key: cord-0005504-jvyq1b3l authors: Myoung, Jinjong; Lee, Jeong Yoon; Min, Kang Sang title: Methyltransferase of a cell culture-adapted hepatitis E inhibits the MDA5 receptor signaling pathway date: 2019-11-22 journal: J Microbiol DOI: 10.1007/s12275-019-9478-8 sha: ff6d9926b159c7997f3b71878a220e89e06fb7e2 doc_id: 5504 cord_uid: jvyq1b3l Hepatitis E virus (HEV) is a causative agent of acute hepatitis and jaundice. The number of human infections is approximated to be over 20 million cases per year. The transmission is mainly via the fecal-oral route and contaminated water and food are considered to be a major source of infection. As a mouse model is not available, a recent development of a cell culture-adapted HEV strain (47832c) is considered as a very important tools for molecular analysis of HEV pathogenesis in cells. Previously, we demonstrated that HEV-encoded methyltransferase (MeT) encoded by the 47832c strain inhibits MDA5- and RIG-I-mediated activation of interferon β (IFN-β) promoter. Here, we report that MeT impairs the phosphorylation and activation of interferon regulatory factor 3 and the p65 subunit of NF-κB in a dose-dependent manner. In addition, the MeT encoded by the 47832c, but not that of HEV clinical or field isolates (SAR-55, Mex-14, KC-1, and ZJ-1), displays the inhibitory effect. A deeper understanding of MeTmediated suppression of IFN-β expression would provide basis of the cell culture adaptation of HEV. Hepatitis E virus (HEV) is a single-stranded positive-sense RNA virus which is found enveloped in blood and non-enveloped in bile and feces (Denner, 2019) . Its genome is known to have 3-4 partially overlapping open reading frames (ORFs): ORF1 encodes 6 non-structural proteins (Koonin et al., 1992; Ropp et al., 2000; Ahola and Karlin, 2015; Kanade et al., 2018; Kang et al., 2018) while ORF2 and ORF3 encode viral capsid protein and an ion channel, respectively (Ding et al., 2017; Kang and Myoung, 2017a; Kang et al., 2018) . Recently, ORF4 has been proposed and Nair et al. (2016) reported that it is expressed under certain conditions, such as ER stress, whose expression seems to be limited to HEV genotype 1. HEV in-fections are known to be associated with the most common cause of acute viral hepatitis, accounting for over 20 million cases world-wide (Kang et al., 2018) . In healthy subjects, HEV infections are mostly self-limiting and asymptomatic. However, high mortality has been reported if HEV infects pregnant women (Jilani et al., 2007; Navaneethan et al., 2008) . Upon virus infections, type I interferons (IFNs) are rapidly induced, subsequently activating down-stream signaling molecules which leads to anti-viral innate immune responses (Theofilopoulos et al., 2005; Xi et al., 2012; Kang and Myoung, 2017a) . The genomes of invading viruses are recognized by pattern recognition receptors Fujita et al., 2007; Medzhitov, 2007) : toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I) and melanoma differentiationassociated protein 5 (MDA5). Although both RIG-I and MDA5 recognize double-stranded RNA's (dsRNA's) (Loo et al., 2008; Takeuchi and Akira, 2010) , there seem to be subtle differences in the nature, length, and structure of dsRNA's they bind. RIG-I has been reported to bind to relatively short dsRNA's which are blunt-ended with 5'-triphosphates (Kato et al., , 2008 . On the contrary, MDA5 does not seem to have strict structural and chemical restrictions, binding to long dsRNA's (Hornung et al., 2006; Pichlmair et al., 2006; Kim and Myoung, 2018; Myoung and Min, 2019) . Upon cognate ligand binding, RLR's undergo conformational changes, releasing auto-inhibited caspase activation and recruitment domain (CARD). CARD domains of the RLR's interact with that of mitochondrial antiviral signaling protein (MAVS), which subsequently induces its oligomerization on the outer membrane of mitochondria. Activated MAVS in turn induces activation of down-stream signaling molecules: TANKbinding kinase 1 (TBK1), IκB kinase ε (IKKε), and interferon regulatory factor 3 (IRF3). Before activation, IRF3 resides in the cytoplasm in an auto-inhibited form. Phosphorylation of IRF3 at the C-terminal regulatory domain induces self-dimerization and nuclear translocation of IRF3. Nuclear IRF3 dimers bind to the promoter of IFN-β, leading to transcription of its mRNA. Previously, we showed that HEV-encoded methyltransferase (MeT) strongly inhibits MDA5-mediated induction of the IFN-β promoter (Myoung and Min, 2019) as well as that of RIG-I (Kang et al., 2018) . Here, we demonstrated that MeT efficiently down-regulates the phosphorylation of IRF3 S396. In addition, NF-κB p65-mediated activation of the NF-κB promoter was strongly suppressed in a dose-responsive manner, likely due to blockade of phosphorylation at S468 of p65. More importantly, only MeT of HEV genotype 3 (47832c strain) displayed inhibitory activity on MDA5-mediated activation of IFN-β. Taken together, MeT seems to be a novel antagonist of the receptor signaling pathway and delineation of its detailed mechanisms will likely pave way to the development of virus-specific therapeutics. Human embryonic kidney 293T (HEK293T) cells were maintained in Dubecco's modified Eagle's medium (DMEM, Wel-Gene) with supplements (10% fetal bovine serum; Gibco and 1% penicillin/streptomycin; Thermo Fisher Scientific) in a humidifying incubator at 37°C with 5% CO2. Polyethyleneimmine (PEI, Signma-Aldrich) was used for transfection of DNA at 1:2 ratio (DNA vs PEI). Antibodies were purchased from various manufacturers: anti-FLAG antibody (M2) from Sigma-Aldrich, anti-phospho-IRF3 (S396) and anti-phospho-p65 (S468) from Cell Signaling, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and antimouse IgG antibody conjugated with horseradish peroxidase (HRP) from Cell Signaling. Construction of expression plasmids of HEV-encoded genes and the host signaling molecules (MDA5, RIG-I, TBK1, IKKε, and IRF3) were described elsewhere (Kang et al., 2018; Kim and Myoung, 2018; Myoung and Min, 2019) . Sequences of MeT's of 4 reference strains were codon optimized and synthesized by Bionics. Accession numbers of each HEV strain are as follows: Sar-55 (genotype 1, AF444003.1), Mex-14 (genotype 2, KX578717.1), Kernow C-1 (genotype 3, JQ679014.1), and ZJ-1 (genotype 4, JQ993308.1). Synthesized MeT gene fragments were cloned into pCMV10-3XFLAG using EcoRI and XbaI. For luciferase assay, IFN-β-luc or NF-κB-luc was co-transfected with plasmids expressing an HEV-encoded gene and a host signaling molecule as indicated. DNA-PEI complex was generated by mixing at 1:2 ratio and incubating at RT for 30 min (Lee et al., 2019a (Lee et al., , 2019b (Lee et al., , 2019c . DNA-PEI complexes were added dropwise onto cells and cells were incubated for 24 h before harvested for luciferase reporter assay or western blotting. To examine if HEV-encoded MeT regulates the MDA5/ RIG-I receptor signaling pathway, luciferase reporter assay was employed as described before (Kang et al., 2018; Kim and Myoung, 2018; Myoung and Min, 2019; Park et al., 2019) . Briefly, MeT expressing plasmid was co-transfected into HEK293T cells with IFN-β-Luc/NF-κB-Luc, β-galactosidase (β-gal) control, and one of signaling molecule gene involved in the pathway as indicated. At 24 h post-transfection, cells were lysed for luciferase assay using Glomax (Promega) following the manufacturer's instructions. Luciferase activities of each experimental group, an indicator of IFN-β promoter induction, were measured and normalized to those of β-gal of each well. Statistical significance was determined by the two-tailed Student's t-test. P value less than 0.05 was considered statistically significant. Data shown are one representative from two independent experiments. Previously, we demonstrated that MeT strongly antagonize RIG-I- (Kang et al., 2018) and MDA5-(Myoung and Min, 2019) mediated activation of the IFN-β promoter. To investigate if MeT regulates other signaling molecules of the IFN-β singling pathway (i.e., MAVS and IKKε) and if NF-κB activation is also affected, HEK293T cells were co-transfected with MeT and signaling molecules as indicated in Fig. 1 . As we previously demonstrated, MeT strongly inhibited RIG-Iand MDA5-mediated activation of the IFN-β promoter ( Fig. 1A and B, left panels). Of note, MeT also antagonized induction of NF-κB promoter as well, which suggests that NF-κB activation and/or nuclear translocation was inhibited ( Fig. 1A and B, right panels). On the other hand, MAVS-and IKKε-mediated activation of the IFN-β signaling pathway was unperturbed, suggesting that MeT-mediated antagonism is largely via inhibition of RLR's. RIG-I and MDA5-mediated induction of the IFN-β signal-ing pathway ultimately leads to phosphorylation and dimerization of IRF3, a key transcription factor of the pathway. Therefore, we tested if IRF3 phosphorylation is regulated by the expression of HEV MeT. HEK293T cells were co-transfected with MeT and IRF3 together with either RIG-I or MDA5 (Fig. 2) . When cells were transfected with IRF3 and RIG-I, IRF3 was efficiently phosphorylated at S396 with total IRF3 levels remaining largely unchanged (Fig. 2, left lanes) . How- ever, when MeT was co-expressed, IRF3 phosphorylation was significantly impaired (Fig. 2, middle lanes) . The same was true to MDA5-mediated activation of IRF3 (Fig. 2, right lanes) . As MDA5 strongly inhibits activation of NF-κB (Fig. 1) , phosphorylation of p65 at the S468 residue was probed. An increasing amount (0, 0.5, 1, 2 μg) of HEV MeT-expressing plasmids were co-transfected with MDA5 and p65 (1 μg) into HEK293T cells and at 24 h post-transfection, cells were harvested for luciferase assay and western blotting. Of note, p65-mediated activation of the NF-κB promoter was significantly diminished by MeT co-expression in a dose-dependent manner (Fig. 3, top panels) , suggesting that HEV MeT is a true antagonist of NF-κB. Interestingly, phosphorylation levels of p65, induced by MDA5, were down-regulated by MeT (Fig. 3 , bottom panels), suggesting that MeT-mediated inhibition of NF-κB activation is likely due to impairment of p65 phosphorylation. The bottlenecks of studies on HEV and HEV-mediated diseases have been the lack of efficient cell culture system and a small animal model. Recently, Shemmerer et al. (2016) reported that a cell culture-adapted HEV strain (HEV 47832c) was successfully isolated and amplified in a subline of A549 (A549/D3). To establish cell culture-adapted HEV 47832c, a clinical isolate of GT3 was passed in A549/D3 over many passages which may have allowed accumulation of mutations for the culture adaptation. To investigate if IFN-β signaling inhibition by HEV 47832c-derived MeT is conserved among those of clinical isolates, MeT's of various HEV strains were cloned and tested (Fig. 4) . Interestingly, only MeT of the 47832c strain showed inhibitory activities against activation of the IFN-β signaling while MeT's, derived from other clinical isolates, had little, if any, effect on the pathway. The lack of an efficient cell culture model and a small animal model has greatly hampered in-depth understanding of HEV-mediated pathologies. In this study, we demonstrated that MeT of HEV 47832c strain, a cell culture-adapted HEV strain, displays strong antagonism of MDA5-/RIG-I-mediated receptor signaling pathway. Previously, we demonstrated that MeT of HEV 47832c strain strongly inhibits MDA5- (Myoung and Min, 2019) or RIG-I- (Kang et al., 2018) mediated induction of the IFN-β signaling pathway. MDA5 and RIG-I are pattern recognition receptors (PRR's) which sense the genomes of invading viral pathogens (Loo et al., 2008; Takeuchi and Akira, 2010; Myoung, 2017a, 2017b) . The notion is consistent with our current findings: MeT strongly antagonized RIG-I-and MDA5-, but not MAVS-or IKKε-, mediated activation of the IFN-β signaling pathway (Fig. 1 ). In addition, MeT-mediated inhibition of PRR's seems to result in low levels of IRF3 phosphorylation (Fig. 2) , leading to low levels of induction of the IFN-β and NF-κB promoters (Fig. 1) . Similarly, MeT inhibited NF-κB activation (Fig. 1) and phosphorylation of the p65 unit of NF-κB was suppressed by the expression of MeT in a dose-dependent manner (Fig. 3) . These results suggest that MeT efficiently blocks activation of IRF3 and NF-κB, presumably due to down-regulation of MDA5-and RIG-Imediated activation of the pathway. Of note, only MeT of HEV 47832c strain demonstrated inhibitory activity on the MDA5-mediated induction of the IFN-β signaling (Fig. 4) while MeT's, derived from Sar-55, Mex-14, ZJ-1 or Kernow-C1, had little, if any, effect. It is even more interesting because HEV 47832c strain is a cell culture-adapted strain (Schemmerer et al., 2016) . A simple hypothesis would be that HEV adaption to cell culture requires effective blockade of the IFN-β signaling. Currently, we are testing this hypothesis by establishing another cell culture adapted HEV strains and by comparing MeT sequences of clinical (original) vs cell culture-adapted HEV strains. Alternatively, cell culture adapted HEV strains have shown host sequence insertions in its genome (Shukla et al., 2011) . It would be very interesting to test if those insertional modifications of the viral genome may be associated with viral antagonisms of IFN-β. Last but not in the least, MeT of HEV 47832c, used in this study, excludes the C-terminal Y domain (so called "iceberg") while MeT's other strains encompass it. The Y domain was initially considered an independent viral protein, but now it is considered an integral part of the MeT protein (Ropp et al., 2000; Ahola and Karlin, 2015; Kanade et al., 2018) . Studies are currently underway to investigate if MeT with/without the Y domain may have differential functions. Taken together, we demonstrated that MeT inhibits phosphorylation of IRF3 and NF-κB, thus blocking efficient induction of the IFN-β promoter. Delineation of detailed molecular mechanisms of MeT-mediated antagonism of the IFN-β pathway may pave way to the development of HEVspecific therapeutics. 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