Impaired K48-polyubiquitination downmodulates mouse norovirus propagation.

Introduction: Noroviruses are small non-enveloped, single stranded positive-sense RNA viruses that belong to the family Methods: In this study, using the persistent murine norovirus S99 strain (MNoV_S99), we have investigated the role in the regulation of anti-noroviral responses of ubiquitination, a post-translational modification that covalently adds one or multiple ubiquitin molecules onto lysine residues of target proteins. To that end, we have first generated RAW264.7 cells overexpressing YFP-Ubiquitin_WT, _K29R, _K48R or_K63R constructs. All non-WT constructs encode a ubiquitin fusion protein with one lysine mutated into an arginine residue, thus preventing the formation of the K29-, K48- or K63-dependent polyubiquitin chains respectively. Results: Upon infection of these cells with MNoV_S99, we unexpectedly observed that only cells expressing the YFP-Ubiquitin_K48R protein showed a significantly impaired expression of several viral markers: NS5, NS7, VP1 and the replication intermediate dsRNA. Consequently, the number of viral genome copies or viral titers were also significantly decreased in the YFP-Ubiquitin_K48R cells compared to the YFP-Ubiquitin_WT cells. This negative regulation cannot be explained by perturbed viral entry, but rather a constitutive hypersecretion of the pro-inflammatory cytokine TNF and downstream upregulation of IκBα phosphorylation and the subsequent NF-κB nuclear translocation. Conclusion: Overall, these consequences combined impose a non-permissive environment for MNoV_S99 replication and propagation.