Effect Of ABHD16A/ABHD17A On Japanese Encephalitis Virus Infection By Dynamically Regulating The Palmitoylation Cycle Of IFITM

Interferon is a cytokine that is induced by innate immune pathways and can resist viral infection by inducing the expression of interferon stimulated genes(ISGs).Interferon-induced transmembrane protein(IFITM)is an antiviral protein encoded by ISG that prevents the fusion of viruses with cell membranes.Studies have shown that the antiviral activity of IFITM depends on the S-type palmitoyl modification of cysteine residues.However,research on key enzymes mediating palmitoyl modification of IFITM is relatively weak.Therefore,it is of great significance to study the regulation of palmitoylation and the effect of palmitoylation status on virus replication.Our previous research team found that ABHD16 A in humans and pigs is a palmitoylase that negatively regulates the level of palmitoylation modification of IFITM and its antiviral ability.In order to further explore the regulatory mechanism of ABHD16 A in the immune pathway,the laboratory constructed a HEK293-abhd16a-/-cell line,and transcriptome sequencing analysis found that the deletion of the adhd16 a gene downregulated the expression of adhd17 a.Previous studies have reported that ABHD17 A can regulate the palmitoylation level of proteins such as N-Ras.Is ABHD17 A involved in the palmitoylation regulation of IFITM? This experiment started with the study of the interaction between mouse ABHD16A(m ABHD16A)and IFITM(m IFITM),further exploring the regulation of human and mouse ABHD17 A on palmitoylation of IFITM,and analyzing its impact on the subcellular localization and antiviral ability of IFITM.This study uses Co-IP and BiFC methods to study interactions;A new palmitoylation detection method,Acyl-PEGyl exchange gel shift(APEGS),was used to detect the palmitoylation level of proteins;Using laser confocal imaging technology to study protein subcellular localization;Further,RT-q PCR and Western Blot techniques were used to detect the copy number and protein of related viruses.The main results of this study are as follows:(1)m ABHD16 A interacts with m IFITM3,but does not interact withIFITM1 and IFITM2.The result of the interaction is that m ABHD16 A reduces the palmitoyl modification level of m IFITM3,thereby negatively regulating the antiviral ability of m IFITM3.(2)hABHD17 A interacts with hIFITM1,and m ABHD17 A interacts with m IFITM3.The result is that ABHD17 A increases the palmitoyl modification level of IFITM,promotes the localization of IFITM on the cell membrane,and thereby enhances the antiviral ability of IFITM.(3)The key active sites of ABHD17 A were identified as cysteine residues at positions 101 and 106.This study is the first to find that ABHD17 A enhances its antiviral ability by increasing the palmitoyl modification level of IFITM.The interaction between m ABHD16 A and m IFITM3 was identified for the first time,but there was no interaction between m ABHD16 A and m IFITM1 and m IFITM2.The results of this study suggest that ABHD16 A and ABHD17 A are likely to dynamically regulate the palmitoylation cycle of IFITM.The regulation of ABHD16A/ABHD17 A on the palmitoylation cycle of IFITM contributes to a deeper understanding of the antiviral immune mechanism of this innate broad-spectrum antiviral protein,and expands the spectrum of proteins involved in palmitoylation modification and antiviral ability regulation of IFITM The research and development of antiviral drugs targeting ABHD17 A and ABHD16 A has certain theoretical significance and application value.