Epigenetic suppression of interferon lambda receptor expression leads to enhanced HuNoV replication in vitro

Human norovirus (HuNoV) is the main cause of gastroenteritis worldwide yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an IFN-cured population of replicon-harboring HGT cells (HGT-cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-cured cells created an environment favouring norovirus replication. Microarray analysis was used to identify genes differentially regulated in HGT-NV and HGT-cured compared to parental HGT cells. We found that the IFN lambda receptor alpha (IFNLR1) expression was highly reduced in HGT-NV and HGT-cured cells. All three cell lines responded to exogenous IFN-β by inducing interferon stimulated genes (ISGs), however, HGT-NV and HGT-cured failed to respond to exogenous IFN-λ. Inhibition of DNA methyltransferase activity with 5-aza-2’-deoxycytidine partially reactivated IFNLR1 expression in HGT-NV and IFN-cured cells suggesting that host adaptation occurred via epigenetic reprogramming. In line with this, ectopic expression of the IFN-λ receptor alpha rescued HGT-NV and HGT-cured cells response to IFN-λ. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. This study unravels for the first time epigenetic reprogramming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signalling is able to control human norovirus replication. Importance Noroviruses are one of the most wide-spread causes of gastroenteritis yet we have no therapeutics for their control and we do not fully understand what cellular processes control viral replication. Recent work has highlighted the importance of type III interferon (IFN) responses in the restriction of viruses that infect the intestine. Here we analysed the adaptive changes required to support long term replication of noroviruses in cell culture and found that the receptor for type III IFN is decreased in its expression. We confirmed that this decreased expression was driven by epigenetic modifications and that cells lacking the type III IFN receptor are more permissive for norovirus replication. This work provides new insights into key host-virus interactions required for the control noroviruses and opens potential novel avenues for their therapeutic control.

RNA to specific cellular environments (Supporting  host cell adaptation, wild-type VPg-linked RNA extracted from BHK-NV cells was re-transfected in 1 3 9 both HGT and HGT-Cured cell lines that were subsequently selected for 5 days with G418. As shown levels further increased at day 6 to yield a 15-fold difference between HGT-Cured and HGT cells. comparable across the three cell lines, evident by the relative ratio with respect to β-actin, we 2 0 0 concluded that PAMP sensing and downstream signalling pathways are functional in these cell lines. The ability to respond to type III but not type I IFN in HGT-NV and HGT-Cured cells is 2 0 2 debilitated.

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We next investigated the ability of type I and type III interferons to elicit ISGs induction. To this end, 2 0 4 cells were incubated with human IFN-β or IFN-λ2 for 16 hours and viperin expression levels were anti-STAT1 phospho-specific antibodies. We observed that although STAT1 was phosphorylated in 2 1 2 all cell lines following IFN-β treatment (Fig. 5C), no STAT1 phosphorylation was detected in HGT-2 1 3 NV and HGT-Cured cells following IFN-λ2 treatment (Fig. 5D). To ensure that the absence of 2 1 4 STAT1 phosphorylation in HGT-NV cells is not due to a potential clonal effect, different clones as represented here by clone 2 (C2) and the polyclonal cell population responded to IFN-β but not to of parental HGT cells corroborating the antiviral activity of both type I and type III IFNs on HuNoV 2 4 0 replication (Fig. 7). Similarly, an increased ability of modified cell lines to promote colony formation induced by the HuNoV replicon was observed (data not shown). The dimeric interferon lambda receptor consists of the ubiquitously expressed IL10RB chain subunit 2 4 4 and the interferon lambda specific chain IFNLR1 whose expression is limited to cells of epithelial were incubated in the presence 5-aza-2'-deoxycytidine (5azadC), a deoxynucleoside analogue that also observed in the case of HGT-Cured cells but to a lower extent (2.8-fold). In contrast, no change 2 5 3 in IL10RB expression was detected when the same cells were incubated with 5azadC ( Fig. 8B). In that the presence of replicating HuNoV did not modify the chromatin structure of the IFNLR1 gene (data not shown). Taken together, these results suggest that the replication of HuNoV in HGT methylation of its promoter.  HuNoV observed in HGT-Cured cells, or that some of the differentially expressed genes identified have a potent yet unknown proviral activity towards the human norovirus. high confidence gene expression datasets provided with this study is expected be useful for the 3 2 4 selection and examination of new targets aimed to antiviral therapy. essential medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 3 3 2 µg/ml streptomycin, 1X non-essential amino acid and 0.5 mg/ml G418 in the case of HGT-NV cells.

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The DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (Sigma-Aldrich, A3656) was diluted in 3 3 4 sterile water and used at a final concentration of 10 μM. appropriate culture media. After four successive passages, total RNA was extracted using TRIzol® RNA was then reverse transcribed using random hexamers and the M-MLV RT enzyme (Promega).

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SYBR green-based quantitative PCR was performed using gene-specific primers listed in  and results are shown as a ratio to levels detected in control cells according to the Δ Δ Ct method (49).

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Additional non-template and non-reverse transcriptase samples were analysed as negative controls.

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Data were collected using a ViiA 7 Real-Time PCR System (Applied Biosystems). Genomic viral RNA was quantified by one step RT-qPCR using GI NV-specific primers. Viral genome copy 3 5 1 numbers were calculated by interpolation from a standard curve generated using serial dilutions of 3 5 2 viral RNA transcribed from the pNV101 plasmid coding for the full-length Norwalk genome (50). in antibiotic-free growth medium at a density of 2×10 5 or 1×10 6 cells per well in 24-or 6-well plates, Cells were plated on 12 mm glass coverslips and allowed to adhere overnight before fixation with 4%  channel scanning to prevent fluorophore bleed-through artefacts due to spectral overlap. Cell lysates were prepared in radio-immuno precipitation assay buffer (RIPA: 150 mM NaCl, 0.5%  unspecific antigens was carried out in 5% non-fat dried milk or 5% BSA in PBST for 1 h at 4°C.

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Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C with gentle rocking  Table 7). Membranes were washed three times in PBST for 5 min at RT. Species-matched IRDye-800CW secondary antibodies were diluted in blocking buffer as before and 3 9 0 incubated at RT for 1 h. Membranes were washed again three times in PBST for 5 min at RT.

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Fluorescent signal was detected through an Odyssey CLx infrared imaging system (Li-COR).  G  e  n  e  r  a  t  i  o  n  o  f  I  F  N  A  R  1  o  r  I  F  N  L  R  1  k  n  o  c  k  o  u  t  H  G  T  c  e  l  l  s   3  9  3 HGT cells knockout for IFNAR1 or IFNLR1 genes were generated using the CRISPR/Cas9 system.

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Lentivirus vectors encoding single-guide RNAs against IFNAR1 or IFNLR1 were generously     replicates. Viral RNA copy numbers were below the limit of detection in both HGT and HGT-Cured  Cured cells. mRNA expression levels were expressed as differences of cycle threshold (C t ) of viperin, relative to to type III IFN and the loss of the receptor enhances viral RNA replication in HGT cells. from one experiment performed in biological triplicate and compared to the replication levels 4 9 1 measured in HGT control at day 0. Statistical significance was determined using the unpaired t test.