Epigenetic Suppression of Interferon Lambda Receptor Expression Leads to Enhanced Human Norovirus Replication In Vitro

Abstract

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 interferon (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 favoring norovirus replication. Microarrays were used to identify genes differentially regulated in HGT-NV and HGT-Cured compared to parental cells. We found that IFN lambda receptor (IFNLR1) expression was highly reduced in HGT-NV and HGT-Cured cells. While all three cell lines responded to exogenous IFN-β by inducing interferon-stimulated genes, HGT-NV and HGT-Cured cells failed to respond to exogenous IFN-λ. Methylation-sensitive PCR showed that an increased methylation of the IFNLR1 promoter and inhibition of DNA methyltransferase activity partially reactivated IFNLR1 expression in HGT-NV and HGT-Cured cells, indicating that host adaptation occurred via epigenetic reprogramming. Moreover, IFNLR1 ectopic expression rescued response to IFN-λ and restricted HuNoV replication in HGT-NV cells. 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 signaling is able to control human norovirus replication. IMPORTANCE Noroviruses are one of the most widespread causes of gastroenteritis, yet no suitable therapeutics are available for their control. Moreover, to date, knowledge of the precise cellular processes that control the replication of the human norovirus remains ill defined. Recent work has highlighted the importance of type III interferon (IFN) responses in the restriction of viruses that infect the intestine. Here, we analyzed 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 of noroviruses and opens potential novel avenues for their therapeutic control.

Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids

“…Before the discovery of the HIE-based HuNoV ex vivo culture system, innate immune responses to HuNoV RNA replication were examined by using human hepatoma (HG23) or gastric tumor (HGT) cell-based GI.1 replicon systems (31,49,50), and human embryonic kidney (293FT) cells transfected with authentic GI.1 HuNoV RNA from stools of infected persons or RNA expressed from a reverse genetics full-length GII.3 plasmid (30). The main limitation to these previous studies is that none represented a complete virus infection cycle; in particular, the viral entry step was missing, which itself can induce innate responses, and the sites of RNA replication may have been in cytoplasmic compartments different from those in an authentic viral infection.…”

Leaked genomic and mitochondrial DNA contribute to the host response to noroviruses in a STING-dependent manner

“…Importantly, this also potentially explains the discrepancy between in vivo and in vitro results from studies on IFN responses to the human norovirus. For example, studies in Huh7 and 293FT cells have shown no IFN responses to HuNoV [51,52], while human challenge studies [53], studies in animal models [54,55], and in vitro studies in organoids [56][57][58], and replicon-containing cell lines [31] have all demonstrated induction of IFNs following infection. Given that both Huh7 and 293FT cells have impaired cGAS-STING pathways [29,59,60], our data demonstrating a role of this pathway in restriction of noroviruses harmonises these various otherwise conflicting data.…”

“…Next, we wanted to see if there is a similar role for STING in the IFN responses against human noroviruses. For this, we made use of the recently described HGT-NV cells [31], a human gastric tumour cell line stably harbouring a GI.I human norovirus replicon. The cells were treated with DMSO or indicated doses of H-151, harvested 24 hours post treatment and subjected to RT-qPCR.…”

Current and Future Antiviral Strategies to Tackle Gastrointestinal Viral Infections

“…IFN type I and II were shown to have an impact on NoV infections by triggering the host innate immune response [ 153 , 154 , 155 ]. Also type III IFN (IFN λ) was shown to protect mice against MNV challenge, therefore this could be explored for the treatment/prophylaxis of NoV infections [ 156 , 157 ].…”