Regulation Of Poly(I：C)-induced Expression Of Multiple Cytokines By MuV N Protein Phase Separation

Mumps virus(MuV)is a single-stranded negative-sense RNA virus that can cause diseases such as mumps and meningitis in children and adolescents.MuV can continue to proliferate in the host after infection and trigger a series of immune responses.Nearly90% of individuals will be infected with mumps virus when not vaccinated,and even in populations that have received preventive vaccination with the MMR vaccine,the re-emergence of mumps is not uncommon.However,research on the inflammatory responses targeted by mumps virus in various cells is not yet sufficiently comprehensive.The mechanism by which mumps virus regulates the expression and activity of host cell cytokines during mumps infection to avoid immune killing is still not clear.Additionally,based on previous literature,various viruses can induce liquid-liquid phase separation(LLPS)within cells to regulate various biochemical processes to evade innate immune responses.However,it has not been reported whether MuV can induce LLPS and whether LLPS is involved in the regulation of host cell innate immune responses.This article used various methods such as in vitro pre-aggregation,intracellular transfection,and fluorescence recovery after photobleaching(FRAP)to verify the phase separation ability of the screened N protein.The downstream cytokine of the TLR3 pathway was detected to study the effect of the N protein on the pathway.Finally,the protein interaction was detected by COIP-MS pulldown of the phase-separated aggregates formed by the N protein.The main research content is as follows:Firstly,the MuV genome was screened for phase-separated proteins,and the ability of the recombinant expressed protein to pre-aggregate in vitro was verified.The amino acid sequences of the seven MuV genome-encoded proteins were from the NCBI protein database,and four different prediction algorithms were used to screen the phaseseparated proteins from various perspectives.The N and P proteins were screened as the most potential phase-separated proteins.The N protein of the MuV was codonoptimized and heterologously expressed and purified in E.coli,while the P protein was available in the lab.The in vitro turbidity experiment verified that the N protein had pre-aggregation ability,while the P protein could not form aggregated particles in vitro.Next,to further explore the effect of the N protein on the innate immune TLR3 pathway,we detected the mRNA expression of six downstream cytokines of the TLR3 pathway,including IL-6,CCL2,IFN-α,TNF-α,CXCL10,and IFN-β,by RT-qPCR.The transcription of these cytokines was upregulated by poly(I:C)induction,but their relative expression levels were downregulated after N protein transfection.Among them,IL-6,CXCL10,and IFN-β were downregulated most significantly.To further verify the regulation of the TLR3 pathway by the phase separation of the N protein,we detected the interferon IFN-β content in the cell supernatant of the experimental group by ELISA.The results confirmed that after the TLR3 pathway was activated by poly(I:C),the transfection of the N protein could downregulate the expression of downstream signaling molecules of the TLR3,indicating that the N protein has an inhibitory effect on the virus’ s innate immune response mechanism.After verifying the pre-aggregation ability of the N protein in vitro,its ability to undergo phase separation was further confirmed.N-p EGFP-N1 and P-pc DNA3.1-m Cherry plasmids were constructed,and transient transfection was performed in host cells.The results showed that N protein could form aggregation points(foci)in cells after single transfection,while P protein could not form aggregation points in cells after single transfection,and the results of co-transfection of N and P proteins also supported this.After that,the FRAP experiment was performed,and a part of the fluorescence was extinguished by laser after the N protein formed droplet-like aggregates.The fluorescence in the extinguished area could recover within a few seconds after bleaching.These results proved that the MuV N protein can indeed undergo phase separation.To further explore the key region that regulates the phase separation process in the N protein,we constructed truncated forms of the N protein and found that the N protein could not undergo phase separation when lacking the IDR structure domain.Finally,CO-IP-MS technology was used to identify and analyze the interacting proteins in the N protein LLPS condensates.The recombinant N protein containing an EGFP tag was immobilized on Anti-GFP magnetic beads.As N protein can undergo phase separation in cells,the proteins in the phase-separated fraction were co-purified with the N protein by the magnetic beads.The protein-bead complex was denatured and the protein bound to the beads was eluted.The eluted proteins were separated by SDSPAGE gel electrophoresis,and the differential bands were cut and analyzed by mass spectrometry to identify the interacting proteins in the LLPS condensates.This study screened the most potential protein with phase separation ability by analyzing the full-length proteins encoded by the MuV viral genome,and first determined that the nucleocapsid protein of mumps virus can undergo phase separation.Moreover,the experimental results confirmed that the N protein can downregulate downstream signaling molecules of TLR3 after activation of the TLR3 pathway by poly(I:C).By combining CO-IP with mass spectrometry,the protein components in the N protein LLPS condensates were analyzed.This study helps to further understand the mechanism of the inherent immune evasion after MuV viral infection.As many viruses undergo LLPS during the infection process and play critical physiological functions,disrupting this phase separation process may interfere with the viral lifecycle,providing a new target for the development of broad-spectrum antiviral drugs.It is of great significance.