Discovery Of SARS-CoV-2 NSP10-11 And A Preliminary Study On Its Interaction With Host

Severe acute respiratory syndrome coronavirus 2(SARS-Co V-2),also known as a novel coronavirus,can induce immune pathological damage characterized by the abnormal elevation of cytokine levels,referred to as a cytokine storm.Cytokine storm is an important indicator of the severity of COVID-19 infection,and its pathophysiological mechanisms are not yet fully elucidated.The main protease of COVID-19,NSP5 plays an important role in the cytokine storm.NSP5 is an endopeptidase,and we speculated that it may have an incomplete cleavage of the NSP10-11 recognition site located at the C-terminus of the polyprotein,resulting in the formation of NSP10-11 dimer.In the preliminary stage of this study,Western blot analysis of the COVID-19 cell culture lysate using an NSP10 protein antibody given two bands,and the molecular weight of the bands was used to preliminarily determine the natural form of NSP10-11 dimer in COVID-19.Further investigation of the interaction between NSP10-11 dimer and host cells will help expand our understanding of the pathogenic mechanisms of SARS-Co V-2.Based on preliminary evidence showing that the SARS-Co V-2 virus naturally exists as an NSP10-11 dimer,this study used a dual plasmid transfection of NSP5 and NSP10-11 in HEK-293 T cells to investigate the forms and distribution of NSP10-11 production.The experiments were performed using NSP10-11 and NSP5 in a ratio of 1:3-3:1,and then tested the NSP5’s cleavage effect on NSP10-11.The experiments used human coronavirus NL63(HCo V-NL63)as a reference and GC376 as an NSP5 enzyme inhibitor.Additionally,the study constructed an m TFP1-S2NSP10-11-Venus eukaryotic expression vector and used fluorescence resonance energy transfer(FRET)to examine NSP5’s cleavage activity on NSP10-11.The results showed that in the natural range of 2:1 NSP5: NSP10-11,the SARSCo V-2 NSP5 enzyme failed completely cleave NSP10-11,resulting in the existence of partially formed NSP10-11 connector.In contrast,NSP10-11 in HCo V-NL63 was entirely cleaved by its NSP5,and NSP10 and NSP11 existed as monomers.FRET analysis of SARSCo V-2 NSP5 activity showed that as the amount of NSP5 gradually increased,the FRET phenomenon only became weaker but still visitable when the NSP5: NSP10-11 ratio was within the natural range.This suggested that NSP5’s activity did entirely cleave NSP10-11,and m TFP1 and Venus continued to undergo energy transfer,indicating that weak NSP5 enzyme activity on the cleavage site between NSP10 and NSP11 may be essential for the existence of SARS-Co V-2 NSP10-11 connector.Based on the preliminary study of the cause of the NSP10-11 dimerization in SARSCo V-2,we further investigated the subcellular localization of NSP10 and NSP10-11.NSP10 was found to be located in the perinuclear region and cytoplasm,while NSP10-11 was found to be located in both the cytoplasm and nucleus.We conducted cell immunofluorescence experiments and used Image J software to analyze the changes in NSP10-11 localization after transfecting cells with NSP10-11 alone,NSP10-11 and NSP5,or NSP10-11 and NSP5 and treating them with GC376 for 12 hours.The results showed that NSP10-11 was located in both the cytoplasm and nucleus,and the uncut N-terminus of NSP10-11 was located in the nucleus and cytoplasm while the C-terminus was consistent with the N-terminus and NSP10 was located in the perinuclear region and cytoplasm.After inhibiting NSP5 enzyme activity with GC376,NSP10-11 was found to be located in both the cytoplasm and nucleus,indicating that NSP10-11 and NSP10 have different subcellular localizations.We continued to investigate the host interaction proteins of NSP10-11 and their impact on host protein function.Using the combination of immunoprecipitation-mass spectrometry(IP-MS)and Co-IP,we found that NSP10 interacted with NKRF,stimulating the expression of IL-8(consistent with previous reports),and knocking down NKRF weakened this effect.NSP10-11 had no interaction with NKRF,but instead,it interacted with FAF2,which induces the upregulation of the expression of endoplasmic reticulum stress factor ATF6,and knocking down FAF2 weakened the effect.In summary,this study preliminarily experimentally verified the unnatural existence of NSP10-11 dimer in SARS-Co V-2 and found that the low enzyme activity of NSP5 is one of the causes for the existence of NSP10-11 dimerization.It also confirmed that NSP10-11 interacts with FAF2 to induce the upregulation of the endoplasmic reticulum stress factor ATF6 expression.Information yielded in this study expands our understanding of the mechanistic interaction between SARS-Co V-2 and host cells.