RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV), an acute infectious disease agent in swine, causes enormous economic losses to the global swine industry. PRRSV nonstructural protein 1ß (nsp1ß) plays a critical role in viral subgenomic mRNA synthesis and host immune regulation. However, the global changes of cellular gene expression in natural target cells regulated by the nsp1ß have not yet been identified. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry were used to quantitatively identify cellular proteins in porcine alveolar macrophage (PAM) 3D4/21 cells transduced with recombinant lentivirus expressing PRRSV nsp1ß that are differentially expressed compared with PAM 3D4/21 cells transduced with recombinant lentivirus expressing GFP. Of the 425 cellular proteins detected as differentially expressed, 186 were upregulated and 239 were downregulated. Based on the identities of the differentially expressed cellular proteins and the essential role of nsp1ß in interferon (IFN) activation and inflammatory factor antagonism during PRRSV infection, we propose a potential mechanism in which nsp1ß inhibits IFN induction and nuclear factor κB (NF-κB) signaling pathways. Our results suggest that mitochondrial antiviral signaling (MAVS) protein and translocases of outer membrane complex 70 (TOM70), involved in type I IFN induction, were downregulated, while protein phosphatase 1A (PPM1A), related to the inhibition of NF-κB pathway activation, was upregulated in nsp1ß-overexpressed PAM 3D4/21 cells. These data provide valuable information for better understanding the potential biological function of nsp1ß during PRRSV infection and the mechanism of virus escape from host immune surveillance of viral replication.
Assuntos
Macrófagos Alveolares/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteoma , Proteínas não Estruturais Virais/fisiologia , Animais , Western Blotting , Linhagem Celular , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Lentivirus/genética , Macrófagos Alveolares/virologia , Fosforilação , Suínos , Fator de Transcrição RelA/metabolismoRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which causes severe reproductive failure in sows, respiratory disease in young and growing pigs, and enormous economic losses to the global swine industry. In this study, SILAC combined with MS/MS was used to quantitatively identify the secretory proteins differentially expressed in PRRSV-infected Marc-145 cells compared with mock-infected controls. In total, we identified 204 secretory proteins showing significant differences in infected cells (163 upregulated, 41 downregulated). Intensive bioinformatic analysis of secretome data revealed that PRRSV infection strongly activated nonclassical protein secretion, especially vesicle-mediated release of exosomal proteins, including different danger-associated molecular pattern molecules and the majority of secreted proteins involved in protein binding and transport, regulation of response to stimulus, metabolic processes, and immune responses. According to the functional proteins analysis, we speculate that proteins functioning in binding, transport, and the immune response are exploited by PRRSV to facilitate virus replication and immune evasion. Our study for the first time analyzes the secretory protein profile of PRRSV-infected Marc-145 cells and provides valuable insight into the host response to PRRSV infection.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteoma/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Proteoma/análise , Proteômica , Suínos , Espectrometria de Massas em TandemRESUMO
Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection.