The Study On Palmitoylation Of Chikungunya Virus NsP1

Palmitoylation is an important form of lipid post-translation modification,which affects the function of protein by regulating its transport,stability and localization.In particular.S-Palmitoylation is a common form of palmitoylation.Chikungunya virus,a member of alpahviruses,Toaviridae,is an enveloped virus with a single positive strand RNA genome.In this study,we used the protein palmitoylation predicted software to predict its palmitoylated sites and found that the palmitoylated sites of nsP1 were located in C417-419.We constructed the plasmids of single-site mutations and three-site mutation of this region,and found that three sites were the key sites of nsP1 palmitoylation by Acyl-Biotin Exchange(ABE)assay.Previous studies have shown that fatty acid synthase(FASN)inhibitors,C75 and cerulenin,could repress the CHIKV replication,however,the mechanism is still not clear.We found that the product of FASN,palmitate,could rescue the inhibition of FASN inhibitors on viral replication.These results indicated that the inhibition of the FASN inhibitors on the virus was due to their influence on nsP1 palmitoylation.Protein palmitoylation is mainly mediated by zinc-finger D-H-H-C domain containing-palmitoyltransferase(ZDHHCs).However which ZDHHC palmitoylate nsP1 is not known.In this study,we tried to screen the ZDHHC that mediates CHIKV nsP1 palmitoylation.Firstly,we overexpressed 23 ZDHHCs and CHIKV nsP1 in 293T cells and found that ZDHHC2 and ZDHHC 19 interact with nsP1 strongly by co-immunoprecipitation assay.Moreover,silcenging ZDHHC2 and ZDHHC 19 with siRNA inhibited nsP1 palmitoylation and CHIKV replication significantly.In summary,our research focused on S-palmitoylation of CHIKV nsP1 and determined its palmitoylation sites.We also clarified that FASN inhibitor could weaken the palmitoylation of nsP1.Preliminary study suggested that ZDHHC2 and ZDHHC 19 were the mainly enzymes catalyzed nsP1 palmitoylation and revealed the mechanism of CHIKV nsP1 palmitoylation.Moreover,we explored the roles of the key components of COPI and COPII in CHIKV replication.