21, 33, 34 In our study, coimmunoprecipitation with anti-STAT1 antibody followed by immunoblotting with HEV anti-ORF3 or ORF2 antibody, showed that

ORF3 protein, but not ORF2 protein, could bind to STAT1 in HEV-A549 cells. HEV ORF3 protein has the ability to optimize the Ceritinib solubility dmso cellular environment for viral infection and replication by interacting with multiple cellular proteins involved in signal transduction, such as mitogen-activated protein kinase (MAPK) phosphatase, CIN85, α-1-microglobulin, and bikunin precursor protein.7, 11, 35-37 In this study, our transfection experiments with HEV ORF3 showed that the STAT1 phosphorylation and IFN-α–stimulated genes PKR, 2′,5′-OAS, and MxA were inhibited in the IFN-α–treated A549 cells. It is thus reasonable to conclude that the binding of HEV ORF3 protein to STAT1 inhibits STAT1 phosphorylation and then suppresses the expression of IFN-α–stimulated mTOR inhibitor genes. Furthermore, we observed some differences in the inhibition

pattern of IFN-α–stimulated genes when HEV ORF3 alone was used compared with the whole virus infection of A549 cells. The expression of target gene MxA was inhibited in HEV ORF3-transfected cells but not in HEV-infected A549 cells and the increased levels of STAT1 were observed in HEV-infected A549 cells but not in HEV ORF3-transfected cells. Further studies are needed to determine more definitively the precise mechanism of IFN signaling inhibition in HEV infection. An intriguing finding was the increased levels of STAT1 during HEV infection. Such increased levels of STAT proteins during viral infection have recently been shown by other RNA viruses, such as human metapneumovirus (hMPV) and respiratory syncytial virus (RSV).38, 39 It is unclear what mechanisms caused these increased levels and what biological relevance, if any, the increased STAT levels may have in viral infections. One potential explanation

could be that expression of the STATs is up-regulated in response to HEV infection in an IFN-independent manner. Viruses have been shown to up-regulate ISGs in such a manner by activation of IRF3.40 A component of the HEV virion could be recognized by a pathogen-associated molecular click here pattern receptor, which then causes STAT protein levels to be increased without dependence on IFN, as previously demonstrated in hantavirus infection.41 Alternatively, the increased levels of STAT1 could be due to the reduction of normal degradation of STAT1. Because the STAT proteins have a relatively long biological half-life of 2 or 3 days,42 the increased levels shown here may be attributed to a gradual build-up of STAT1 during the course of our experiments. In conclusion, the data from our study show that IFN-α signal pathway plays an important role in HEV replication in host cells, and point to the role of type I IFN and STAT1 in protecting the host cells from HEV infection.