RESUMO
Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-ß, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.
Assuntos
Vírus da Encefalite Equina Venezuelana , alfa Carioferinas , Animais , Cavalos , Humanos , alfa Carioferinas/química , alfa Carioferinas/metabolismo , Vírus da Encefalite Equina Venezuelana/metabolismo , Simulação de Dinâmica Molecular , Ligantes , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Núcleo Celular/metabolismo , Sítios de Ligação , Ligação ProteicaRESUMO
Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/virologia , Proteínas do Envelope Viral/imunologia , Replicação Viral/efeitos dos fármacos , Alphavirus/imunologia , Infecções por Alphavirus/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Chlorocebus aethiops , Reações Cruzadas , Encefalomielite Equina Venezuelana/terapia , Glicoproteínas/imunologia , Imunoterapia , Camundongos , Ligação Proteica , Células Vero , Proteínas do Envelope Viral/metabolismo , Vírion/imunologia , Vírion/metabolismoRESUMO
Venezuelan equine encephalitis virus (VEEV), a New World alphavirus of the Togaviridae family of viruses causes periodic outbreaks of disease in humans and equines. Disease following VEEV infection manifests as a febrile illness with flu-like symptoms, which can progress to encephalitis and cause permanent neurological sequelae in a small number of cases. VEEV is classified as a category B select agent due to ease of aerosolization and high retention of infectivity in the aerosol form. Currently, there are no FDA-approved vaccines or therapeutics available to combat VEEV infection. VEEV infection in vivo is characterized by extensive systemic inflammation that can exacerbate infection by potentially increasing the susceptibility of off-site cells to infection and dissemination of the virus. Hence, a therapeutic targeting both the infection and associated inflammation represents an unmet need. We have previously demonstrated that host defense peptides (HDPs), short peptides that are key components of the innate immune response, exhibit antiviral activity against a multitude of viruses including VEEV. In this study, we designed synthetic peptides derived from indolicidin, a naturally occurring HDP, and tested their efficacy against VEEV. Two candidate synthetic peptides inhibited VEEV replication by approximately 1000-fold and decreased the expression of inflammatory mediators such as IL1α, IL1ß, IFNγ, and TNFα at both the gene and protein expression levels. Furthermore, an increase in expression levels of genes involved in chemotaxis of leukocytes and anti-inflammatory genes such as IL1RN was also observed. Overall, we conclude that our synthetic peptides inhibit VEEV replication and the inflammatory burden associated with VEEV infection.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Vírus da Encefalite Equina Venezuelana/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Cavalos , Humanos , Inflamação , Camundongos , Células VeroRESUMO
Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans1. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.
Assuntos
Vírus da Encefalite Equina Venezuelana/metabolismo , Receptores de LDL/metabolismo , Receptores Virais/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/prevenção & controle , Encefalomielite Equina Venezuelana/virologia , Feminino , Teste de Complementação Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores Virais/genética , Ligação Viral , Internalização do VírusRESUMO
Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKß inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKß and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKß kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKß can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.
Assuntos
Antivirais/farmacologia , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/metabolismo , Quinase I-kappa B/metabolismo , Proteínas não Estruturais Virais/metabolismo , Aedes , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana , Humanos , Mutação , NF-kappa B/metabolismo , Fosforilação , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
Protein phosphorylation plays an important role during the life cycle of many viruses. Venezuelan equine encephalitis virus (VEEV) capsid protein has recently been shown to be phosphorylated at four residues. Here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. Phosphorylation site prediction software suggests that Protein Kinase C (PKC) is responsible for phosphorylation of VEEV capsid. VEEV capsid co-immunoprecipitated with PKCδ, but not other PKC isoforms and siRNA knockdown of PKCδ caused a decrease in viral replication. Furthermore, knockdown of PKCδ by siRNA decreased capsid phosphorylation. A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. RNA:capsid binding was significantly increased in the mutant virus, confirming these results. Finally, VEEV CPD is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. Collectively our data support a model in which PKCδ mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis.
Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/enzimologia , Proteína Quinase C-delta/metabolismo , RNA Viral/metabolismo , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/virologia , Feminino , Cavalos , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Endogâmicos C3H , Fosforilação , Ligação Proteica , Proteína Quinase C-delta/genética , RNA Viral/genéticaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that is vectored by mosquitos and cycled in rodents. It can cause disease in equines and humans characterized by a febrile illness that may progress into encephalitis. Like the capsid protein of other viruses, VEEV capsid is an abundant structural protein that binds to the viral RNA and interacts with the membrane-bound glycoproteins. It also has protease activity, allowing cleavage of itself from the growing structural polypeptide during translation. However, VEEV capsid protein has additional nonstructural roles within the host cell functioning as the primary virulence factor for VEEV. VEEV capsid inhibits host transcription and blocks nuclear import in mammalian cells, at least partially due to its complexing with the host CRM1 and importin α/ß1 nuclear transport proteins. VEEV capsid also shuttles between the nucleus and cytoplasm and is susceptible to inhibitors of nuclear trafficking, making it a promising antiviral target. Herein, the role of VEEV capsid in viral replication and pathogenesis will be discussed including a comparison to proteins of other alphaviruses.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/patogenicidade , RNA Viral/metabolismo , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Vírus da Encefalite Equina do Leste , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/metabolismo , Vírus da Encefalite Equina do Oeste , Cavalos , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Virulência , Replicação Viral/genética , Proteína Exportina 1RESUMO
The mosquito-borne New World alphavirus, Venezuelan equine encephalitis virus (VEEV) is a Category B select agent with no approved vaccines or therapies to treat infected humans. Therefore it is imperative to identify novel targets that can be targeted for effective therapeutic intervention. We aimed to identify and validate interactions of VEEV nonstructural protein 3 (nsP3) with host proteins and determine the consequences of these interactions to viral multiplication. We used a HA tagged nsP3 infectious clone (rTC-83-nsP3-HA) to identify and validate two RNA helicases: DDX1 and DDX3 that interacted with VEEV-nsP3. In addition, DDX1 and DDX3 knockdown resulted in a decrease in infectious viral titers. Furthermore, we propose a functional model where the nsP3:DDX3 complex interacts with the host translational machinery and is essential in the viral life cycle. This study will lead to future investigations in understanding the importance of VEEV-nsP3 to viral multiplication and apply the information for the discovery of novel host targets as therapeutic options.
Assuntos
RNA Helicases DEAD-box/metabolismo , Vírus da Encefalite Equina Venezuelana/metabolismo , Interações Hospedeiro-Patógeno , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Vírus da Encefalite Equina Venezuelana/química , Técnicas de Silenciamento de Genes , Humanos , RNA Helicases/metabolismo , Células Vero , Carga Viral , Replicação ViralRESUMO
Venezuelan equine encephalitis (VEE) virus causes an acute central nervous system infection in human and animals. Melatonin (MLT), minocycline (MIN) and ascorbic acid (AA) have been shown to have antiviral activities in experimental infections; however, the mechanisms involved are poorly studied. Therefore, the aim of this study was to determine the effects of those compounds on the viral titers, NO production and lipid peroxidation in the brain of mice and neuroblastoma cultures infected by VEE virus. Infected mouse (10 LD50) were treated with MLT (500 µg/kg bw), MIN (50mg/kg bw) or AA (50mg/kg bw). Infected neuroblastoma cultures (MOI: 1); MLT: 0.5, 1, 5mM, MIN: 0.1, 0.2, 2 µM or AA: 25, 50, 75 µM. Brains were obtained at days 1, 3 and 5. In addition, survival rate of infected treated mice was also analyzed. Viral replication was determined by the plaque formation technique. NO and lipid peroxidation were measured by Griess׳ reaction and thiobarbituric acid assay respectively. Increased viral replication, NO production and lipid peroxidation were observed in both, infected brain and neuroblastoma cell cultures compared with uninfected controls. Those effects were diminished by the studied treatments. In addition, increased survival rate (50%) in treated infected animals compared with untreated infected mice (0%) was found. MLT, MIN and AA have an antiviral effect involving their anti-oxidant properties, and suggesting a potential use of these compounds for human VEE virus infection.
Assuntos
Antivirais/farmacologia , Ácido Ascórbico/farmacologia , Encefalomielite Equina Venezuelana/tratamento farmacológico , Melatonina/farmacologia , Minociclina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/mortalidade , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Camundongos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Taxa de Sobrevida , Resultado do Tratamento , Carga ViralRESUMO
Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/ß-fold.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Vírus da Encefalite Equina Venezuelana/metabolismo , Ressonância Magnética Nuclear Biomolecular , Espectroscopia de Prótons por Ressonância Magnética , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Isótopos de Nitrogênio , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The outbreaks of chikungunya (CHIKV) and venezuelan equine encephalitis (VEEV) viral infections in humans have emerged or re-emerged in various countries of "Africa and southeast Asia", and "central and south America", respectively. At present, no drug or vaccine is available for the treatment and therapy of both viral infections, but the non-structural protein, nsP3, is a potential target for the design of potent inhibitors that fit at the adenosine-binding site of its macro domain. Here, so as to understand the fundamental basis of the particular interactions between the ADP-ribose bound to the nsP3 amino acid residues at the binding site, molecular dynamics simulations were applied. The results show that these two nsP3 domains share a similar binding pattern for accommodating the ADP-ribose. The ADP-ribose phosphate unit showed the highest degree of stabilization through hydrogen bond interactions with the nsP3 V33 residue and the consequent amino acid residues 110-114. The adenine base of ADP-ribose was specifically recognized by the conserved nsP3 residue D10. Additionally, the ribose and the diphosphate units were found to play more important roles in the CHIKV nsP3-ADP-ribose complex, while the ter-ribose was more important in the VEEV complex. The slightly higher binding affinity of ADP-ribose toward the nsP3 macro domain of VEEV, as predicted by the simulation results, is in good agreement with previous experimental data. These simulation results provide useful information to further assist in drug design and development for these two important viruses.
Assuntos
Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Vírus Chikungunya/química , Vírus da Encefalite Equina Venezuelana/química , Vírus da Encefalite Equina Venezuelana/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Animais , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Termodinâmica , Proteínas não Estruturais Virais/genéticaRESUMO
Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alpha- and flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187-210, and MAB 4F6 interacts with the fragment of residues 263-278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187-210 and 263-278) as well as the central beta-folded region turning into the alpha-helical site including residues 200-216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.
Assuntos
Vírus da Encefalite Equina Venezuelana/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Receptores de Laminina/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Anticorpos Anti-Idiotípicos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Chlorocebus aethiops , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores de Laminina/química , Receptores de Laminina/genética , Receptores Virais/química , Receptores Virais/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) were used to model the initial phase of VEE-induced encephalitis in the mouse brain. VRP can target and infect cells as VEE, but VRP do not propagate beyond the first infected cell due to the absence of the structural genes. Direct intracranial inoculation of VRP into mice induced acute encephalitis with signs similar to the neuronal phase of wild-type VEE infection and other models of virus-induced encephalitis. Using the previously established VRP-mRNP tagging system, a new method to distinguish the host responses in infected cells from those in uninfected bystander cell populations, we detected a robust and rapid innate immune response in the central nervous system (CNS) by infected neurons and uninfected bystander cells. Moreover, this innate immune response in the CNS compromised blood-brain barrier integrity, created an inflammatory response, and directed an adaptive immune response characterized by proliferation and activation of microglia cells and infiltration of inflammatory monocytes, in addition to CD4(+) and CD8(+) T lymphocytes. Taken together, these data suggest that a naïve CNS has an intrinsic potential to induce an innate immune response that could be crucial to the outcome of the infection by determining the composition and dynamics of the adaptive immune response. Furthermore, these results establish a model for neurotropic virus infection to identify host and viral factors that contribute to invasion of the brain, the mechanism(s) whereby the adaptive immune response can clear the infection, and the role of the host innate response in these processes.
Assuntos
Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/crescimento & desenvolvimento , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/virologia , Vírion/crescimento & desenvolvimento , Animais , Proliferação de Células , Citocinas/metabolismo , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/patologia , Feminino , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microglia/metabolismo , Microglia/patologia , RNA Viral/genética , Vírion/genéticaRESUMO
BACKGROUND: The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. CONCLUSIONS/SIGNIFICANCE: We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.
Assuntos
Vírus da Encefalite Equina Venezuelana/metabolismo , Replicon , Animais , Encefalomielite Equina Venezuelana/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Genoma , Glicoproteínas/química , Sistema Imunitário , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The encephalitogenic New World alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses, constitute a continuing public health threat in the United States. They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that these viruses have developed the ability to interfere with cellular transcription and use it as a means of downregulating a cellular antiviral response. The results of the present study suggest that the N-terminal, approximately 35-amino-acid-long peptide of VEEV and EEEV capsid proteins plays the most critical role in the downregulation of cellular transcription and development of a cytopathic effect. The identified VEEV-specific peptide C(VEE)33-68 includes two domains with distinct functions: the alpha-helix domain, helix I, which is critically involved in supporting the balance between the presence of the protein in the cytoplasm and nucleus, and the downstream peptide, which might contain a functional nuclear localization signal(s). The integrity of both domains not only determines the intracellular distribution of the VEEV capsid but is also essential for direct capsid protein functioning in the inhibition of transcription. Our results suggest that the VEEV capsid protein interacts with the nuclear pore complex, and this interaction correlates with the protein's ability to cause transcriptional shutoff and, ultimately, cell death. The replacement of the N-terminal fragment of the VEEV capsid by its Sindbis virus-specific counterpart in the VEEV TC-83 genome does not affect virus replication in vitro but reduces cytopathogenicity and results in attenuation in vivo. These findings can be used in designing a new generation of live, attenuated, recombinant vaccines against the New World alphaviruses.
Assuntos
Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/farmacologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Proteínas/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Proteínas do Capsídeo/genética , Sobrevivência Celular , Cricetinae , Efeito Citopatogênico Viral , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/mortalidade , Encefalomielite Equina Venezuelana/patologia , Encefalomielite Equina Venezuelana/virologia , Feminino , Imunização , Camundongos , Mutação , Proteínas/genéticaRESUMO
Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-alpha1 but not with karyopherin-alpha2, -3, or -4, suggesting that karyopherin-alpha1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.
Assuntos
Núcleo Celular/metabolismo , Vírus da Encefalite Equina Venezuelana/metabolismo , Sinais de Localização Nuclear/genética , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Núcleo Celular/química , Citoplasma/química , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , alfa Carioferinas/metabolismoRESUMO
Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.
Assuntos
Encéfalo/virologia , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Recombinação Genética , Sindbis virus/genética , Vacinas Virais/administração & dosagem , Replicação Viral , Animais , Encéfalo/patologia , Cricetinae , Replicação do DNA , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/patologia , Encefalomielite Equina Venezuelana/virologia , Feminino , Humanos , Masculino , Mesocricetus , Camundongos , Sindbis virus/imunologia , Sindbis virus/metabolismo , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Proteínas Estruturais Virais/metabolismo , Vacinas Virais/genéticaRESUMO
Alphaviruses are regarded as attractive systems for expression of heterologous genes and development of recombinant vaccines. Venezuelan equine encephalitis virus (VEE)-based vectors are particularly promising because of their specificity to lymphoid tissues and strong resistance to interferon. To improve understanding of the VEE genome packaging and optimize application of this virus as a vector, we analyzed in more detail the mechanism of packaging of the VEE-specific RNAs. The presence of the RNAs in the VEE particles during serial passaging in tissue culture was found to depend not only on the presence of packaging signal(s), but also on the ability of these RNAs to express in cis nsP1, nsP2 and nsP3 in the form of a P123 precursor. Packaging of VEE genomes into infectious virions was also found to be more efficient compared to that of Sindbis virus, in spite of lower levels of RNA replication and structural protein production.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/metabolismo , Regulação Viral da Expressão Gênica , RNA Viral/metabolismo , Proteínas não Estruturais Virais/biossíntese , Montagem de Vírus/fisiologia , Animais , Linhagem Celular , Cricetinae , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Especificidade por SubstratoRESUMO
The purpose of this work was to determine the effect of melatonin on the nitric oxide levels in murine splenocytes cultured with the Venezuelan equine encephalomyelitis virus. After incubation, nitric oxide levels were measured by the diazotization assay. Those cultures with the Venezuelan equine encephalomyelitis virus increased nitric oxide levels. Splenocytes infected and treated with 100 and 150 microg/ml of melatonin, decreased significantly the nitric oxide levels when compared to infected and non-treated splenocytes. These findings show that splenocytes infected with the Venezuelan equine encephalomyelitis virus generate important amounts of nitric oxide and suggest that melatonin protects the mice infected with the Venezuelan equine encephalomyelitis virus by a mechanism involving the decreasing of nitric oxide concentrations in tissue.
Assuntos
Vírus da Encefalite Equina Venezuelana/metabolismo , Melatonina/metabolismo , Óxido Nítrico/metabolismo , Baço/citologia , Animais , Células Cultivadas , Masculino , Melatonina/administração & dosagem , Camundongos , Baço/virologiaRESUMO
Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.