PB1-F2 protein of highly pathogenic avian influenza A (H7N9) virus suppresses MAVS-mediated antiviral responses through enhancing MAVS degradation

Abstract

Influenza A viruses (IAVs) are a major pathogen causing human respiratory disease. Historically, IAV has impacted human population with great disease burden that seasonal IAV epidemic, highly pathogenic IAV epidemic and even global IAV pandemic happened through years to kill life. Understanding the underlying mechanism of IAV pathogenesis provides clues to cure the disease. Avian influenza A viruses (AIV) are enzootic to avian species. Human infection by AIV was shown to be highly pathogenic. The best representatives were the H5N1 virus that caused over 60% mortality and the newly emerged H7N9 virus that caused about 40% mortality. Severe complications including pneumonia, acute respiratory distress syndrome (ARDS) and multi-organ failure were found in H5N1 and H7N9 virus infected patients. Recently, it has been demonstrated that the PB1-F2 protein expressed from the second open reading frame of the PB1 segment determined IAV pathogenicity by suppressing early MAVS-mediated type I interferon (IFN) response. It is therefore possible that the PB1-F2 proteins of H5N1 and H7N9 viruses can suppress type I IFN response to mediate the pathogenesis of H5N1 and H7N9 viruses. In the current study, the H7N9 PB1-F2 was characterized. Indeed, the H7N9 PB1-F2 showed potent suppressive effect against MAVS-mediated type I IFN response. The H7N9 PB1-F2 was also shown to suppress MAVS-mediated NLRP3 inflammasome maturation to produce interleukin 1β (IL-1β). When recombinant influenza A virus carrying H7N9 PB1-F2 was used to infect the human monocyte-derived macrophages (hMDMs) and the human lung fibroblast (HFL) or A549 cells, type I IFN and IL-1β/IL-18 responses were found to be suppressed only in hMDMs but not in HFL nor A549 cells. The cell type specific suppressive effect against MAVS-mediated antiviral responses (the combined responses of type I IFN and IL-1β/IL-18) were correlated to enhanced MAVS degradation in hMDMs. Indeed, the H7N9 PB1-F2 protein expression enhanced MAVS degradation when MAVS was activated by Sendai virus infection or MAVS overexpression. The MAVS aggregate was targeted by H7N9 PB1-F2 for degradation as shown by Semi-denaturing agarose gel electrophoresis (SDD-AGE). The MAVS signalosome with TBK-1 or NLRP3 was thus broken down as demonstrated by co-immunoprecipitation. Ubiquitination assay showed that both WSN PB1-F2 and H7N9 PB1-F2 enhanced the polyubiquitination of MAVS while only the latter signaled MAVS for degradation. Finally, the H7N9 PB1-F2 mediated MAVS degradation was shown to be inhibited when the proteasome inhibitor MG132 and the autophagic inhibitor Bafilomycin A1 were applied together, suggesting that the degradation effect was non-selective to proteasome or autophagic degradation pathways of MAVS protein. Taken together, the H7N9 PB1-F2 was reported to be able to suppress MAVS-mediated antiviral responses in human macrophages by enhancing MAVS protein degradation.