotoxicity and genotoxicity were assessed for the bioactive plant extracts. METHODS: Eighteen plants were selected on the basis of their traditional uses and extracted with 70% ethanol and water to obtain thirty-six extracts. The obtained extracts were screened using different in vitro bioassays namely, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, α-glucosidase inhibitory and pancreatic lipase inhibitory assays. Furthermore, the active plant extracts were investigated for their cytotoxicity and genotoxicity on HeLa cell line using HCS DNA Damage Assay. RESULTS: Both 70% ethanol and water extracts of Acacia nilotica, Ziziphus spina-christi, Abrus precatorius, and Geigeria alata along with the 70% ethanol extract of Martynia annua showed potent free radical scavenging activity. Regarding the α-glucosidase inhibition assay, both extracts of Acacia nilotica, Ziziphus spina-christi, Geigeria alata, and Cyperus rotundus showed potent activity. In general, 70% ethanol extracts were more potent compared to water extracts with exception of Cordia sinensis and Cymbopogon proximus, for which water extracts also showed potent enzyme inhibitory activity. Similarly, water extracts of Acacia nilotica and Ziziphus spina-christi showed potent inhibitory activity against pancreatic lipase enzyme. Some of the extracts also showed significant genotoxicity and cytotoxicity at the concentration range used for bioactivities. CONCLUSION: The extracts of Acacia nilotica, Ziziphus spina-christi, Geigeria alata, Martynia annua and Abrus precatorius exhibited an appreciable range of activity on antioxidant and enzyme inhibitory assays.
Assuntos
Antioxidantes/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Lipase/antagonistas & inibidores , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Compostos de Bifenilo , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Picratos , Extratos Vegetais/toxicidade , SudãoRESUMO
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
Polyethylene glycol (PEG) modification (PEGylation) is one of the best approaches to improve the stabilities and blood half-lives of protein drugs; however, PEGylation dramatically reduces the bioactivities of protein drugs. Here, we present "self-assembly PEGylation retaining activity" (SPRA) technology via a host-guest interaction between PEGylated ß-cyclodextrin (PEG-ß-CyD) and adamantane-appended (Ad) proteins. PEG-ß-CyD formed stable complexes with Ad-insulin and Ad-lysozyme to yield SPRA-insulin and SPRA-lysozyme, respectively. Both SPRA-proteins showed high stability against heat and trypsin digest, comparable with that of covalently PEGylated protein equivalents. Importantly, the SPRA-lysozyme possessed ca. 100% lytic activity, whereas the activity of the covalently PEGylated lysozyme was ca. 23%. Additionally, SPRA-insulin provided a prolonged and peakless blood glucose profile when compared with insulin glargine. It also showed no loss of activity. In contrast, the covalently PEGylated insulin showed a negligible hypoglycemic effect. These findings indicate that SPRA technology has potential as a generic method, surpassing conventional PEGylation methods for proteins.
Assuntos
Polietilenoglicóis/química , Proteínas/química , Animais , Hipoglicemiantes/química , Insulina/análogos & derivados , Insulina/química , Masculino , Muramidase/química , Ratos , Ratos Wistar , Tripsina/química , beta-Ciclodextrinas/químicaRESUMO
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.