Effect Of PA-X Protein Of H1N1 Influenza Virus On Caspase-1 Mediated Inflammatory Pathway

Influenza A virus(IAV)is an important human-animal virus.after IAV invades the host,the organism develops a systemic or local inflammatory response by synthesizing and secreting inflammatory and chemokines to suppress and clear the virus from the organism.Caspase-1-mediated inflammatory responses were found to play an important role in the host’s resistance to influenza virus infection and clearance of infected cells.PA-X protein suppresses the host’s natural immune response during viral infection.the PA-X protein is of inconsistent length in avian and mammalian influenza and is suspected to be involved in the immune adaptation process of the virus to a new host during interspecies transmission.To investigate the effect of PA-X protein on Caspase-1-mediated inflammatory pathway in influenza virus infection,two recombinant viral strains expressing different lengths of PA-X protein(pdm09＿PAX＿41,truncated and pdm09＿PAX＿61,full-length)were successfully rescued by reverse genetic techniques and subjected to in vitro mononuclear macrophage(RAW264.7)infection assay and Caspase-1 knockout mice infection assay.1.After infection with RAW264.7 cells,it was detected using fluorescent quantitative PCR that at 12 h,24 h,36 h,and 48 h after viral infection,both the pdm09＿PAX＿61 strain and the pdm09＿PAX＿41 strain induced the inflammatory factors IL-1β,IL-18,and TNF-α,and the NLRP3 pathway-related NLRP3,ASC and Caspase-1,but pdm09＿PAX＿61 caused more significant upregulation of mRNA transcript levels than pdm09＿PAX＿41,indicating that in monocytes,pdm09＿PAX＿61strain was able to induce more transcript production of inflammatory factor genes than pdm09＿PAX＿41 strain,resulting in a more severe inflammatory response.2.18 h after virus infection of RAW264.7 cells,extracellular IL-1β secretion was significantly increased by ELISA,and IL-1β secretion in the pdm09＿PAX＿61 group was significantly higher than that in the pdm09＿PAX＿41 group;the results of protein immunoblotting experiments showed that the pro-Caspase-1 expression level was lower than that of the pdm09＿PAX＿41 group,while pro-IL-1β and IL-1β expression levels were higher than that of the pdm09＿PAX＿41 group,indicating that the pdm09＿PAX＿61 strain promoted more NLRP3 inflammatory vesicles synthesized by pro-Caspase-1 protein than the pdm09＿PAX＿41 strain,further promoting more pro-IL-1β protein synthesis and IL-1β protein maturation and secretion.In contrast,the addition of Caspase-1 inhibitor could effectively inhibit Caspase-1 production and IL-1β maturation and secretion.3.Compared with infected wild-type mice,both recombinant strains caused more severe clinical signs and exhibited higher mortality in Caspase-1 knockout mice,with faster and earlier weight loss,slower and longer weight recovery time,and caused more severe lung tissue damage.In contrast,the pdm09＿PAX＿61 strain caused more severe damage in mice than the pdm09＿PAX＿41 strain,showing the same pattern as infecting wild-type mice,indicating that the pdm09＿PAX＿61 strain was more pathogenic to mice than the pdm09＿PAX＿41 strain and that the virus remained in mice for a longer period of time.4.The assay of relevant proteins and inflammatory factors in lung tissues showed that the transcript levels of Caspase-1 and IL-1β mRNA in the pdm09＿PAX＿61 group were lower than those in the pdm09＿PAX＿41 group after viral infection of wild-type mice,while the synthetic Caspase-1 protein and IL-1β protein were significantly higher than those in the pdm09＿PAX＿41 group.suggesting that the pdm09＿PAX＿61 strain was more able to promote Caspase-1 and IL-1β translation and processing in the lung than the pdm09＿PAX＿41 strain,forming mature proteins that were secreted into the lung to participate in the inflammatory response,whereas only a small amount of IL-1β protein was detected in the knockout Caspase-1 gene mice;Caspase-1-mediated inflammatory pathway The trends of changes such as related pro-Caspase-1 protein and pro-IL-1β protein were consistent with the findings of cellular experiments.The present study demonstrated that the expression of PA-X protein of different lengths by pdm09 H1N1 subtype of swine influenza virus affected the expression of related proteins and cytokines in the caspase-1-mediated inflammatory pathway to different degrees.By gaining insight into the specific regulatory role of PA-X protein on the host antiviral inflammatory response,we can help us better understand the strategies of influenza viruses to evade host immune responses and adapt to new hosts,and provide a new basis for the treatment of influenza virus inflammatory storms.