RESUMEN
In recent years, intrinsically disordered proteins (IDPs) and disordered domains have attracted great attention. Many of them contain linear motifs that mediate interactions with other factors during formation of multicomponent protein complexes. NMR spectrometry is a valuable tool for characterizing this type of interactions on both amino acid (aa) and atomic levels. Alphaviruses encode a nonstructural protein nsP3, which drives viral replication complex assembly. nsP3 proteins contain over 200-aa-long hypervariable domains (HVDs), which exhibits no homology between different alphavirus species, are predicted to be intrinsically disordered and appear to be critical for alphavirus adaptation to different cells. Previously, we have shown that nsP3 HVD of chikungunya virus (CHIKV) is completely disordered with low tendency to form secondary structures in free form. In this new study, we used novel NMR approaches to assign the spectra for the nsP3 HVD of Venezuelan equine encephalitis virus (VEEV). The HVDs of CHIKV and VEEV have no homology but are both involved in replication complex assembly and function. We have found that VEEV nsP3 HVD is also mostly disordered but contains a short stable α-helix in its C-terminal fragment, which mediates interaction with the members of cellular Fragile X syndrome protein family. Our NMR data also suggest that VEEV HVD has several regions with tendency to form secondary structures.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/enzimología , Espectroscopía de Resonancia Magnética , Dominios y Motivos de Interacción de Proteínas , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Animales , Fraccionamiento Químico , Proteínas Intrínsecamente Desordenadas/química , Unión Proteica , Solubilidad , Relación Estructura-Actividad , Proteínas no Estructurales Virales/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteínas no Estructurales Virales/genética , Replicación Viral/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Sitios de Unión , Línea Celular , Virus Chikungunya/metabolismo , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Encefalomielitis Equina Venezolana/virología , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Ratones , Alineación de Secuencia , Proteínas no Estructurales Virales/química , Dominios Homologos srcRESUMEN
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
Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. This genus is divided into the Old World and New World alphaviruses, which demonstrate profound differences in pathogenesis, replication, and virus-host interactions. VEEV is a representative member of the New World alphaviruses. The biology of this virus is still insufficiently understood, particularly the function of its nonstructural proteins in RNA replication and modification of the intracellular environment. One of these nonstructural proteins, nsP3, contains a hypervariable domain (HVD), which demonstrates very low overall similarity between different alphaviruses, suggesting the possibility of its function in virus adaptation to different hosts and vectors. The results of our study demonstrate the following. (i) Phosphorylation of the VEEV nsP3-specific HVD does not play a critical role in virus replication in cells of vertebrate origin but is important for virus replication in mosquito cells. (ii) The VEEV HVD is not required for viral RNA replication in the highly permissive BHK-21 cell line. In fact, it can be either completely deleted or replaced by a heterologous protein sequence. These variants require only one or two additional adaptive mutations in nsP3 and/or nsP2 proteins to achieve an efficiently replicating phenotype. (iii) However, the carboxy-terminal repeat in the VEEV HVD is indispensable for VEEV replication in the cell lines other than BHK-21 and plays a critical role in formation of VEEV-specific cytoplasmic protein complexes. Natural VEEV variants retain at least one of the repeated elements in their nsP3 HVDs.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/fisiología , Variación Genética , Proteínas no Estructurales Virales/genética , Replicación Viral/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Cricetinae , Culicidae , Electroporación , Técnica del Anticuerpo Fluorescente , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Células 3T3 NIH , Fosforilación , Plásmidos/genética , Estructura Terciaria de Proteína/genética , Especificidad de la Especie , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
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
Venezuelan equine encephalitis virus (VEEV) is a significant human and animal pathogen. The highlight of VEEV replication in vitro, in cells of vertebrate origin, is the rapid development of cytopathic effect (CPE), which is strongly dependent upon the expression of viral capsid protein. Besides being an integral part of virions, the latter protein is capable of (i) binding both the nuclear import and nuclear export receptors, (ii) accumulating in the nuclear pore complexes, (iii) inhibiting nucleocytoplasmic trafficking, and (iv) inhibiting transcription of cellular ribosomal and messenger RNAs. Using our knowledge of the mechanism of VEEV capsid protein function in these processes, we designed VEEV variants containing combinations of mutations in the capsid-coding sequences. These mutations made VEEV dramatically less cytopathic but had no effect on infectious virus production. In cell lines that have defects in type I interferon (IFN) signaling, the capsid mutants demonstrated very efficient persistent replication. In other cells, which have no defects in IFN production or signaling, the same mutants were capable of inducing a long-term antiviral state, downregulating virus replication to an almost undetectable level. However, ultimately, these cells also developed a persistent infection, characterized by continuous virus replication and beta IFN (IFN-beta) release. The results of this study demonstrate that the long-term cellular antiviral state is determined by the synergistic effects of type I IFN signaling and the antiviral reaction induced by replicating viral RNA and/or the expression of VEEV-specific proteins. The designed mutants represent an important model for studying the mechanisms of cell interference with VEEV replication and development of persistent infection.
Asunto(s)
Proteínas de la Cápside/genética , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/virología , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de la Cápside/fisiología , Células Cultivadas , Cricetinae , Efecto Citopatogénico Viral/genética , Efecto Citopatogénico Viral/fisiología , ADN Viral/genética , Virus de la Encefalitis Equina Venezolana/inmunología , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/inmunología , Genes Virales , Enfermedades de los Caballos/inmunología , Enfermedades de los Caballos/virología , Caballos , Humanos , Interferón Tipo I/inmunología , Ratones , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Homología de Secuencia de Aminoácido , Transducción de Señal/inmunología , Virus Sindbis/genética , Virus Sindbis/patogenicidad , Virus Sindbis/fisiología , Replicación ViralRESUMEN
Development of the cellular antiviral response requires nuclear translocation of multiple transcription factors and activation of a wide variety of cellular genes. To counteract the antiviral response, several viruses have developed an efficient means of inhibiting nucleocytoplasmic traffic. In this study, we demonstrate that the pathogenic strain of Venezuelan equine encephalitis virus (VEEV) has developed a unique mechanism of nuclear import inhibition. Its capsid protein forms a tetrameric complex with the nuclear export receptor CRM1 and the nuclear import receptor importin alpha/beta. This unusual complex accumulates in the center channel of the nuclear pores and blocks nuclear import mediated by different karyopherins. The inhibitory function of VEEV capsid protein is determined by a short 39-amino-acid-long peptide that contains both nuclear import and supraphysiological nuclear export signals. Mutations in these signals or in the linker peptide attenuate or completely abolish capsid-specific inhibition of nuclear traffic. The less pathogenic VEEV strains contain a wide variety of mutations in this peptide that affect its inhibitory function in nuclear import. Thus, these mutations appear to be the determinants of this attenuated phenotype. This novel mechanism of inhibiting nuclear transport also shows that the nuclear pore complex is vulnerable to unusual cargo receptor complexes and sheds light on the importance of finely adjusted karyopherin-nucleoporin interactions for efficient cargo translocation.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus de la Encefalitis Equina Venezolana/patogenicidad , Carioferinas/metabolismo , Poro Nuclear/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Datos de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteína Exportina 1RESUMEN
The development of infectious cDNA for different alphaviruses opened an opportunity to explore their attenuation by extensively modifying the viral genomes, an approach that might minimize or exclude the reversion to the wild-type, pathogenic phenotype. Moreover, the genomes of such alphaviruses can be engineered to contain RNA elements that would be functional only in cells of vertebrate, but not insect, origin. In the present study, we developed a recombinant VEEV that is more attenuated than TC-83 and capable of replicating only in vertebrate cells. This phenotype was achieved by rendering the translation of the viral structural proteins, and ultimately viral replication, dependent on the internal ribosome entry site of encephalomyocarditis virus (EMCV IRES). This recombinant virus was viable, but required additional, adaptive mutations in nsP2 that strongly increased its replication rates. In spite of efficient replication in cultured vertebrate cells, the genetically modified VEEV demonstrated a highly attenuated phenotype in newborn mice, and yet induced protective immunity against VEEV infection.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/fisiología , Adaptación Fisiológica , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cricetinae , Culicidae , Virus de la Encefalitis Equina Venezolana/patogenicidad , Ratones , Mutación , Células 3T3 NIH , Fenotipo , ARN Viral/genética , Recombinación Genética , Proteínas no Estructurales Virales/genética , Virulencia/genética , Replicación Viral/genéticaRESUMEN
Venezuelan equine encephalitis virus (VEEV) represents a continuous public health threat in the United States. It has the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that replicating VEEV interferes with cellular transcription and uses this phenomenon as a means of downregulating a cellular antiviral response. VEEV capsid protein was found to play a critical role in this process, and its approximately 35-amino-acid-long peptide, fused with green fluorescent protein, functioned as efficiently as did the entire capsid. We detected a significant fraction of VEEV capsid associated with nuclear envelope, which suggested that this protein might regulate nucleocytoplasmic trafficking. In this study, we demonstrate that VEEV capsid and its N-terminal sequence efficiently inhibit multiple receptor-mediated nuclear import pathways but have no effect on the passive diffusion of small proteins. The capsid protein of the Old World alphavirus Sindbis virus and the VEEV capsid, with a previously defined frameshift mutation, were found to have no detectable effect on nuclear import. Importantly, the VEEV capsid did not noticeably interfere with nuclear import in mosquito cells, and this might play a critical role in the ability of the virus to develop a persistent, life-long infection in mosquito vectors. These findings demonstrate a new aspect of VEEV-host cell interactions, and the results of this study are likely applicable to other New World alphaviruses, such as eastern and western equine encephalitis viruses.
Asunto(s)
Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , Virus de la Encefalitis Equina Venezolana/fisiología , Transporte Activo de Núcleo Celular , Animales , Proteínas de la Cápside/genética , Línea Celular , Cricetinae , Culicidae , Mutación del Sistema de Lectura , Humanos , Mamíferos , Ratones , Proteínas Mutantes/metabolismo , Proteínas de Transporte Nucleocitoplasmático/antagonistas & inhibidores , Virus Sindbis/fisiologíaRESUMEN
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.