Potential conflict of interest: Nothing to report.
The interferon (IFN) system is integral to the host response against viruses, and many viruses have developed strategies to overcome its antiviral effects. The effects of hepatitis E virus (HEV), the causative agent of hepatitis E, on IFN signaling have not been investigated primarily because of the nonavailability of an efficient in vitro culture system or small animal models of infection. We report here the generation of A549 cell lines persistently infected with genotype 3 HEV, designated as HEV-A549 cells and the effects HEV has on IFN-α–mediated Janus kinase–signal transducer and activator of transcription (JAK–STAT) signaling. Treatment of HEV-A549 cells with 250, 500, and 1000 U/mL of IFN-α for 72 hours showed a dose-dependent reduction in HEV RNA levels by 10%, 20%, and 50%, respectively. IFN-α–stimulated genes coding for the antiviral proteins dsRNA-activated protein kinase (PKR) and 2′,5′-oligoadenylate synthetase (2′,5′-OAS) were down-regulated in IFN-α–treated HEV-A549 cells. HEV infection also prevented IFN-α–induced phosphorylation of STAT1. Regulation of STAT1 by HEV was specific, as phosphorylation of STAT2, tyrosine kinase (Tyk) 2, and Jak1 by IFN-α was unaltered. Additionally, STAT1 levels were markedly increased in HEV-A549 cells compared with naive A549 cells. Furthermore, binding of HEV open reading frame (ORF)3 protein to STAT1 in HEV-A549 cells was observed. HEV ORF3 protein alone inhibited IFN-α–induced phosphorylation of STAT1 and down-regulated the IFN-α–stimulated genes encoding PKR, 2′,5′-OAS, and myxovirus resistance A. Conclusion: HEV inhibits IFN-α signaling through the regulation of STAT1 phosphorylation in A549 cells. These findings have implications for the development of new strategies against hepatitis E. (HEPATOLOGY 2012 )
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The interferon system is an important component of the host response against viruses.1, 2 Acute viral infection of susceptible host cells initiates a type I interferon (IFN) response that is composed predominantly of interferon-α and -β (IFN-α/β) signaling through the IFN-α receptor. IFN-α/β receptor binding results in receptor subunit dimerization and activation through tyrosine phosphorylation of two tyrosine kinases of the Janus family, Janus kinase 1(Jak1) and tyrosine kinase 2 (Tyk2), which then phosphorylate signal transducer and activator of transcription (STAT) 1 and STAT2 on a single tyrosine residue, leading to STAT1–STAT2 heterotrimerization with interferon regulatory factor (IRF) 9 followed by nuclear localization.1 In the nucleus these proteins serve to transactivate the interferon-stimulated response element (ISRE) found in the promoter of interferon-stimulated genes (ISGs). A number of these ISGs encode the antiviral proteins, the best known of which include myxovirus resistance A (MxA), the double-stranded RNA-activated protein kinase (PKR), and the 2′,5′-oligoadenylate synthetase (2′,5′-OAS).1, 2 Viruses have evolved different mechanisms to inhibit type I IFN response and block various aspects of the signaling pathway, thus escaping the host immune response and causing infection.
Hepatitis E, caused by hepatitis E virus (HEV), is an emerging public health problem in both developing and developed countries. Hepatitis E mostly manifests as a self-limiting, icteric hepatitis in most individuals. However, substantially high mortality rates of as much as 20% are observed in pregnant women.3 Furthermore, organ transplant recipients, as well as human immunodeficiency virus (HIV)-infected and other immunocompromised individuals, run the risk of developing chronic liver disease when infected with HEV.4 Treatment of chronically infected patients with pegylated interferon-α2a/α2b or ribavirin for 3-12 months has been shown to achieve a sustained virological response for 3-6 months after completion of the therapy.5, 6
HEV is a nonenveloped virus with a single positive-sense RNA genome of approximately 7.2 kb and is classified in the Hepevirus genus within the family Hepeviridae.3 The viral RNA consists of a short 5′ noncoding region (NCR), three open reading frames (ORFs), and a 3′ NCR. A cap structure has been identified at the 5′ end of the viral genome, which may play a role in the initiation of HEV replication.7 ORF1, located at the 5′ end of the genome, encodes nonstructural polyproteins that are involved in viral replication.8, 9 ORF2, located at the 3′ end of the genome, encodes the viral capsid protein, which plays an important role in viral immune response.10 ORF3 encodes a cytoskeleton-associated phosphoprotein11 that is responsible for virion egress from infected cells and is essential for virus infectivity in vivo, although it is not required for virus infection in vitro.12
Viruses have been reported to influence the IFN regulatory pathway,13, 14 but effects of HEV on IFN signaling have not been studied so far because of the lack of an efficient HEV cell culture system or a small animal model of infection. Propagation and production of HEV in vitro have been attempted in many cell lines, but culturing HEV has proven to be difficult.15 Recently, successful propagation of HEV in A549 cells, a human lung adenocarcinoma epithelial cell line, was reported.15, 16 In this study, we describe the generation of an A549 cell line (HEV-A549) persistently infected with the genotype 3 HEV strain. Using this cell line, we investigated whether HEV inhibits IFN-α signaling.
Stool specimens previously collected from a kidney transplant patient chronically infected with HEV genotype 3 strain were stored in our laboratory (GenBank accession number: JN837481). Stool suspensions were prepared in 0.01 M phosphate-buffered saline (PBS; 10% [wt/vol]). The suspension was centrifuged at 10,000g at 4°C for 20 minutes, and supernatants were filtered through 0.22-μm filters (Millipore, Billerica, MA); after clarification, they were aliquoted and stored at −80°C. The HEV RNA level pooled from these virus stocks was determined to be 6.28 × 106 copies/mL.
The generation of a monoclonal antibody, 5G5, which was raised in mice by inoculation of HEV ORF2 proteins expressed in E. coli, has been described previously.17 Mouse polyclonal antibody to HEV ORF3 protein was purchased from Abbiotec, LLC (San Diego, CA). Mouse monoclonal antibody to β-actin and STAT1, and rabbit polyclonal antibodies to STAT2, Jak1, Tyk2, phosphotyrosin 701-STAT1 (pY-STAT1), phosphotyrosin 690-STAT2 (pY-STAT2), phosphotyrosin 1022/1023-Jak1 (pY-Jak1), and phosphotyrosin 1054/1055-Tyk2 (pY-Tyk2) were purchased from Cell Signaling Technology (Danvers, MA). Recombinant human IFN-α was purchased from Invitrogen (Carlsbad, CA).
Cell Culture and Virus Infection.
Virus infection was carried out as previously described with slight modifications.16 The A549 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C, 5% CO2, and 100% relative humidity. For virus infection, monolayers of confluent A549 cells in a 25-cm2 flask were washed three times with PBS and inoculated with 0.5 mL of stool suspension containing 3.14 × 106 copies of HEV RNA that had been diluted with PBS containing 0.2% (wt/vol) bovine serum albumin (BSA) and filtered through a 0.22-μm filter. After inoculation, the cells were incubated at room temperature for 1 hour and the medium was replaced with 6 mL of maintenance medium, which contained DMEM with 2% FBS and 30 mM MgCl2, other supplements being the same as those in the growth medium. All cultures were performed at 37°C in a humidified 5% CO2 atmosphere.
One day after inoculation, the cells were washed five times with PBS, and 6 mL of maintenance medium was added. Subsequently, 3 mL of the culture medium was replaced with fresh maintenance medium every other day, and harvested media were stored at −80°C. The infected cells were examined daily for specific cytopathic effect (CPE). For passaging, one flask of HEV-infected A549 cells, designated hereafter as HEV-A549, were split into three flasks and maintained as described above. Up to eight passages were made with HEV-A549 cells. The harvested media were stored at −80°C.
Detection of HEV RNA: Real-Time RT-PCR.
The levels of HEV RNA were determined by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay, already described, with slight modifications.18 Briefly, total RNA was extracted from 100 μL of stool suspension or culture medium, which was then subjected to real-time RT-PCR with the One-Step Platinum qRT-PCR kit (Invitrogen) using a sense primer (5′- ACCCTGTTTAATCTTGCTGATAC-3′), an antisense primer (5′-ACAGTCGGCTCGCCAT TGG-3′), and a probe (5′-FAM-CCGACAGAATTGATTTCGTCGGC-BHQ-3′) on the Mx3005 Real-Time PCR System (Agilent Technologies, Santa Clara, CA). The thermal cycling conditions were 50°C for 30 minutes, 95°C for 15 minutes, and 50 cycles of 94°C for 15 seconds, 56°C for 30 seconds, and 72°C for 30 seconds.
Detection of HEV Proteins: Immunofluorescence Assay (IFA).
Briefly, monolayer cultures of A549 cells and HEV-A549 cells were fixed with 100% methanol for 2 hours, and then incubated with HEV ORF2 monoclonal antibody 5G5 at 37°C for 1 hour. After three washes with PBS, cells were incubated for 1 hour at 37°C with an Alexa Fluor 488–conjugated goat anti-mouse antibody (Invitrogen). After extensive washing with PBS, cells were viewed with an epifluorescence microscope (Axiovert 200, Carl Zeiss, Germany). Images were acquired with an Axiocam MRc5 camera (Carl Zeiss).
Treatment of HEV-A549 Cells With IFN-α.
The effects of IFN-α on the replication of HEV in the HEV-A549 cells were examined in the presence of different concentrations of IFN-α (10, 50, 100, 250, 500, and 1000 U/mL). Various concentrations of IFN-α were added to the HEV-A549 cell culture supernatant containing approximately 4.16 × 104 HEV-RNA copies/mL. After 72 hours of treatment, the levels of HEV RNA were quantitated by RT-PCR as described above. All samples were assayed in triplicate.
Detection of IFN-α–induced Gene Expression.
IFN-α–induced gene expression levels were quantitated by real-time RT-PCR according to the methods described, with slight modifications.19 In brief, total RNA was isolated using the MagNA Pure LC (Roche Applied Science, Indianapolis, IN) and subsequently treated with deoxyribonuclease I (Roche Applied Science). RNA integrity was assessed using an ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE), and then subjected to real-time RT-PCR with the following Human SYBR Green QuantiTect Primer Assays (Qiagen, Valencia, CA): double-stranded RNA-activated protein kinase (PKR, no. QT00022960), MXA (no. QT00090895), and OAS1 (no. QT00099134). Reactions were set up in 96-well plates using the Mx3005 Real-Time PCR System. All samples were assayed in triplicate. The endogenous control genes eukaryotic translation elongation factor 1α (EF1A; no. QT01669934) and β-glucuronidase (no. QT00371623; Human SYBR Green QuantiTect Primer Assays, Qiagen) were used to normalize expression levels of target genes.
Immunoprecipitation and Immunoblotting.
Immunoblotting was performed as described.20 For total protein extracts, cells were washed three times with ice-cold PBS and scraped from culture dishes in the presence of NP40 lysis buffer (25 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1% NP40, 2 mM ethylene diamine tetraacetic acid [EDTA], 1 mM phenylmethylsulfonyl fluoride [PMSF], 5 mM NaVO4, 10% glycerol) supplemented with protease inhibitor cocktail (Roche Applied Science). Equal amounts of protein extracts (50 μg) were run on 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad Laboratories Inc, Hercules, CA). The nonspecific antibody-binding sites were blocked with 5% nonfat milk in TBS-T (25 mM Tris, 0.8% NaCl, and 2.68 mM KCl [pH 7.4], with 0.1% Tween 20) before addition of the primary antibody. The blots were then treated with a horseradish peroxidase–conjugated secondary antibody (KPL Inc, Gaithersburg, MA) and developed with an ECL system (GE Healthcare Life Science, Piscataway, NJ). For reblotting, the membrane was washed with stripping solution (Thermo-Scientific) for 15 minutes at room temperature. The membrane was then blocked with 5% nonfat milk in TBS-T for 1 hour, followed by treatment with the primary antibody. For immunoprecipitation, 400 μg of total protein was incubated with 100 ng of mouse anti-STAT1 monoclonal antibody overnight at 4°C. Protein complexes were precipitated with the protein A/G Plus Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at 4°C. Immunoprecipitates were washed three times with NP-40 lysis buffer and boiled in 2X SDS sample buffer. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting analysis.
Plasmid Construction and Transfection Experiments.
Full-length HEV ORF3 was amplified from the same stool suspension containing HEV genotype 3, as described above, using a sense primer 5′-GACGACGACAAGATGGGATCACCATGCGCC-3′ and an antisense primer 5′-GAGGAGAAGCCCGGTCAGCGGCGCAGCCCCAG-3′ and cloned into pTriEx-4 vector by using the pTriEx-4 EK/LIC Vector kit (Novagen, San Diego, CA). Transfection experiments were conducted in monolayers of A549 cells grown in 6-well plates to 50%-70% confluency. The cells were transfected with either HEV ORF3 construct, designated pTriEx-4/ORF3, or control plasmid, pTriEx-4, using 1 μg of DNA and 3 μL FuGENE 6 Transfection Reagent (Roche Applied Science, Indianapolis, IN). Twenty-four hours after transfection, the cells transfected with pTriEx-4 and pTriEx-4/ORF3 vector were induced with IFN-α (1000 U/mL) for 15 or 30 minutes or left untreated, respectively. Relative gene expression levels and protein levels were examined by real-time PCR and immunoblotting as described above.
Results of experiments with IFN were expressed as mean ± standard deviation of three independent experiments. For statistical comparison, significance was evaluated using Student t test.
Infection of A549 Cells With HEV.
As shown in Fig. 1A, HEV RNA appeared in the culture medium of A549 cells inoculated with HEV genotype 3 stool suspension containing 3.14 × 106 copies of HEV RNA on day 40 after inoculation. The levels of HEV RNA in the culture medium were 1.98 × 102 copies/mL; these levels continued to increase thereafter, reaching a maximum level of 4.35 × 105 copies/mL on day 100 after inoculation. No CPE was observed in HEV-A549 cells. To determine whether HEV was stably generated from HEV-A549 cells, the cells were split for subsequent passage at a ratio of 1:3 when HEV RNA reached the peak titer of 4.35 × 105 copies/mL in culture medium. Figure 1B illustrates that HEV RNA could be detected in the culture medium harvested from HEV-A549 cells at the second passage. The viral titers were maintained at approximately 3-4 × 104 copies/mL up to the 16th day of passage. IFA showed that ORF2 protein was detectable in the cytoplasm of the HEV-A549 cells (Fig. 1C,D).
Effect of IFN-α on HEV.
HEV-A549 cells generating an HEV RNA titer of 4.16 × 104 copies/mL into the culture medium were treated with increasing concentrations of human IFN-α (10, 50, 100, 250, 500, and 1000 U/mL). As shown in Fig, 2, the average reduction rates (as a percentage of the rate of the control) of the HEV RNA in culture supernatants were only about 10%, 20%, and 50% in the presence of IFN-α at concentrations of 250, 500, and 1000 U/mL, respectively, after 72 hours of incubation. Lower doses of IFN-α (10, 50, and 100 U/mL) did not result in any appreciable reduction in HEV RNA levels (data not shown). Furthermore, subsequent experiments showed that HEV replication was not completely inhibited by IFN-α even at a concentration of 5000 U/mL (approximately 50% reduction, data not shown).
HEV Down-regulates IFN-α–induced Gene Expression.
To investigate how HEV resists IFN-α–mediated responses, three IFN-stimulated response element–controlled cellular genes, PKR, MxA, and 2′,5′-OAS, were analyzed by real-time PCR in both HEV-A549 cells and A549 cells with and without IFN-α. In the absence of stimulation by IFN-α, no significant difference was found in the expression of any of these genes in A549 cells compared with HEV-A549 cells (Fig. 3). Addition of IFN-α resulted in a significant induction of PKR (∼126-fold increase) and 2′,5′-OAS (∼20-fold). Similarly, an increase in induction of PKR and 2′,5′-OSA was observed after IFN-α treatment of HEV-A549 cells that was significantly weaker than observed in A549 cells (P < 0.005). The difference in activation of MxA was not significant between A549 cells and HEV-A549 cells with and without IFN-α treatment.
HEV Blocks IFN-α–induced STAT1 Phosphorylation.
Many viruses inhibit IFN-α signaling by interfering with the normal activities of STAT1 in the Jak/STAT signal transduction pathway.21 Therefore, steady-state protein level and phosphorylation of STAT1 in response to IFN-α in uninfected A549 cells were determined and compared with HEV-infected HEV-A549 cells. As shown in Fig. 4, STAT1 levels were markedly increased in HEV-A549 cells compared with A549 cells. Furthermore, IFN-α induced a rapid STAT1 phosphorylation in A549 cells, as assessed by immunoblotting with a phospho-STAT1–specific antibody. STAT1 phosphorylation was detectable 15 minutes after addition of IFN-α and was further increased at 30 minutes, but was not detectable in uninfected, IFN-α–untreated A549 cells. In contrast, the levels of tyrosine phosphorylated STAT1 were dramatically reduced in IFN-α–treated HEV-A549 cells compared with uninfected A549 cells.
Effect of HEV on Phosphorylation of STAT2, Tyk2, and Jak1.
To determine whether events upstream of STAT1 phosphorylation are altered during HEV infection, STAT2, Tyk2, and Jak1 were evaluated for abundance and phosphorylation. Immunoblot analysis showed there was no significant difference in STAT2 levels between A549 cells and HEV-A549 cells. Although very low levels of phosphorylated STAT2 were detectable in unstimulated A549 cells, infection with HEV or stimulation with IFN-α for 15 minutes significantly increased levels of phosphorylated STAT2 (Fig. 5). Furthermore, phosphorylated STAT2 was readily detectable in IFN-α–stimulated and HEV-A549 cells, indicating that in contrast to STAT1, HEV infection in A549 cells did not prevent STAT2 phosphorylation. To assess whether HEV can alter Tyk2 or Jak1 phosphorylation, immunoblotting with phospho-Tyk2– or phospho-Jak1–specific antibodies was performed. Naive A549 cells not treated with IFN-α displayed a basal level of phosphorylation of both Tyk2 and Jak1 (Fig. 5). However, stimulation with IFN-α for 15 or 30 minutes was sufficient to induce increased phosphorylation of both proteins. The phosphorylation of Tyk2 or Jak1 by IFN-α was not inhibited by HEV infection in HEV-A549 cells.
Binding of HEV ORF3 Protein to STAT1 in HEV-A549 Cells.
To investigate the possible mechanism of inhibition of STAT1 phosphorylation in HEV-infected A549 cells, HEV-A549 cell lysates (400 μg of total protein) were immunoprecipitated with the anti-STAT1 monoclonal antibody and analyzed by immunoblotting with anti-ORF2 or anti-ORF3 antibodies. As controls, HEV ORF2 and ORF3 proteins were analyzed on immunoblots without immunoprecipitation by anti-STAT1 antibody. As shown in Fig. 6, ORF3 protein but not ORF2 protein was detected in immunoprecipitated STAT1, indicating that the ORF3 protein could bind to STAT1 in HEV-infected A549 cells.
HEV ORF3 Protein Blocks IFN-α–induced STAT1 Phosphorylation and Down-regulates IFN- α–induced Gene Expression.
To investigate whether HEV ORF3 alone can block IFN-α–induced STAT1 phosphorylation and its effect on IFN-α–stimulated genes, A549 cells were transfected with either pTriEx-4 or pTriEx-4/ORF3 vector. As shown in Fig. 7A, STAT1 phosphorylation was inhibited in IFN-α–treated pTrix-4/ORF3–transfected cells, but not in pTriEx-4 vector–transfected A549 cells. However, there was no significant difference in STAT1 levels between A549 cells transfected with either pTriEx-4 or pTriEx-4/ORF3 vector. Moreover, three IFN-α–stimulated genes, PKR, MxA, and 2′,5′-OAS, were analyzed by real-time PCR in A549 cells transfected with pTriEx-4 or pTriEx-4/ORF3 vector with and without IFN-α. As shown in Fig. 7B, all three target genes, PKR, 2′,5′-OAS, and MxA, were inhibited in pTriEx-4/ORF3–transfected A549 cells compared with the cells transfected with pTriEx-4 vector (P < 0.01).
Although hepatocytes are recognized as the main sites of HEV replication, the detection of a replicative strand of HEV RNA in cell types other than liver cells shows that the extrahepatic replication of HEV does occur. In experimentally infected SPF pigs, HEV RNA has been detected by RT-PCR in peripheral blood, feces, bile, and numerous tissues including liver, mesenteric lymph nodes, stomach, spleen, and lung.22 In naturally infected pigs, evidence of HEV replication can be detected in liver, lymph nodes, spleen, tonsils, kidney, and small and large intestines by immunohistochemistry and in situ hybridization.23 Propagation of HEV in the human lung epithelial A549 cells was recently reported.16 In the present study we generated an HEV-A549 cell line that could stably excrete HEV in cell culture supernatant, and using this cell culture system, we have demonstrated that the IFN-induced JAK–STAT signaling pathway is inhibited during HEV infection.
It is believed that most, if not all, viruses have the ability to attenuate the IFN response during infection to ensure that the virus has sufficient time to successfully replicate, be packaged in, and released from host cells.24 Previous studies have reported that hepatitis A, B, C, and D viruses use various strategies to inhibit IFN-α–stimulated host defense mechanisms. Hepatitis A virus protein 2B suppresses IFN-β gene transcription by interfering with IFN regulatory factor 3 activation.25, 26 Hepatitis B virus (HBV) suppresses IFN-α response by the inhibition of STAT1 methylation.27 Moreover, the HBV regulatory protein HBx can bind to adaptor protein interferon promoter stimulator 1 (IPS-1) and inhibit the activation of IFN-β.28 A number of reports indicate that the hepatitis C virus core, NS3 and NS5A proteins impair IFN responses through blocking different aspects of the IFN-α signal pathway.29-32 Hepatitis D virus has also been shown to have the ability to block the IFN-α signal pathway in vitro.19 However, the effects of HEV on IFN-α signaling have not been investigated so far. By generating HEV-A549 cells, we report here that, during replication in A549 cells, HEV suppressed IFN-α–stimulated gene activation (PKR and 2′,5′-OSA). Moreover, HEV replication was not completely inhibited by IFN-α treatment in vitro, and IFN-α–mediated phosphorylation of STAT1 was prevented by HEV in A549 cells. Further investigation of the upstream signaling components in the IFN-α signal cascade revealed that the ability of Tyk2, Jak1, and STAT2 to be phosphorylated in response to IFN-α stimulation was not affected by HEV infection. These results suggest that HEV was able to abolish type I IFN signaling through mechanisms regulating STAT1 phosphorylation.
The exact mechanism by which HEV inhibits JAK–STAT signaling is not yet known. Previous studies showed that some viral proteins, such as Nipah virus V and W proteins and Rinderpest virus P and C proteins, can bind to STAT1 or STAT2 and thus inhibit the type I IFN signaling pathway.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 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 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 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. Moreover, HEV ORF3 protein alone has the ability to inhibit STAT1 phosphorylation and to reduce IFN-α–stimulated gene expression. An understanding of the precise mechanisms of how HEV inhibits the IFN-α signaling pathway will be important for designing better antiviral strategies against hepatitis E.
We thank Jan Drobeniuc, Tracy Greene-Montforte, and Ngoc-Thao Le for their assistance with this study.