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1.
Mol Ther ; 32(7): 2316-2327, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38734901

RESUMO

HIV-1 infection remains a public health problem with no cure. Although antiretroviral therapy (ART) is effective for suppressing HIV-1 replication, it requires lifelong drug administration due to a stable reservoir of latent proviruses and may cause serious side effects and drive the emergence of drug-resistant HIV-1 variants. Gene therapy represents an alternative approach to overcome the limitations of conventional treatments against HIV-1 infection. In this study, we constructed and investigated the antiviral effects of an HIV-1 Tat-dependent conditionally replicating adenovirus, which selectively replicates and expresses the diphtheria toxin A chain (Tat-CRAds-DTA) in HIV-1-infected cells both in vitro and in vivo. We found that Tat-CRAds-DTA could specifically induce cell death and inhibit virus replication in HIV-1-infected cells mediated by adenovirus proliferation and DTA expression. A low titer of progeny Tat-CRAds-DTA was also detected in HIV-1-infected cells. In addition, Tat-CRAds-DTA showed no apparent cytotoxicity to HIV-1-negative cells and demonstrated significant therapeutic efficacy against HIV-1 infection in a humanized mouse model. The findings in this study highlight the potential of Tat-CRAds-DTA as a new gene therapy for the treatment of HIV-1 infection.


Assuntos
Adenoviridae , Toxina Diftérica , Terapia Genética , Vetores Genéticos , Infecções por HIV , HIV-1 , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Humanos , HIV-1/genética , Toxina Diftérica/genética , Animais , Adenoviridae/genética , Infecções por HIV/terapia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Camundongos , Terapia Genética/métodos , Vetores Genéticos/genética , Modelos Animais de Doenças , Linhagem Celular , Células HEK293 , Expressão Gênica , Fragmentos de Peptídeos
2.
Mol Ther ; 32(3): 689-703, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38268188

RESUMO

Passive delivery of antibodies to mucosal sites may be a valuable adjunct to COVID-19 vaccination to prevent infection, treat viral carriage, or block transmission. Neutralizing monoclonal IgG antibodies are already approved for systemic delivery, and several clinical trials have been reported for delivery to mucosal sites where SARS-CoV-2 resides and replicates in early infection. However, secretory IgA may be preferred because the polymeric complex is adapted for the harsh, unstable external mucosal environment. Here, we investigated the feasibility of producing neutralizing monoclonal IgA antibodies against SARS-CoV-2. We engineered two class-switched mAbs that express well as monomeric and secretory IgA (SIgA) variants with high antigen-binding affinities and increased stability in mucosal secretions compared to their IgG counterparts. SIgAs had stronger virus neutralization activities than IgG mAbs and were protective against SARS-CoV-2 infection in an in vivo murine model. Furthermore, SIgA1 can be aerosolized for topical delivery using a mesh nebulizer. Our findings provide a persuasive case for developing recombinant SIgAs for mucosal application as a new tool in the fight against COVID-19.


Assuntos
Anticorpos Neutralizantes , COVID-19 , Animais , Camundongos , Humanos , Imunoglobulina A Secretora , SARS-CoV-2/genética , Vacinas contra COVID-19 , COVID-19/prevenção & controle , Anticorpos Monoclonais , Imunoglobulina G , Anticorpos Antivirais
3.
Small ; 20(42): e2402482, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38855997

RESUMO

Hydrogel as a solar evaporator shows great potential in freshwater production. However, hydrogels often lead to an imbalance between solar energy input and water supply management due to their excessively high saturated water content. Thus, achieving a stable water-energy-balance in hydrogel evaporators remains challenging. Here, by tortuosity engineering designed water transport channels, a seamless high-tortuosity/low-tortuosity/high-tortuosity structured hydrogel (SHLH structure hydrogel) evaporator is developed, which enables the hydrogel with customized water transport rate, leading to the controlled water supply at the evaporator interface. An excellent equilibrium between the photothermal conversion and water supply is established, and the maximum utilization of solar energy is realized, thereby achieving an excellent evaporation rate of 3.64 kg m-2 h-1 under one solar illumination. This tortuosity engineering controlled SHLH structured evaporator provides a novel strategy to attain water-energy-balance and expands new approaches for constructing hydrogel-based evaporators with tailored water transportation capacity.

4.
BMC Emerg Med ; 24(1): 174, 2024 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-39333918

RESUMO

BACKGROUND: Rapid sequence intubation (RSI) is a crucial step in the resuscitation process for critically ill patients, and the judicious use of sedative drugs during RSI significantly influences the clinical outcomes of patients. Ketamine is a commonly used anesthetic sedative; however, its impact on the mortality of patients undergoing RSI has yielded inconsistent findings. Therefore, we conducted a systematic review and meta-analysis investigating ketamine's role in RSI to provide insights into selecting appropriate sedatives for critically ill patients. METHODS: In this systematic review and meta-analysis, we conducted a systematic search on MEDLINE (PubMed), Embase, and Cochrane Central Register of Controlled Trials, without restricting to randomized controlled trials (RCTs) or cohort studies. The search was performed from inception until Dec 12, 2023, with no language restrictions. All studies comparing the use of sedatives, including ketamine, and documenting in-hospital mortality were included in this study. RESULTS: A total of 991 studies were identified, out of which 15 studies (5 RCTs and 10 cohort studies) involving 16,807 participants fulfilled the inclusion criteria. No significant impact on in-hospital mortality was observed with the use of ketamine compared to other drugs during RSI (OR 0.90, 95%CI 0.72 to 1.12). Low-quality evidence suggested that ketamine might reduce mortality within the first seven days of hospitalization (OR 0.42, 95%CI 0.19 to 0.93), but it may also have a potential effect on prolonging ICU-free days at day 28 (MD -0.71, 95%CI -1.38 to -0.05). There were no significant differences in the results of the other RSI-related outcomes, such as physiological function and adverse events. CONCLUSIONS: Based on existing studies, ketamine showed no significant difference compared to other sedatives in terms of in-hospital mortality, physiological impact, and side effects following RSI. However, it may reduce mortality within 7 days while probably prolong the length of stay in the ICU. TRIAL REGISTRATION: CRD42023478020.


Assuntos
Ketamina , Indução e Intubação de Sequência Rápida , Humanos , Anestésicos Dissociativos/administração & dosagem , Anestésicos Dissociativos/efeitos adversos , Estado Terminal , Mortalidade Hospitalar , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/efeitos adversos , Ketamina/administração & dosagem , Ketamina/efeitos adversos , Indução e Intubação de Sequência Rápida/efeitos adversos , Indução e Intubação de Sequência Rápida/métodos
5.
J Immunol ; 206(12): 2852-2861, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34049972

RESUMO

NF-κB plays a crucial role in regulating cell proliferation, inflammation, apoptosis, and immune responses. HSV type 2 (HSV-2) is one of the most predominant sexually transmitted pathogens worldwide, and its infection increases the risk of HIV type 1 (HIV-1) acquisition and transmission. HSV-2 glycoprotein D (gD), highly homologous to HSV-1 gD, is essential for viral adhesion, fusion, entry, and spread. It is known that HSV-1 gD can bind herpesvirus entry mediator (HVEM) to trigger NF-κB activation and thereby facilitate viral replication at the early stage of infection. In this study, we found that purified HSV-2 gD triggered NF-κB activation at the early stage of infection, whereas ectopic expression of HSV-2 gD significantly downregulated TNF-α-induced NF-κB activity as well as TNF-α-induced IL-6 and IL-8 expression. Mechanistically, HSV-2 gD inhibited NF-κB, but not IFN-regulatory factor 3 (IRF3), activation and suppressed NF-κB activation mediated by overexpression of TNFR-associated factor 2 (TRAF2), IκB kinase α (IKKα), IKKß, or p65. Coimmunoprecipitation and binding kinetic analyses demonstrated that HSV-2 gD directly bound to the NF-κB subunit p65 and abolished the nuclear translocation of p65 upon TNF-α stimulation. Mutational analyses further revealed that HSV-2 gD interacted with the region spanning aa 19-187 of p65. Findings in this study together demonstrate that HSV-2 gD interacts with p65 to regulate p65 subcellular localization and thereby prevents NF-κB-dependent gene expression, which may contribute to HSV-2 immune evasion and pathogenesis.


Assuntos
Herpesvirus Humano 2/imunologia , Fator de Transcrição RelA/imunologia , Proteínas do Envelope Viral/imunologia , Células HEK293 , Células HeLa , Humanos
6.
Int J Clin Pract ; 2023: 6733465, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36743822

RESUMO

Sepsis is a high-incidence disease and demands intensive care. Finding effective treatment is the key to cure sepsis. Studies have shown a lower level of vitamin C in patients with sepsis. Therefore, vitamin C supplementation has become one of the measures to treat sepsis. However, the clinical studies of vitamin C in the treatment of sepsis have been controversial. We performed a meta-analysis to evaluate vitamin C's efficacy and safety in the treatment of sepsis. We searched four electronic databases: PubMed, Embase, Web of Science, and the Cochrane Library, and two researchers independently screened 24 eligible RCTs published in English. Our review demonstrates that intravenous (IV) vitamin C might improve short-term mortality (RR, 0.82; 95% CI, 0.65-1.02; P=0.07; and I 2 = 45%) and overall mortality (RR, 0.86; 95% CI, 0.74-1.01; P=0.06; and I 2 = 51%) of patients with sepsis. Moreover, the SOFA score of patients with sepsis improved significantly after treatment with vitamin C for over 72 hours (RR, 0.26; 95% CI, 0.09-0.42; P=0.002; and I 2 = 0%). The main results of our study were moderate-quality evidence. More high-quality, multicenter RCTs are needed to provide more substantial evidence on the efficacy and safety of IV vitamin C for sepsis.


Assuntos
Ácido Ascórbico , Sepse , Humanos , Ácido Ascórbico/uso terapêutico , Vitaminas/uso terapêutico , Sepse/tratamento farmacológico , Estudos Multicêntricos como Assunto
7.
Molecules ; 28(12)2023 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-37375411

RESUMO

Pentagalloyl glucose (PGG) is a natural hydrolyzable gallotannin abundant in various plants and herbs. It has a broad range of biological activities, specifically anticancer activities, and numerous molecular targets. Despite multiple studies available on the pharmacological action of PGG, the molecular mechanisms underlying the anticancer effects of PGG are unclear. Here, we have critically reviewed the natural sources of PGG, its anticancer properties, and underlying mechanisms of action. We found that multiple natural sources of PGG are available, and the existing production technology is sufficient to produce large quantities of the required product. Three plants (or their parts) with maximum PGG content were Rhus chinensis Mill, Bouea macrophylla seed, and Mangifera indica kernel. PGG acts on multiple molecular targets and signaling pathways associated with the hallmarks of cancer to inhibit growth, angiogenesis, and metastasis of several cancers. Moreover, PGG can enhance the efficacy of chemotherapy and radiotherapy by modulating various cancer-associated pathways. Therefore, PGG can be used for treating different human cancers; nevertheless, the data on the pharmacokinetics and safety profile of PGG are limited, and further studies are essential to define the clinical use of PGG in cancer therapies.


Assuntos
Glucose , Taninos Hidrolisáveis , Humanos , Taninos Hidrolisáveis/farmacologia , Taninos Hidrolisáveis/metabolismo
8.
J Immunol ; 205(5): 1281-1292, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32699158

RESUMO

Type I IFNs play an important role in innate immunity against viral infections by inducing the expression of IFN-stimulated genes (ISGs), which encode effectors with various antiviral functions. We and others previously reported that HSV type 2 (HSV-2) inhibits the synthesis of type I IFNs, but how HSV-2 suppresses IFN-mediated signaling is less understood. In the current study, after the demonstration of HSV-2 replication resistance to IFN-ß treatment in human epithelial cells, we reveal that HSV-2 and the viral protein ICP22 significantly decrease the expression of ISG54 at both mRNA and protein levels. Likewise, us1 del HSV-2 (ICP22-deficient HSV-2) replication is more sensitive to IFN-ß treatment, indicating that ICP22 is a vital viral protein responsible for the inhibition of type I IFN-mediated signaling. In addition, overexpression of HSV-2 ICP22 inhibits the expression of STAT1, STAT2, and IFN regulatory factor 9 (IRF9), resulting in the blockade of ISG factor 3 (ISGF3) nuclear translocation, and mechanistically, this is due to ICP22-induced ubiquitination of STAT1, STAT2, and IRF9. HSV-2 ICP22 appears to interact with STAT1, STAT2, IRF9, and several other ubiquitinated proteins. Following further biochemical study, we show that HSV-2 ICP22 functions as an E3 ubiquitin protein ligase to induce the formation of polyubiquitin chains. Taken together, we demonstrate that HSV-2 interferes with type I IFN-mediated signaling by degrading the proteins of ISGF3, and we identify HSV-2 ICP22 as a novel E3 ubiquitin protein ligase to induce the degradation of ISGF3. Findings in this study highlight a new mechanism by which HSV-2 circumvents the host antiviral responses through a viral E3 ubiquitin protein ligase.


Assuntos
Herpes Genital/imunologia , Herpesvirus Humano 2/imunologia , Proteínas Imediatamente Precoces/imunologia , Interferon beta/imunologia , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas Virais/imunologia , Antivirais/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Herpesvirus Humano 1/imunologia , Humanos , Imunidade Inata/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT2/imunologia , Ubiquitinação/imunologia
9.
Ren Fail ; 44(1): 1150-1159, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35834360

RESUMO

BACKGROUND: Acute kidney injury (AKI) is a serious complication related to cardiac surgery. Several studies have been conducted to investigate the effect of dexmedetomidine administration on AKI prevention. OBJECTIVE: To assess if dexmedetomidine is associated with a protective effect of renal function after cardiac surgery. And the aim of conducting this meta-analysis is to summarize the literature and determine the clinical utility of dexmedetomidine administration in patients undergoing cardiac surgery. METHODS: PubMed, Cochrane Library, and EMBASE databases were comprehensively searched for all randomized controlled trials (RCTs) published before 1 December, 2021 that investigated the effect of dexmedetomidine on AKI prevention. RESULTS: Our analysis included 16 studies involving 2148 patients. Compared with the control group, dexmedetomidine administration significantly reduced AKI incidence (OR, 0.47; 95% CI, 0.36-0.61; p < 0.00001; I2 = 26%) and the length of stay in the intensive care unit (ICU) but did not alter mortality rate, length of stay in the hospital, and mechanical ventilation time. Furthermore, the incidence of delirium among patients treated with dexmedetomidine was significantly decreased. CONCLUSION: Dexmedetomidine administration has a positive effect on preventing AKI and postoperative delirium after cardiac surgery and significantly reduces the length of stay in the ICU.


Assuntos
Injúria Renal Aguda , Procedimentos Cirúrgicos Cardíacos , Delírio , Dexmedetomidina , Injúria Renal Aguda/epidemiologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/prevenção & controle , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Delírio/induzido quimicamente , Delírio/tratamento farmacológico , Dexmedetomidina/uso terapêutico , Humanos , Unidades de Terapia Intensiva
10.
Emerg Infect Dis ; 27(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33256890

RESUMO

We investigated the dynamics of seroconversion in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. During March 29-May 22, 2020, we collected serum samples and associated clinical data from 177 persons in London, UK, who had SARS-CoV-2 infection. We measured IgG against SARS-CoV-2 and compared antibody levels with patient outcomes, demographic information, and laboratory characteristics. We found that 2.0%-8.5% of persons did not seroconvert 3-6 weeks after infection. Persons who seroconverted were older, were more likely to have concurrent conditions, and had higher levels of inflammatory markers. Non-White persons had higher antibody concentrations than those who identified as White; these concentrations did not decline during follow-up. Serologic assay results correlated with disease outcome, race, and other risk factors for severe SARS-CoV-2 infection. Serologic assays can be used in surveillance to clarify the duration and protective nature of humoral responses to SARS-CoV-2 infection.


Assuntos
COVID-19/sangue , COVID-19/imunologia , Imunoglobulina G/sangue , SARS-CoV-2 , Soroconversão , Adulto , Idoso , Anticorpos Antivirais/sangue , COVID-19/fisiopatologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
J Immunol ; 201(1): 53-68, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29760190

RESUMO

Tick-borne encephalitis virus (TBEV) is one of the flaviviruses that targets the CNS and causes encephalitis in humans. The mechanism of TBEV that causes CNS destruction remains unclear. It has been reported that RANTES-mediated migration of human blood monocytes and T lymphocytes is specifically induced in the brain of mice infected with TBEV, which causes ensuing neuroinflammation and may contribute to brain destruction. However, the viral components responsible for RANTES induction and the underlying mechanisms remain to be fully addressed. In this study, we demonstrate that the NS5, but not other viral proteins of TBEV, induces RANTES production in human glioblastoma cell lines and primary astrocytes. TBEV NS5 appears to activate the IFN regulatory factor 3 (IRF-3) signaling pathway in a manner dependent on RIG-I/MDA5, which leads to the nuclear translocation of IRF-3 to bind with RANTES promoter. Further studies reveal that the activity of RNA-dependent RNA polymerase (RdRP) but not the RNA cap methyltransferase is critical for TBEV NS5-induced RANTES expression, and this is likely due to RdRP-mediated synthesis of dsRNA. Additional data indicate that the residues at K359, D361, and D664 of TBEV NS5 are critical for RdRP activity and RANTES induction. Of note, NS5s from other flaviviruses, including Japanese encephalitis virus, West Nile virus, Zika virus, and dengue virus, can also induce RANTES expression, suggesting the significance of NS5-induced RANTES expression in flavivirus pathogenesis. Our findings provide a foundation for further understanding how flaviviruses cause neuroinflammation and a potential viral target for intervention.


Assuntos
Quimiocina CCL5/biossíntese , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Encefalite Transmitida por Carrapatos/patologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular Tumoral , Quimiocina CCL5/genética , Chlorocebus aethiops , Proteína DEAD-box 58/metabolismo , Células HEK293 , Células HeLa , Humanos , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Regiões Promotoras Genéticas/genética , Receptores Imunológicos , Células Vero , Proteínas não Estruturais Virais/genética
12.
J Virol ; 92(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29925661

RESUMO

Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.


Assuntos
Códon/genética , Vacinas Virais/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Camundongos , Genética Reversa/métodos , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Células Vero , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Virulência/genética , Replicação Viral/genética , Replicação Viral/imunologia , Zika virus/genética , Zika virus/patogenicidade , Infecção por Zika virus/imunologia
13.
J Gen Virol ; 99(1): 73-85, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29182509

RESUMO

Enterovirus A71 (EV-A71) is a positive-strand RNA virus that causes hand-foot-mouth disease and neurological complications in children and infants. Although the underlying mechanisms remain to be further defined, impaired immunity is thought to play an important role. The host zinc-finger antiviral protein (ZAP), an IFN-stimulated gene product, has been reported to specifically inhibit the replication of certain viruses. However, whether ZAP restricts the infection of enteroviruses remains unknown. Here, we report that EV-A71 infection upregulates ZAP mRNA in RD and HeLa cells. Moreover, ZAP overexpression rendered 293 T cells resistant to EV-A71 infection, whereas siRNA-mediated depletion of endogenous ZAP enhanced EV-A71 infection. The EV-A71 infection stimulated site-specific proteolysis of two ZAP isoforms, leading to the accumulation of a 40 kDa N-terminal ZAP fragment in virus-infected cells. We further revealed that the 3C protease (3Cpro) of EV-A71 mediates ZAP cleavage, which requires protease activity. Furthermore, ZAP variants with single amino acid substitutions at Gln-369 were resistant to 3Cpro cleavage, implying that Gln-369 is the sole cleavage site in ZAP. Moreover, although ZAP overexpression inhibited EV-A71 replication, the cleaved fragments did not show this effect. Our results indicate that an equilibrium between ZAP and enterovirus 3Cpro controls viral infection. The findings in this study suggest that viral 3Cpro mediated ZAP cleavage may represent a mechanism to escape host antiviral responses.


Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/enzimologia , Interações Hospedeiro-Patógeno , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Proteases Virais 3C , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Enterovirus Humano A/genética , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Células Musculares/metabolismo , Células Musculares/virologia , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Células Sf9/imunologia , Células Sf9/virologia , Transdução de Sinais , Spodoptera , Proteínas Virais/genética
14.
J Neuroinflammation ; 15(1): 275, 2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-30241539

RESUMO

BACKGROUND: Many viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo. METHODS: Mice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo. RESULTS: ZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased. CONCLUSION: Findings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/virologia , Proteínas Serina-Treonina Quinases/genética , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Proteína 1 de Ligação a X-Box/genética , Zika virus/genética , Zika virus/fisiologia
15.
J Gen Virol ; 98(9): 2351-2361, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28809154

RESUMO

Herpes simplex virus type 2 (HSV-2) increases human immunodeficiency virus type 1 (HIV-1) acquisition and transmission via unclear mechanisms. Herpesvirus entry mediator (HVEM), an HSV-2 entry receptor, is highly expressed on HIV-1 target cells (CD4+ T cells) and may be incorporated into HIV-1 virions, while HSV-2 glycoproteins can be present on the infected cell surface. Since HVEM-gD interaction together with gB/gH/gL is essential for HSV-2 entry, HVEM-bearing HIV-1 (HIV-1/HVEM) may enter HSV-2-infected cells through such interactions. To test this hypothesis, we first confirmed the presence of HVEM on HIV-1 virions and glycoproteins on the HSV-2-infected cell surface. Additional studies showed that HIV-1/HVEM bound to the HSV-2-infected cell surface in an HSV-2 infection-time-dependent manner via HVEM-gD interaction. HIV-1/HVEM entry of HSV-2-infected cells was dependent on HVEM-gD interaction and the presence of gB/gH/gL, and was inhibited by azidothymidine. Furthermore, peripheral blood mononuclear cell-derived HIV-1 infected HSV-2-infected primary foreskin epithelial cells and the infection was inhibited by anti-HVEM/gD antibodies. Together, our results indicate that HIV-1 produced from CD4+ T cells bears HSV-2 receptor HVEM and can bind to and enter HSV-2-infected epithelial cells depending on HVEM-gD interaction and the presence of gB/gH/gL. Our findings provide a potential new mechanism underlying HSV-2 infection-enhanced HIV-1 mucosal transmission and may shed light on HIV-1 prevention.


Assuntos
Células Epiteliais/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Herpes Simples/metabolismo , Herpesvirus Humano 2/fisiologia , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Linfócitos T CD4-Positivos/virologia , Células CHO , Cricetulus , Células Epiteliais/virologia , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Herpes Simples/genética , Herpesvirus Humano 2/genética , Humanos , Camundongos , Ligação Proteica , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Receptores Virais/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus
16.
J Virol ; 90(10): 5141-5151, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26962213

RESUMO

UNLABELLED: The NF-κB signaling network, which is an ancient signaling pathway, plays a pivotal role in innate immunity and constitutes a first line of defense against invading pathogens, including viruses. However, numerous viruses possess evolved strategies to antagonize the activation of the NF-κB signaling pathway. Our previous study demonstrated that the nonstructural protein 2C of enterovirus 71 (EV71), which is the major pathogen of hand, foot, and mouth disease, inhibits tumor necrosis factor alpha (TNF-α)-mediated activation of NF-κB by suppressing IκB kinase ß (IKKß) phosphorylation. Nevertheless, the mechanism underlying the inhibition of IKKß phosphorylation by EV71 2C remains largely elusive. We demonstrate that EV71 2C interacts with all isoforms of the protein phosphatase 1 (PP1) catalytic subunit (the PP1α, PP1ß, and PP1γ isoforms) through PP1-docking motifs. EV71 2C has no influence on the subcellular localization of PP1. In addition, the PP1-binding-deficient EV71 2C mutant 3E3L nearly completely lost the ability to suppress IKKß phosphorylation and NF-κB activation was markedly restored in the mutant, thereby indicating that PP1 binding is efficient for EV71 2C-mediated inhibition of IKKß phosphorylation and NF-κB activation. We further demonstrate that 2C forms a complex with PP1 and IKKß to dephosphorylate IKKß. Notably, we reveal that other human enteroviruses, including poliovirus (PV), coxsackie A virus 16 (CVA16), and coxsackie B virus 3 (CVB3), use 2C proteins to recruit PP1, leading to the inhibition of IKKß phosphorylation. Our findings indicate that enteroviruses exploit a novel mechanism to inhibit IKKß phosphorylation by recruiting PP1 and IKKß to form a complex through 2C proteins, which ultimately results in the inhibition of the NF-κB signaling pathway. IMPORTANCE: The innate antiviral immunity system performs an essential function in recognizing and eliminating invading viruses. Enteroviruses include a number of important human pathogens, including poliovirus (PV), EV71, and coxsackieviruses (CVs). As 2C is the most conserved and complex nonstructural protein of enteroviruses, its biological function is largely unclear, whereas the 2A and 3C proteinases of enteroviruses are well characterized. We reveal that EV71 2C forms a complex with PP1 and IKKß to maintain IKKß in an unphosphorylated and inactive state, resulting in the inactivation of the TNF-α-mediated NF-κB signaling pathway. We provide evidence that the 2C proteins of the enteroviruses PV, CVA16, and CVB3 suppress IKKß phosphorylation through the same mechanism involving PP1. We demonstrate that enteroviruses exploit a novel mechanism involving PP1 to regulate innate antiviral immunity, and our findings may be particularly important for understanding the pathogenicity of enteroviruses.


Assuntos
Proteínas de Transporte/metabolismo , Enterovirus/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas de Transporte/genética , Enterovirus/química , Enterovirus/genética , Enterovirus Humano A/química , Enterovirus Humano A/metabolismo , Enterovirus Humano B/metabolismo , Células HeLa , Humanos , Fosforilação , Poliovirus/química , Poliovirus/metabolismo , Ligação Proteica , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Proteínas não Estruturais Virais/genética
17.
J Virol ; 90(23): 10472-10485, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27630238

RESUMO

Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCE: Infection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication.


Assuntos
Enterovirus Humano A/fisiologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Chlorocebus aethiops , Enterovirus Humano A/genética , Estabilidade Enzimática , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sumoilação , Ubiquitinação , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral/fisiologia
18.
J Immunol ; 195(1): 329-38, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25994965

RESUMO

There is a lack of an HSV-2 vaccine, in part as the result of various factors that limit robust and long-term memory immune responses at the mucosal portals of viral entry. We previously demonstrated that chemokine CCL19 augmented mucosal and systemic immune responses to HIV-1 envelope glycoprotein. Whether such enhanced immunity can protect animals against virus infection remains to be addressed. We hypothesized that using CCL19 in a fusion form to direct an immunogen to responsive immunocytes might have an advantage over CCL19 being used in combination with an immunogen. We designed two fusion constructs, plasmid (p)gBIZCCL19 and pCCL19IZgB, by fusing CCL19 to the C- or N-terminal end of the extracellular HSV-2 glycoprotein B (gB) with a linker containing two (Gly4Ser)2 repeats and a GCN4-based isoleucine zipper motif for self-oligomerization. Following immunization in mice, pgBIZCCL19 and pCCL19IZgB induced strong gB-specific IgG and IgA in sera and vaginal fluids. The enhanced systemic and mucosal Abs showed increased neutralizing activity against HSV-2 in vitro. Measurement of gB-specific cytokines demonstrated that gB-CCL19 fusion constructs induced balanced Th1 and Th2 cellular immune responses. Moreover, mice vaccinated with fusion constructs were well protected from intravaginal lethal challenge with HSV-2. Compared with pgB and pCCL19 coimmunization, fusion constructs increased mucosal surface IgA(+) cells, as well as CCL19-responsive immunocytes in spleen and mesenteric lymph nodes. Our findings indicate that enhanced humoral and cellular immune responses can be achieved by immunization with an immunogen fused to a chemokine, providing information for the design of vaccines against mucosal infection by HSV-2 and other sexually transmitted viruses.


Assuntos
Quimiocina CCL19/imunologia , Herpes Genital/prevenção & controle , Herpesvirus Humano 2/imunologia , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Motivos de Aminoácidos , Animais , Quimiocina CCL19/administração & dosagem , Quimiocina CCL19/genética , Feminino , Herpes Genital/imunologia , Herpes Genital/mortalidade , Herpes Genital/patologia , Herpesvirus Humano 2/química , Imunidade Celular/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Imunização , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos/administração & dosagem , Plasmídeos/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Equilíbrio Th1-Th2 , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vagina/imunologia , Vagina/patologia , Vagina/virologia , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
19.
J Immunol ; 194(7): 3102-15, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25712217

RESUMO

HSV-2 is the major cause of genital herpes, and its infection increases the risk of HIV-1 acquisition and transmission. After initial infection, HSV-2 can establish latency within the nervous system and thus maintains lifelong infection in humans. It has been suggested that HSV-2 can inhibit type I IFN signaling, but the underlying mechanism has yet to be determined. In this study, we demonstrate that productive HSV-2 infection suppresses Sendai virus (SeV) or polyinosinic-polycytidylic acid-induced IFN-ß production. We further reveal that US1, an immediate-early protein of HSV-2, contributes to such suppression, showing that US1 inhibits IFN-ß promoter activity and IFN-ß production at both mRNA and protein levels, whereas US1 knockout significantly impairs such capability in the context of HSV-2 infection. US1 directly interacts with DNA binding domain of IRF-3, and such interaction suppresses the association of nuclear IRF-3 with the IRF-3 responsive domain of IFN-ß promoter, resulting in the suppression of IFN-ß promoter activation. Additional studies demonstrate that the 217-414 aa domain of US1 is critical for the suppression of IFN-ß production. Our results indicate that HSV-2 US1 downmodulates IFN-ß production by suppressing the association of IRF-3 with the IRF-3 responsive domain of IFN-ß promoter. Our findings highlight the significance of HSV-2 US1 in inhibiting IFN-ß production and provide insights into the molecular mechanism by which HSV-2 evades the host innate immunity, representing an unconventional strategy exploited by a dsDNA virus to interrupt type I IFN signaling pathway.


Assuntos
Regulação da Expressão Gênica , Herpesvirus Humano 2/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/biossíntese , Interferon beta/genética , Regiões Promotoras Genéticas , Animais , Linhagem Celular , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Fator Regulador 3 de Interferon/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais
20.
J Neuroinflammation ; 13(1): 209, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27576490

RESUMO

BACKGROUND: Tick-borne encephalitis virus (TBEV) is one of the most important flaviviruses that targets the central nervous system (CNS) and causes encephalitides in humans. Although neuroinflammatory mechanisms may contribute to brain tissue destruction, the induction pathways and potential roles of specific chemokines in TBEV-mediated neurological disease are poorly understood. METHODS: BALB/c mice were intracerebrally injected with TBEV, followed by evaluation of chemokine and cytokine profiles using protein array analysis. The virus-infected mice were treated with the CC chemokine antagonist Met-RANTES or anti-RANTES mAb to determine the role of RANTES in affecting TBEV-induced neurological disease. The underlying signaling mechanisms were delineated using RANTES promoter luciferase reporter assay, siRNA-mediated knockdown, and pharmacological inhibitors in human brain-derived cell culture models. RESULTS: In a mouse model, pathological features including marked inflammatory cell infiltrates were observed in brain sections, which correlated with a robust up-regulation of RANTES within the brain but not in peripheral tissues and sera. Antagonizing RANTES within CNS extended the survival of mice and reduced accumulation of infiltrating cells in the brain after TBEV infection. Through in vitro studies, we show that virus infection up-regulated RANTES production at both mRNA and protein levels in human brain-derived cell lines and primary progenitor-derived astrocytes. Furthermore, IRF-3 pathway appeared to be essential for TBEV-induced RANTES production. Site mutation of an IRF-3-binding motif abrogated the RANTES promoter activity in virus-infected brain cells. Moreover, IRF-3 was activated upon TBEV infection as evidenced by phosphorylation of TBK1 and IRF-3, while blockade of IRF-3 activation drastically reduced virus-induced RANTES expression. CONCLUSIONS: Our findings together provide insights into the molecular mechanism underlying RANTES production induced by TBEV, highlighting its potential importance in the process of neuroinflammatory responses to TBEV infection.


Assuntos
Quimiocina CCL5/biossíntese , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Encefalite Transmitida por Carrapatos/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Transdução de Sinais/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/virologia , Linhagem Celular Tumoral , Quimiocina CCL5/genética , Quimiocinas/biossíntese , Quimiocinas/genética , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/genética , Feminino , Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Carga Viral/tendências
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