| Influenza A virus (IAV) can infect diverse host species and remains severe threat to public health. Pathogenicity of IAV varies greatly among different virus subtypes due to its genetic variability. Moreover, IAV can translate multiple viral proteins from a single gene segment which may increase the complexity of its pathogenesis. In 1970s, the genome of the IAV was mapped for the first time and 10 viral proteins were indentified, namely PB2, PBI, PA, HA, NP, NA, M1, M2, NS1 and NS2. Later, in 2001, the first accessory protein, PB1-F2, which was translated from the alternative open reading frame (ORF), was discovered. Since then, another six viral proteins, PB1-N40, PA-X, M42,NS3, PA-N155 and PA-N182 have also been identified.The PA-X protein is a 29-kD frameshifting product that is encoded by the segment 3 of the IAV genome. Jagger et al. has shown that PA-X decreases the pathogenicity of the 1918 H1N1 virus in mice and inhibits the innate immune response, notably the inflammatory and apoptotic response. Desmet et al. found that the PA-X protein contributes to the host-cell shut off and inhibits host antiviral response. Khaperskyy et al. found that PA-X strongly inhibits the formation of the stress granule (SG) associated with the host antiviral mechanisms that detect the stress of the virus infection and arrest cap-dependent mRNA translation. Recently, Gao et al. clarified that PA-X can suppress the virulence of both 2009 H1N1 pandemic and H5N1 HPAI viruses in mice. Our previous study revealed that PA-X attenuates the pathogenicity of HPIAV H5N1 subtype virus in mice, ducks and chickens and probably decreases the viral virulence in avian species by inhibiting viral replication and host immune response.1. Role of PA-X in the pathogenicity of H5N1 HPAI virus in mice.To investigate the role of PA-X in the pathogenicity of HPAIV H5N1 virus in mammals, we used an H5N1 HPAI virus A/Chicken/Jiangsu/k0402/2010(CK10) as the parental virus, and generated two recombinant viruses CK-PAX3 and CK-PAX5 by reverse genetics in which PA-X expressions were reduced to different levels. By Western Blotting analysis, we confirmed that the PA-X expressions of the two mutants were authentically decreased when compared with that of the parental virus. The CK-PAX3 and CK-PAX5 showed 53% and 15% lower PA-X expression respectively than the r-CK10. Later in the animal experiments, we found that loss of PA-X expression enhanced the virulence of H5N1 AIV in mice, which was mainly manifested by body weight loss, survival rate and MLD50 value. The MLD50 values of CK-PAX3 and CK-PAX5 were 7.5-and 3.5-folds lower than that of r-CK10, respectively. These results futher verified the finding of Jagger et al. that PA-X may play the same roles in different subtypes of AIV.To further investigate the potential pathogenesis associated with PA-X protein, we systematically compared the histopathological changes of lungs in mice infected with parent or recombinant virus. On day 1 post infection, the pathological changes in CK-PAX3 or CK-PAX5 infected mice were significantly more severe than those in r-CK10 infected mice. The CK-PAX3-infected mice exhibited severe interstitial pneumonia and bronchopneumonia with a pathological score of 1.71 ± 0.49, whereas the pathological score for r-CK10-infected mouse lung was 1 ± 0 at this time point, mainly characterized by mild bronchopneumonia.2. Mechanism of virulence attenuation associated with PA-X in miceWe then explored the mechanism of virulence attenuation associated with this protein in a mouse model. Since in vivo study showed that the pathological changes of the mouse lungs induced by the two recombinant viruses were significantly different from those induced by the parent virus, we then verify whether they are different in inducing cell injury in vitro model by measuring the cell death in MDCK and A549 cells. By using the annexin V and PI double-staining, we found that loss of PA-X expression significantly enhanced cell death both in MDCK and A549 cells with increased apoptosis and necrosis. To further investigate the potential mechanism of the different histopathological changes caused by the recombinants, we then systematically compared the viral replication both in vivo and in vitro. In vitro, the CK.-PAX3 virus replicates to significantly higher viral titers in multiple mammalian cells (including A549, Vero and RAW cells) than the r-CK10 did. In vivo, overall, the PA-X deficient viruses replicated more efficiently in multiple organs than the parent virus. The PA-X deficient viruses were recovered in more mice and induced significantly higher viral titers when compared with r-CK10.To assess why reduced PA-X expression increased viral replication, we next investigate whether mutations at the sites affect PA nuclear accumulation and polymerase activity. We found that loss of PA-X expression enhances PA nuclear accumulation and RNP polymerase activity which may be associated with the increased viral replication observed in vivo and in vitro. To further investigate the potential mechanism of the difference in the histopathology of lungs caused by the mutant and parent visuses, we evaluated the role of PA-X on inflammatory response by testing the expression of some representative cytokine genes in mouse lung. In general, the mutants, especially the CK-PAX3 virus, induced a stronger cytokine response than r-CK10. Cytokines, such as CCL-3, CCL-4, CCL-5,CXCL-9, CXCL-10, IFN-a, IFN-β, IL-6, and TNF-a induced by CK-PAX3 at Id p.i. were significantly higher than those induced by the parent virus while the expression of CCL-4, CCL-5, CXCL-9, IFN-β, and IL-6 activated by CK-PAX5 were significantly higher than that by r-CK10 at this time point. These results provide a positive connection of the cytokine/chemokine response levels with the severity of lung pathology and viral pathogenicity.In summary, our study clearly demonstrated that PA-X suppresses the virulence of the H5N1 subtype HPAIV in mice. Reduced PA-X expression mediated higher viral replication, faster PA nuclear accumulation, accelerated cell death, and elevated inflammatory immune response contributes to the increased virulence of the H5N1 virus in the mouse model. Thereby, our study indicated that the novel PA-X protein is a virulence regulatory factor of influenza virus and may provide useful information for understanding the variable pathogenesis of IAV. |
