RESUMO
The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely alpha-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and approximately 7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative alpha-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.
Assuntos
RNA Viral/metabolismo , Vírus Sincicial Respiratório Humano/química , Proteínas Virais/química , Proteínas Estruturais Virais/metabolismo , Sítios de Ligação , Dicroísmo Circular , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
Human and bovine respiratory syncytial viruses (HRSV and BRSV) are two closely related, worldwide prevalent viruses that are the leading cause of severe airway disease in children and calves, respectively. Efficacy of commercial bovine vaccines needs improvement and no human vaccine is licensed yet. We reported that nasal vaccination with the HRSV nucleoprotein produced as recombinant ring-shaped nanoparticles (N(SRS)) protects mice against a viral challenge with HRSV. The aim of this work was to evaluate this new vaccine that uses a conserved viral antigen, in calves, natural hosts for BRSV. Calves, free of colostral or natural anti-BRSV antibodies, were vaccinated with N(SRS) either intramuscularly, or both intramuscularly and intranasally using Montanide ISA71 and IMS4132 as adjuvants and challenged with BRSV. All vaccinated calves developed anti-N antibodies in blood and nasal secretions and N-specific cellular immunity in local lymph nodes. Clinical monitoring post-challenge demonstrated moderate respiratory pathology with local lung tissue consolidations for the non-vaccinated calves that were significantly reduced in the vaccinated calves. Vaccinated calves had lower viral loads than the non-vaccinated control calves. Thus N(SRS) vaccination in calves provided cross-protective immunity against BRSV infection without adverse inflammatory reaction.
Assuntos
Doenças dos Bovinos/prevenção & controle , Nucleoproteínas/imunologia , Infecções por Vírus Respiratório Sincicial/veterinária , Vacinas contra Vírus Sincicial Respiratório/imunologia , Proteínas Virais/imunologia , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Bovinos , Doenças dos Bovinos/imunologia , Proteção Cruzada , Imunidade Celular , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Dados de Sequência Molecular , Nanopartículas , Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Bovino/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Carga ViralRESUMO
The respiratory syncytial virus (RSV) is an important human pathogen, yet neither a vaccine nor effective therapies are available to treat infection. To help elucidate the replication mechanism of this RNA virus, we determined the three-dimensional (3D) crystal structure at 3.3 A resolution of a decameric, annular ribonucleoprotein complex of the RSV nucleoprotein (N) bound to RNA. This complex mimics one turn of the viral helical nucleocapsid complex, which serves as template for viral RNA synthesis. The RNA wraps around the protein ring, with seven nucleotides contacting each N subunit, alternating rows of four and three stacked bases that are exposed and buried within a protein groove, respectively. Combined with electron microscopy data, this structure provides a detailed model for the RSV nucleocapsid, in which the bases are accessible for readout by the viral polymerase. Furthermore, the nucleoprotein structure highlights possible key sites for drug targeting.
Assuntos
Proteínas do Nucleocapsídeo/química , RNA Viral/química , Vírus Sinciciais Respiratórios/química , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Viral/metabolismo , Vírus Sinciciais Respiratórios/metabolismoRESUMO
BACKGROUND: Bronchiolitis caused by the respiratory syncytial virus (RSV) in infants less than two years old is a growing public health concern worldwide, and there is currently no safe and effective vaccine. A major component of RSV nucleocapsid, the nucleoprotein (N), has been so far poorly explored as a potential vaccine antigen, even though it is a target of protective anti-viral T cell responses and is remarkably conserved between human RSV A and B serotypes. We recently reported a method to produce recombinant N assembling in homogenous rings composed of 10-11 N subunits enclosing a bacterial RNA. These nanoparticles were named sub-nucleocapsid ring structure (N SRS). METHODOLOGY AND PRINCIPAL FINDINGS: The vaccine potential of N SRS was evaluated in a well-characterized and widely acknowledged mouse model of RSV infection. BALB/c adult mice were immunized intranasally with N SRS adjuvanted with the detoxified E. coli enterotoxin LT(R192G). Upon RSV challenge, vaccinated mice were largely protected against virus replication in the lungs, with a mild inflammatory lymphocytic and neutrophilic reaction in their airways. Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8(+) T cells and IFN-gamma-producing CD4(+) T cells. CONCLUSIONS/SIGNIFICANCE: This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV.
Assuntos
Nanopartículas , Vírus Sinciciais Respiratórios/imunologia , Vacinas Virais/administração & dosagem , Administração Intranasal , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia Eletrônica , Mutagênese Sítio-Dirigida , Vacinas Virais/imunologiaRESUMO
The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in Escherichia coli. Pull-down assays performed with a series of truncated forms of P fused to glutathione S-transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100-160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161-241) fused to GST pulled down exclusively RNA-N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22,900 and 24,820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5' and 3' ends.
Assuntos
Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/fisiologia , Vírus Sinciciais Respiratórios/química , Ribonucleoproteínas/química , Substituição de Aminoácidos , Espectrometria de Massas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/química , RNA Bacteriano/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/metabolismoRESUMO
Bovine respiratory syncytial virus (BRSV) is able to counteract the alpha/beta interferon (IFN-alpha/beta)-mediated antiviral response for efficient replication in a host-specific manner. Mice models have been developed for experimental infection with human, but not bovine, respiratory syncytial virus strains. Here, it is shown that BRSV can replicate efficiently on primary cell cultures derived from type I IFN receptor-deficient, but not from wild-type IFN-competent, mice. However, BRSV infection was not enhanced in mice devoid of the type I IFN receptor. These results show that type I IFN is a major host-range determinant for infection at the cellular level, but that other factors control virus replication and pathology in vivo.
Assuntos
Proteínas de Membrana/fisiologia , Receptores de Interferon/fisiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Bovino/fisiologia , Replicação Viral , Animais , Células Cultivadas , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Receptor de Interferon alfa e beta , Receptores de Interferon/genéticaRESUMO
The RNA-dependent RNA polymerase complex of respiratory syncytial virus (RSV) is composed of the large polymerase (L), the phosphoprotein (P), the nucleocapsid protein (N) and the co-factors M2-1 and M2-2. The P protein plays a central role within the replicase-transcriptase machinery, forming homo-oligomers and complexes with N and L. In order to study P-P and N-P complexes, and the role of P phosphorylation in these interactions, the human RSV P and N proteins were expressed in E. coli as His-tagged or GST-fusion proteins. The non-phosphorylated status of recombinant P protein was established by mass spectrometry. GST-P and GST-N fusion proteins were able to interact with RSV proteins extracted from infected cells in a GST pull-down assay. When co-expressed in bacteria, GST-P and His-P were co-purified by glutathione-Sepharose affinity, showing that the RSV P protein can form oligomers within bacteria. This result was confirmed by chemical cross-linking experiments and gel filtration studies. The P oligomerization domain was investigated by a GST pull-down assay using a series of P deletion constructs. This domain was mapped to a small region situated in the central part of P (aa 120-150), which localized in a computer-predicted coiled-coil domain. When co-expressed in bacteria, RSV N and P proteins formed a soluble complex that prevented non-specific binding of N to bacterial RNA. Therefore, RSV P protein phosphorylation is not required for the formation of P-P and N-P complexes, and P controls the RNA binding activity of N.