Insights Into The Function Of Rotavirus NSP1:Innate Immunity Inhibition And Cellular Protein Expression Modulation

Rotavirus (RV) is the major cause of acute diarrhea in children less than5years of age, leading to approximately600,000deaths annually. Better understanding of the mechanisms of rotavirus pathogenesis, especially the viral strategies to subvert and evade host’s antiviral responses, will help us to identify novel strategies for developing antiviral reagents and vaccines.The function of RV nonstructural protein1(NSPl) has not been elucidated. It has been shown that the interaction between NSP1and host is essential for subverting innate immune response. NSP1represses the innate immunity by at least2mechanisms. First, NSP1can induce proteasome-dependent degradation of the interferon transcription factors (IRF3, IRF7, and IRF5) to inhibite the IFN response. Second, NSP1can inhibit nuclear factor-KB (NF-κB) activation by inducing proteasome-dependent degradation of β-TrCP. However, it is reported NSP1is the least conserved protein encoded by the rotavirus genome in different strains. Furthermore, current evidences indicated that the NSP1effect to subvert innate immunity is rotavirus strain-specific. For examples, the NSP1from OSU strain can not induce IRF3degradation.Taken together, we question if NSP1from different rotavirus strains may target a range of cellular substrates of antiviral signaling pathways. In this study, we indentified the new targets of NSP1to better understand the strategies for RV to antagonize innate immunity. We also investigated the host cell proteins modulated by NSP1overexpression using a quantitative proteomics technology to add understandings to the functions of NSP1.1. Rotavirus NSPl antagonizes innate immunity by interacting with RIG-I Previous studies have shown that RV SA11strain NSP1inhibits IFN response by binding and degradating IRFs through its IRF3binding domain. However, we found that the truncated NSP1of SA11strain lacking IRF3binding domain reserve its ability to inhibite RIG-I-induced IFN-β promoter activity, indicating that there may exist other targets by which NSP1inhibit IFN response. Immunoprecipitation assay showed that NSP1binds and degrades RIG-I, a crucial pathogen recognition receptor to sensor pathogen invasion and induce IFN response. Similar results were obtained in RVs strains from other species, human (WA) and porcine (OSU) in addation to simian RV. We also found that during RV infection, RIG-I expression decreased with rotavirus replication. It was predicted that there is a RING domain in NSP1. We proved that the NSP1is a viral E3ubiquitin ligase but the degradation of RIG-I is not through the ubiquitin-proteasome pathway. Bioinformatics analysis showed that there is a caspase1cleavage site in RIG-I. Whether NSP1might cleave RIG-I by active caspase1needs to be further investigated.2. Influence on host cell proteome by rotavirus NSP1In order to further explore the function of NSP1, by using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) techniques, we analyze the proteomes changes of host cells by over expressing NSP1.A total of12quantitatively altered proteins were identified. Among them, expression of8proteins decresed, while expression of4proteins increased.GO analysis showed that these proteins involved in components of the cytoskeleton, signal transduction, molecular chaperone and transcription. We verified the down-regulate expression of proteins, prohibitin and ERP29by western blot, which is in agreement with DIGE analysis. Then we studied the interaction between Vimentin, an expression increase protein belongs to cytoskeleton, and NSP1. RV infection leads to rearrangement of vimentin which serves virus replication and assemble. We found that expression of NSP1also lead to the rearrangement of vimentin and can co-localized with RV VP6, which indicates vimentin may associate with RV replication and assemble. NSP1maybe is one of the reasons responsible for vimentin rearrangement during RV infection.In summary, our study reveal that rotavirus NSP1can inhibit innate immunity by antagonizing RIG-I, and can modulate the expression and rearrangement of vimentin for the first time. These findings add insights into RV replication and the mechanism by which RV subvert host innate immunity.