RESUMO
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 neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18â¯h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.
Assuntos
Morte Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Encefalomielite Equina Venezuelana/virologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , eIF-2 Quinase/metabolismo , Apoptose , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/virologia , Sobrevivência Celular , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/patologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Transdução de Sinais , Replicação Viral , eIF-2 Quinase/genéticaRESUMO
The New World alphaviruses -Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV respectively) - cause a febrile disease that is often lethal in equines and children and leads to long-term neurological sequelae in survivors. Endemic to the Americas, epizootic outbreaks of the three viruses occur sporadically in the continental United States. All three viruses aerosolize readily, replicate to high titers in cell culture, and have low infectious doses. Additionally, there are no FDA-approved vaccines or therapeutics for human use. To address the therapeutic gap, a high throughput assay utilizing a luciferase reporter virus, TC83-luc, was performed to screen a library of commercially available, FDA-approved drugs for antiviral activity. From a group of twenty compounds found to significantly decrease luminescence, the carcinoma therapeutic sorafenib inhibited replication of VEEV-TC83 and TrD in vitro. Additionally, sorafenib inhibited replication of EEEV and two Old World alphaviruses, Sindbis virus and chikungunya virus, at 8 and 16â¯h post-infection. Sorafenib caused no toxicity in Vero cells, and coupled with a low EC50 value, yielded a selectivity index of >19. Mechanism of actions studies suggest that sorafenib inhibited viral translation through dephosphorylation of several key proteins, including eIF4E and p70S6K, leading to a reduction in viral protein production and overall viral replication.
Assuntos
Alphavirus/efeitos dos fármacos , Antineoplásicos/farmacologia , Antivirais/farmacologia , Reposicionamento de Medicamentos , Sorafenibe/farmacologia , Replicação Viral/efeitos dos fármacos , Alphavirus/crescimento & desenvolvimento , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Ensaios de Triagem em Larga Escala , Luciferases/análise , Luciferases/genética , Medições Luminescentes , Genética ReversaRESUMO
Protein phosphatase 1 (PP1) is a serine/threonine phosphatase which has been implicated in the regulation of a number of viruses, including HIV-1, Ebolavirus, and Rift Valley fever virus. Catalytic subunits of PP1 (PP1α, PP1ß, and PP1γ) interact with a host of regulatory subunits and target a wide variety of cellular substrates through a combination of short binding motifs, including an RVxF motif present in the majority of PP1 regulatory subunits. Targeting the RVxF-interacting site on PP1 with the small molecule 1E7-03 inhibits HIV-1, Ebolavirus, and Rift Valley fever virus replication. In this study, we determined the effect of PP1 on Venezuelan equine encephalitis virus (VEEV) replication. Treatment of VEEV-infected cells with 1E7-03 decreased viral replication by more than 2 logs (50% effective concentration [EC50] = 0.6 µM). 1E7-03 treatment reduced viral titers starting at 8 h postinfection. Viral replication was also decreased after treatment with PP1α-targeting small interfering RNA (siRNA). Confocal microscopy demonstrated that PP1α shuttles toward the cytosol during infection with VEEV and that PP1α colocalizes with VEEV capsid. Coimmunoprecipitation experiments confirmed VEEV capsid interaction with PP1α. Furthermore, immunoprecipitation and mass spectrometry data showed that VEEV capsid is phosphorylated and that phosphorylation is moderated by PP1α. Finally, less viral RNA is associated with capsid after treatment with 1E7-03. Coupled with data showing that 1E7-03 inhibits several alphaviruses, this study indicates that inhibition of the PP1α RVxF binding pocket is a promising therapeutic target and provides novel evidence that PP1α modulation of VEEV capsid phosphorylation influences viral replication.IMPORTANCE Venezuelan equine encephalitis virus (VEEV) causes moderate flu-like symptoms and can lead to severe encephalitic disease and potentially death. There are currently no FDA-approved therapeutics or vaccines for human use, and understanding the molecular underpinning of host-virus interactions can aid in the rational design of intervention strategies. The significance of our research is in identifying the interaction between protein phosphatase 1 (PP1) and the viral capsid protein. This interaction is important for viral replication, as inhibition of PP1 results in decrease viral replication. Inhibition of PP1 also inhibited multiple biomedically important alphaviruses, indicating that PP1 may be a potential therapeutic target for alphavirus-induced disease.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/fisiologia , Proteína Fosfatase 1/metabolismo , Replicação Viral/fisiologia , Animais , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Fosforilação/genética , Proteína Fosfatase 1/genética , Células VeroRESUMO
Venezuelan equine encephalitis virus (VEEV) is a positive sense, single-stranded RNA virus and member of the New World alphaviruses. It causes a biphasic febrile illness that can be accompanied by central nervous system involvement and moderate morbidity in humans and severe mortality in equines. The virus has a history of weaponization, lacks FDA-approved therapeutics and vaccines in humans, and is considered a select agent. Like other RNA viruses, VEEV replicates in the cytoplasm of infected cells and eventually induces apoptosis. The capsid protein, which contains a nuclear localization and a nuclear export sequence, induces a shutdown of host transcription and nucleocytoplasmic trafficking. Here we show that infection with VEEV causes a dysregulation of cell cycling and a delay in the G0/G1 phase in Vero cells and U87MG astrocytes. Cells infected with VEEV encoding a capsid NLS mutant or treated with the capsid-importin α interaction inhibitor G281-1485 were partially rescued from this cell cycle dysregulation. Pathway analysis of previously published RNA-sequencing data from VEEV infected U87MG astrocytes identified alterations of canonical pathways involving cell cycle, checkpoint regulation, and proliferation. Multiple cyclins including cyclin D1, cyclin A2 and cyclin E2 and other regulators of the cell cycle were downregulated in infected cells in a capsid NLS dependent manner. Loss of Rb phosphorylation, which is a substrate for cyclin/cdk complexes was also observed. These data demonstrate the importance of capsid nuclear localization and/or importin α binding for inducing cell cycle arrest and transcriptional downregulation of key cell cycle regulators.
RESUMO
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.