RESUMO
Venezuelan equine encephalitis virus (VEEV) is one of the important human and animal pathogens. It forms replication enzyme complexes (RCs) containing viral nonstructural proteins (nsPs) that mediate the synthesis of virus-specific RNAs. The assembly and associated functions of RC also depend on the presence of a specific set of host proteins. Our study demonstrates that the hypervariable domain (HVD) of VEEV nsP3 interacts with the members of the FXR family of cellular proteins and also binds the Src homology 3 (SH3) domain-containing proteins CD2AP and SH3KBP1. Interactions with FXR family members are mediated by the C-terminal repeating peptide of HVD. A single short, minimal motif identified in this study is sufficient for driving efficient VEEV replication in the absence of HVD interactions with other host proteins. The SH3 domain-containing proteins bind to another fragment of VEEV HVD. They can promote viral replication in the absence of FXR-HVD interactions albeit less efficiently. VEEV replication can be also switched from an FXR-dependent to a chikungunya virus-specific, G3BP-dependent mode. The described modifications of VEEV HVD have a strong impact on viral replication in vitro and pathogenesis. Their effects on viral pathogenesis depend on mouse age and the genetic background of the virus.IMPORTANCE The replication of alphaviruses is determined by specific sets of cellular proteins, which mediate the assembly of viral replication complexes. Some of these critical host factors interact with the hypervariable domain (HVD) of alphavirus nsP3. In this study, we have explored binding sites of host proteins, which are specific partners of nsP3 HVD of Venezuelan equine encephalitis virus. We also define the roles of these interactions in viral replication both in vitro and in vivo A mechanistic understanding of the binding of CD2AP, SH3KBP1, and FXR protein family members to VEEV HVD uncovers important aspects of alphavirus evolution and determines new targets for the development of alphavirus-specific drugs and directions for viral attenuation and vaccine development.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Mutação , Domínios e Motivos de Interação entre Proteínas , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Linhagem Celular , Vírus Chikungunya/metabolismo , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Encefalomielite Equina Venezuelana/virologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Alinhamento de Sequência , Proteínas não Estruturais Virais/química , Domínios de Homologia de srcRESUMO
Eastern, Venezuelan and western equine encephalitis viruses (EEEV, VEEV, and WEEV) are mosquito-borne viruses that cause substantial disease in humans and other vertebrates. Vaccines are limited and current treatment options have not proven successful. In this report, we vaccinated outbred mice with lipid-antigen-nucleic acid-complexes (LANACs) containing VEEV E1+WEEV E1 antigen and characterized protective efficacy against lethal EEEV, VEEV, and WEEV challenge. Vaccination resulted in complete protection against EEEV, VEEV, and WEEV in CD-1 mice. Measurements of bioluminescence and plaque reduction neutralization tests (PRNTs) indicate that LANAC VEEV E1+WEEV E1 vaccination is sterilizing against VEEV and WEEV challenge; whereas immunity to EEEV is not sterilizing. Passive transfer of rabbit VEEV E1+WEEV E1 immune serum to naive mice extended the mean time to death (MTD) of EEEV challenged mice and provided significant protection from lethal VEEV and WEEV challenge.
Assuntos
Alphavirus/imunologia , Antígenos Virais/imunologia , Reações Cruzadas/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina do Oeste/imunologia , Proteínas Virais/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/mortalidade , Infecções por Alphavirus/prevenção & controle , Infecções por Alphavirus/virologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Linhagem Celular , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Vírus da Encefalite Equina do Oeste/genética , Vírus da Encefalite Equina do Oeste/patogenicidade , Feminino , Expressão Gênica , Genes Reporter , Imunidade Humoral , Imunização , Lipossomos , Camundongos , Ácidos Nucleicos , Homologia de Sequência , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Virulência/genética , Replicação ViralRESUMO
UNLABELLED: Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. IMPORTANCE: VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.