RESUMO
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.
Assuntos
Proteínas do Capsídeo/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/prevenção & controle , Mutação , Transcrição Gênica , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Animais , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Feminino , Camundongos , Fenótipo , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Vacinas Virais/efeitos adversos , VirulênciaRESUMO
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.
Assuntos
Vírus da Encefalite Equina Venezuelana/fisiologia , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus , Adaptação Biológica , Animais , Linhagem Celular , Efeito Citopatogênico Viral , Análise Mutacional de DNA , Mutação de Sentido Incorreto , RNA Viral/metabolismoRESUMO
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.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Cricetinae , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Montagem de Vírus , Liberação de Vírus , Replicação ViralRESUMO
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.
Assuntos
Proteínas do Capsídeo/genética , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/virologia , Doença Aguda , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas do Capsídeo/fisiologia , Células Cultivadas , Cricetinae , Efeito Citopatogênico Viral/genética , Efeito Citopatogênico Viral/fisiologia , DNA Viral/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Encefalomielite Equina Venezuelana/imunologia , Genes Virais , Doenças dos Cavalos/imunologia , Doenças dos Cavalos/virologia , Cavalos , Humanos , Interferon Tipo I/imunologia , Camundongos , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Homologia de Sequência de Aminoácidos , Transdução de Sinais/imunologia , Sindbis virus/genética , Sindbis virus/patogenicidade , Sindbis virus/fisiologia , Replicação ViralRESUMO
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.
Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/patogenicidade , Carioferinas/metabolismo , Poro Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteína Exportina 1RESUMO
Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. The pathogenesis of this virus depends strongly on the sequences of the structural proteins and on the mutations in the RNA promoter encoded by the 5' untranslated region (5'UTR) of the viral genome. In this study, we performed a detailed investigation of the structural and functional elements of the 5'-terminal promoter and analyzed the effect of multiple mutations introduced into the VEEV 5'UTR on virus and RNA replication. The results of this study demonstrate that RNA replication is determined by two synergistically functioning RNA elements. One of them is a very 5'-terminal AU dinucleotide, which is not involved in the stable RNA secondary structure, and the second is a short, G-C-rich RNA stem. An increase or decrease in the stem's stability has deleterious effects on virus and RNA replication. In response to mutations in these RNA elements, VEEV replicative machinery was capable of developing new, compensatory sequences in the 5'UTR either containing 5'-terminal AUG or AU repeats or leading to the formation of new, heterologous stem-loops. Analysis of the numerous compensatory mutations suggested that at least two different mechanisms are involved in their generation. Some of the modifications introduced into the 5' terminus of the viral genome led to an accumulation of the mutations in the VEEV nsPs, which suggested to us that there is a direct involvement of these proteins in promoter recognition. Furthermore, our data provide new evidence that the 3' terminus of the negative-strand viral genome in the double-stranded RNA replicative intermediate is represented by a single-stranded RNA. Both the overall folding and the sequence determine its efficient function as a promoter for VEEV positive-strand RNA genome synthesis.
Assuntos
Regiões 5' não Traduzidas , Vírus da Encefalite Equina Venezuelana/genética , Genoma Viral , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , Vírus da Encefalite Equina Venezuelana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Mutação Puntual , Ensaio de Placa Viral , Replicação ViralRESUMO
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.
Assuntos
Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Vírus da Encefalite Equina Venezuelana/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Cricetinae , Culicidae , Mutação da Fase de Leitura , Humanos , Mamíferos , Camundongos , Proteínas Mutantes/metabolismo , Proteínas de Transporte Nucleocitoplasmático/antagonistas & inibidores , Sindbis virus/fisiologiaRESUMO
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
Replication of alphaviruses strongly depends on the promoters located in the plus- and minus-strands of virus-specific RNAs. The most sophisticated promoter is encoded by the 5' end of the viral genome. This RNA sequence is involved in the initiation of translation of viral nsPs, and synthesis of both minus- and plus-strands of the viral genome. Part of the promoter, the 51-nt conserved sequence element (CSE), is located in the nsP1-coding sequence, and this limits the spectrum of possible mutations that can be performed. We designed a recombinant Venezuelan equine encephalitis virus genome, in which the promoter and nsP1-coding sequences are separated. This modification has allowed us to perform a wide variety of genetic manipulations, without affecting the amino acid sequence of the nsPs, and to further investigate 51-nt CSE functioning. The results of this study suggest a direct interaction of the amino terminal domain of nsP2 with the 5' end of the viral genome.