RESUMEN
UNLABELLED: Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, for which no safe and efficient vaccines or therapeutic means have been developed. Viral particle assembly and budding processes represent potential targets for therapeutic intervention. However, our understanding of the mechanistic process of VEEV assembly, RNA encapsidation, and the roles of different capsid-specific domains in these events remain to be described. The results of this new study demonstrate that the very amino-terminal VEEV capsid-specific subdomain SD1 is a critical player in the particle assembly process. It functions in a virus-specific mode, and its deletion, mutation, or replacement by the same subdomain derived from other alphaviruses has strong negative effects on infectious virus release. VEEV variants with mutated SD1 accumulate adaptive mutations in both SD1 and SD2, which result in a more efficiently replicating phenotype. Moreover, efficient nucleocapsid and particle assembly proceeds only when the two subdomains, SD1 and SD2, are derived from the same alphavirus. These two subdomains together appear to form the central core of VEEV nucleocapsids, and their interaction is one of the driving forces of virion assembly and budding. The similar domain structures of alphavirus capsid proteins suggest that this new knowledge can be applied to other alphaviruses. IMPORTANCE: Alphaviruses are a group of human and animal pathogens which cause periodic outbreaks of highly debilitating diseases. Despite significant progress made in understanding the overall structure of alphavirus and VEEV virions, and glycoprotein spikes in particular, the mechanistic process of nucleocapsid assembly, RNA encapsidation, and the roles of different capsid-specific domains in these processes remain to be described. Our new data demonstrate that the very amino-terminal subdomain of Venezuelan equine encephalitis virus capsid protein, SD1, plays a critical role in the nucleocapsid assembly. It functions synergistically with the following SD2 (helix I) and appears to form a core in the center of nucleocapsid. The core formation is one of the driving forces of alphavirus particle assembly.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus de la Encefalitis Equina Venezolana/fisiología , Nucleocápside/metabolismo , Virión/metabolismo , Ensamble de Virus , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/genética , Línea Celular , Cricetinae , Análisis Mutacional de ADN , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/ultraestructura , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ensayo de Placa Viral , Virión/ultraestructuraRESUMEN
UNLABELLED: Alphaviruses represent a significant public health threat worldwide. They are transmitted by mosquitoes and cause a variety of human diseases ranging from severe meningoencephalitis to polyarthritis. To date, no efficient and safe vaccines have been developed against any alphavirus infection. However, in recent years, significant progress has been made in understanding the mechanism of alphavirus replication and virus-host interactions. These data have provided the possibility for the development of new rationally designed alphavirus vaccine candidates that combine efficient immunogenicity, high safety, and inability to revert to pathogenic phenotype. New attenuated variants of Venezuelan equine encephalitis virus (VEEV) designed in this study combine a variety of characteristics that independently contribute to a reduction in virulence. These constructs encode a noncytopathic VEEV capsid protein that is incapable of interfering with the innate immune response. The capsid-specific mutations strongly affect neurovirulence of the virus. In other constructs, they were combined with changes in control of capsid translation and an extensively mutated packaging signal. These modifications also affected the residual neurovirulence of the virus, but it remained immunogenic, and a single immunization protected mice against subsequent infection with epizootic VEEV. Similar approaches of attenuation can be applied to other encephalitogenic New World alphaviruses. IMPORTANCE: Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, which causes periodic outbreaks of highly debilitating disease. Despite a continuous public health threat, no safe and efficient vaccine candidates have been developed to date. In this study, we applied accumulated knowledge about the mechanism of VEEV replication, RNA packaging, and interaction with the host to design new VEEV vaccine candidates that demonstrate exceptionally high levels of safety due to a combination of extensive modifications in the viral genome. The introduced mutations did not affect RNA replication or structural protein synthesis but had deleterious effects on VEEV neuroinvasion and virulence. In spite of dramatically reduced virulence, the designed mutants remained highly immunogenic and protected mice against subsequent infection with epizootic VEEV. Similar methodologies can be applied for attenuation of other encephalitogenic New World alphaviruses.
Asunto(s)
Proteínas de la Cápside/genética , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/prevención & control , Mutación , Transcripción Genética , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Animales , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina Venezolana/genética , Femenino , Ratones , Fenotipo , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/inmunología , Vacunas Virales/efectos adversos , VirulenciaRESUMEN
Alphaviruses are one of the most geographically widespread and yet often neglected group of human and animal pathogens. They are capable of replicating in a wide variety of cells of both vertebrate and insect origin and are widely used for the expression of heterologous genetic information both in vivo and in vitro. In spite of their use in a range of research applications and their recognition as a public health threat, the biology of alphaviruses is insufficiently understood. In this study, we examined the evolution process of one of the alphaviruses, Venezuelan equine encephalitis virus (VEEV), to understand its adaptation mechanism to the inefficient packaging of the viral genome in response to serial mutations introduced into the capsid protein. The new data derived from this study suggest that strong alterations in the ability of capsid protein to package the viral genome leads to accumulation of adaptive mutations, not only in the capsid-specific helix I but also in the nonstructural protein nsP2. The nsP2-specific mutations were detected in the protease domain and in the amino terminus of the protein, which was previously proposed to function as a protease cofactor. These mutations increased infectious virus titers, demonstrated a strong positive impact on viral RNA replication, mediated the development of a more cytopathic phenotype, and made viruses capable of developing a spreading infection. The results suggest not only that packaging of the alphavirus genome is determined by the presence of packaging signals in the RNA and positively charged amino acids in the capsid protein but also that nsP2 is either directly or indirectly involved in the RNA encapsidation process.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/fisiología , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus , Adaptación Biológica , Animales , Línea Celular , Efecto Citopatogénico Viral , Análisis Mutacional de ADN , Mutación Missense , ARN Viral/metabolismoRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/patogenicidad , Vacunas de Partículas Similares a Virus/genética , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Cricetinae , Virus de la Encefalitis Equina Venezolana/inmunología , Virus de la Encefalitis Equina Venezolana/fisiología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Ensamble de Virus , Liberación del Virus , Replicación ViralRESUMEN
Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.