Mechanism Of SPCS1 Regulates The Replication Of Japanese Encephalitis Virus

Japanese encephalitis virus(JEV)is a zoonotic mosquito-transmitted arbovirus belonging to the genus Flavivirus in the family Flaviviridae.In addition to JEV,many other flaviviruses are important human pathogens,such as West Nile virus(WNV),Dengue virus(DENV),Yellow fever virus(YFV),and Zika virus(ZIKV).JEV is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen.Its clinical symptoms include fever,encephalitis,even to death.There are no specific antiviral treatments for JE.In this study,we found SPCS1 is crucial for virus replication which could be a potential target for the development of novel therapeutics.We also found the host factor SPCS1 regulates flavivirus assembly through interaction with hydrophobic transmembrane domains of JEV NS2 B protein.With siRNA interference technique,we verified that host factor SPCS1 plays an important role in the replication of JEV.Then useing SPCS1 KO HEK-293 cell line,we further surveyed the effects of SPCS1 on JEV replication.And then we assessed cell entry of JEV using single-round infectious GFP expressing reporter replicon particles,and SPCS1 knockout cells showed the similar infectivity to WT cells.Quantitative comparison of JEV genome RNA in SPCS1 knockout and WT cells during the early stages of infection revealed no significant differences.When infected with JEV at a high MOI,structural(prM and E)and non-structural(NS1)proteins were detected in cell lysates of both WT and SPCS1 KO cells.But in SPCS1 KO cells,high molecular mass bands of respective proteins were detected,which means that the post-translational polyproein processing were impaired.Using a single-round infectious chimeric JEV reporter replicon particle packaging system,we revealed that SPCS1 knockout impaired the assembly of infectious virus particles The results suggest SPCS1 is most likely involved in the assembly of infectious viruses rather than cell entry,RNA replication,or viral proteins expression during the JEV lifecycle.To dissect the molecular mechanisms by which SPCS1 modulates viral assembly,we employed the Venus-based BiFC system,and found that the JEV non-structural protein NS2 B interacts with the host factor SPCS1,and confirmed interactions between SPCS1 and NS2 B from WNV and ZIKV,suggesting the interaction is conserved throughout the flavivirus genus.Then we verified the interaction results of SPCS1 and NS2 B with co-immunoprecipitation.Through serial deletion mutation of JEV NS2 B potein,it was found that there are two transmembrane domains NS2B(1-49)and NS2B(84-131)interact with SPCS1 respectively.Further mutagenesis analysis at the flavivirus conserved residues with two SPCS1 interaction domains of NS2 B demonstrated that G12 A,G37A,G47 A in NS2B(1-49)and P112 A in NS2B(84-131)weakened the interaction with SPCS1.Through serial deletion mutation of SPCS1,it was found that SPCS1(91-169)interact with NS2B(1-49)and NS2B(84-131)respectively.Therefore,the data in this study demonstrate that SPCS1 affects viral replication by interacting with NS2 B that affects the post-translational processing of the JEV viral proteins and the assembly of virions.