RESUMO
Rabies virus (RABV) is a neurotropic virus that causes fatal neurological disease, raising serious public health issues and attracting extensive attention in society. To elucidate the molecular mechanism of RABV-induced neuronal damage, we used hematoxylin-eosin staining, transmission electron microscopy, transcriptomics analysis, and immune response factor testing to investigate RABV-infected neurons. We successfully isolated the neurons from murine brains. The specificity of the isolated neurons was identified by a monoclonal antibody, and the viability of the neurons was 83.53-95.0%. We confirmed that RABV infection induced serious damage to the neurons according to histochemistry and transmission electron microscope (TEM) scanning. In addition, the transcriptomics analysis suggested that multiple genes related to the pyroptosis pathway were significantly upregulated, including gasdermin D (Gsdmd), Nlrp3, caspase-1, and IL-1ß, as well as the chemokine genes Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl12, and Cxcl10. We next verified this finding in the brains of mice infected with the rRC-HL, GX074, and challenge virus standard strain-24 (CVS-24) strains of RABV. Importantly, we found that the expression level of the Gsdmd protein was significantly upregulated in the neurons infected with different RABV strains and ranged from 691.1 to 5764.96 pg/mL, while the basal level of mock-infected neurons was less than 100 pg/mL. Taken together, our findings suggest that Gsdmd-induced pyroptosis is involved in the neuron damage caused by RABV infection.
Assuntos
Neurônios , Proteínas de Ligação a Fosfato , Piroptose , Vírus da Raiva , Raiva , Animais , Neurônios/virologia , Neurônios/metabolismo , Neurônios/patologia , Vírus da Raiva/patogenicidade , Vírus da Raiva/fisiologia , Raiva/virologia , Raiva/patologia , Raiva/metabolismo , Camundongos , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Encéfalo/virologia , Encéfalo/patologia , Encéfalo/metabolismo , GasderminasRESUMO
African swine fever virus (ASFV) infection causes African swine fever (ASF), a highly contagious and fatal disease that poses severe threat to swine production. To gain insights into the host responses to ASFV, we generated recombinant adenovirus Ad5 expressing viral membrane proteins p54, p17, and pB117L individually and infected an alveolar cell line, 3D4/21, with these recombinant viruses. Then, the cell lysates were analyzed using label-free quantification proteomic analysis method. A total of 2158 differentially expressed proteins (DEPs) were identified, of which 817, 466, and 875 proteins were from Ad5-p54-, Ad5-p17-, Ad5-pB117L-infected 3D4/21 cells, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed distinct yet interconnecting patterns of protein interaction networks. Specifically, the Ad5-p54 virus infection enriched the DEPs primarily involved in the metabolic pathways, endocytosis, adherens junction, and SNARE interactions in vesicular transport. The Ad5-p17 virus infection enriched the DEPs in endocytosis, ubiquitin-mediated proteolysis, N-Glycan biosynthesis, and apoptosis, while the Ad5-pB117L virus infection enriched the DEPs in metabolic pathways, endocytosis, oxidative phosphorylation, and focal adhesion. In summary, these results provide a comprehensive proteinomics analysis of the cellular responses to three ASFV membrane proteins, thus facilitating our understanding of ASFV pathogenesis.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Proteômica , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia , Vírus da Febre Suína Africana/metabolismo , Animais , Suínos , Proteômica/métodos , Linhagem Celular , Febre Suína Africana/virologia , Febre Suína Africana/metabolismo , Interações Hospedeiro-Patógeno , Mapas de Interação de Proteínas , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/genéticaRESUMO
Classical swine fever (CSF) is caused by the classical swine fever virus (CSFV), which poses a threat to swine production. The activation of host innate immunity through linker proteins such as tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) is crucial for the induction of the NF-κB pathway. Recent research has revealed the involvement of mitochondrial antiviral-signaling protein (MAVS) in the interaction with TRAF2, 3, 5, and 6 to activate both the NF-κB and IRF3 pathways. This study revealed that CSFV infection led to the upregulation of TRAF1 mRNA and protein levels; moreover, TRAF1 overexpression inhibited CSFV replication, while TRAF1 knockdown promoted replication, highlighting its importance in the host response to CSFV infection. Additionally, the expression of RIG-I, MAVS, TRAF1, IRF1, and ISG15 were detected in PK-15 cells infected with CSFV, revealing that TRAF1 plays a role in regulating IRF1 and ISG15 within the RIG-I pathway. Furthermore, Co-IP, GST pull-down, and IFA analyses demonstrated that TRAF1 interacted with MAVS and co-localized in the cytoplasm during CSFV infection. Ultimately, TRAF1 acted as a novel member of the TRAF family, bound to MAVS as a linker molecule, and functioned as a mediator downstream of MAVS in the RIG-I/MAVS pathway against CSFV replication.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Vírus da Febre Suína Clássica , Fator Regulador 1 de Interferon , Fator 1 Associado a Receptor de TNF , Regulação para Cima , Animais , Vírus da Febre Suína Clássica/fisiologia , Fator 1 Associado a Receptor de TNF/metabolismo , Fator 1 Associado a Receptor de TNF/genética , Suínos , Regulação para Cima/genética , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Transdução de Sinais , Peste Suína Clássica/virologia , Peste Suína Clássica/metabolismo , Peste Suína Clássica/genética , Replicação Viral , Linhagem Celular , Citocinas/metabolismo , Ligação ProteicaRESUMO
Nanoparticle vaccines displaying mosaic receptor-binding domains (RBDs) or spike (S) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or other sarbecoviruses are used in preparedness against potential zoonotic outbreaks. Here, we describe a self-assembling nanoparticle using lumazine synthase (LuS) as the scaffold to display RBDs from different sarbecoviruses. Mosaic nanoparticles induce sarbecovirus cross-neutralizing antibodies comparable to a nanoparticle cocktail. We find mosaic nanoparticles elicit a B cell receptor repertoire using an immunodominant germline gene pair of IGHV14-3:IGKV14-111. Most of the tested IGHV14-3:IGKV14-111 monoclonal antibodies (mAbs) are broadly cross-reactive to clade 1a, 1b, and 3 sarbecoviruses. Using mAb competition and cryo-electron microscopy, we determine that a representative IGHV14-3:IGKV14-111 mAb, M2-7, binds to a conserved epitope on the RBD, largely overlapping with the pan-sarbecovirus mAb S2H97. This suggests mosaic nanoparticles expand B cell recognition of the common epitopes shared by different clades of sarbecoviruses. These results provide immunological insights into the cross-reactive responses elicited by mosaic nanoparticles against sarbecoviruses.
Assuntos
Nanopartículas , Nanopartículas/química , Animais , Humanos , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Camundongos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Reações Cruzadas/imunologia , Formação de Anticorpos/imunologia , COVID-19/imunologia , COVID-19/virologia , Domínios Proteicos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/imunologia , Feminino , Epitopos Imunodominantes/imunologiaRESUMO
This study aimed to explore the mechanism of how African swine fever virus (ASFV) I226R protein inhibits the cGAS-STING signaling pathway. We observed that I226R protein (pI226R) significantly inhibited the cGAS-STING-mediated type â interferons and the interferon-stimulated genes production by dual-luciferase reporter assay system and real-time quantitative PCR. The results of co-immunoprecipitation assay and confocal microscopy showed that pI226R interacted with cGAS. Furthermore, pI226R promoted cGAS degradation through autophagy-lysosome pathway. Moreover, we found that pI226R decreased the binding of cGAS to E3 ligase tripartite motif protein 56 (TRIM56), resulting in the weakened monoubiquitination of cGAS, thus inhibiting the activation of cGAS and cGAS-STING signaling. In conclusion, ASFV pI226R suppresses the antiviral innate immune response by antagonizing cGAS, which contributes to an in-depth understanding of the immune escape mechanism of ASFV and provides a theoretical basis for the development of vaccines.
Assuntos
Vírus da Febre Suína Africana , Animais , Suínos , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Imunidade Inata , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Transdução de Sinais/genéticaRESUMO
In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.