The Role Of P53 In Regulating IFITM3 Palmitoylation Against Japanese Encephalitis Virus

Japanese encephalitis virus(JEV) is a mosquito borne flavivirus, which has ability to induce encephalitis in humans and reproductive disorder in pigs, and is a very important emerging pathogens of public health significance. Tumor suppressor p53 has been proved to regulate various biological process, such as cell cycle arrest, cellular metabolism, cellular apoptosis and innate antiviral immunity. Compared to p53 WT mice, JEV-infected p53 KO exhibited higher viremia, mortality rate and more serious inflammation in brain, which indicates the important role of p53 in inhibit JEV infection.Except the p53-dependent enhancement of IFN signaling and p53-induced apoptosis in virus infection, maybe there are some unknown antiviral mechanism needs to be discovered. To investigate the anti-JEV mechanism of p53 and host-JEV interaction, these studies were performed. Basing the differential transcriptomic profile of mouse brain infected with JEV, IFITM3 expression obviously upregulated in JEV-infected p53 WT mouse comparing to p53 KO mouse, and displayed a similar expression trend with p53-regulated innate immunity molecules, such as TLR3,IRF9 and GBP1.. In order to investigate the relationship between transcriptional factor p53 and antiviral protein IFITM3, we first want to clarify whether p53 affects IFITM3 transcription. The mRNA level of IFITM3 exhibited no remarkable relationship with different p53 status, which indicated the transcriptional expression of IFITM3 wasn’t regulated by p53. Surprisingly, there was an obvious positive correlation between IFITM3 protein abundance and p53 status. Protein palmitoylation influences protein trafficking, protein stability and protein aggregation, and is regulated by protein acyltransferases(PATs) and acylprotein thioesterases(APTs). We asked whether IFITM3 palmitoylation is the bridge between p53 different status and IFITM3 protein expression. After treatment with 2-bromopalmitate(2BP), the most commonly used palmitoylation inhibitor, the function that p53 increased IFITM3 protein abundance was abolished. 2-BP obviously decreased IFITM3 protein expression and didn’t affect the transcription of IFITM3. The lysosomal degradation pathway is the primary site of IFITM3, and the half-life of IFITM3 was significantly reduced to 2h after 2-BP treatment fron 5h in ethonal treatment conditions, but increased to 10 h when incubated with leupeptin. These results clearly shows that palmitoylation enhances the protein stability of IFITM3.Protein S-palmitoylation is regulated by PATs and APTs reversibly. Since palmitoylation was a critical bond between p53 and IFITM3, so we wanted to identify the potential members of ZDHHC and APTs families regulated by p53, which played a role to modify IFITM3 palmitoylation and enhance IFTM3 stability. ZDHHC1 expression was found to be regulated by p53 both at mRNA and protein level. To further investigate the contribution of ZDHHC1 to IFITM3 protein stability, ZDHHC1 overexpressed in different cells increased IFITM3 protein expression but didn’t affect IFITM3 gene transcription, which illuminated that ZDHHC1 enhanced IFITM3 protein stability. Further study proved that ZDHHC1 and IFITM3 protein expression exhibited coincident expression pattern with p21 following p53 different status. To estimate antiviral activity of p53-ZDHHC1-IFITM3 regulatory pathway, JEV replication ability was determined in p53, ZDHHC1 and IFITM3 knockdown alone or combinated Nutlin-3 treatment in A549 cells. The results exhibited anti-JEV effect of p53-ZDHHC1-IFITM3 pathway and established a novel antiviral mechanism of p53 that p53 enhances IFITM3 protein stability by regulating palmitoyl transferases ZDHHC1 expression to exhibit anti-JEV effect. Meanwhile, JEV also evolved with evasion mechanism to antagonize antiviral role of p53, NS4 A was able to detain p53 into the cytoplasm and inhibt transcriptional activity of p53, which contributed to JEV replication.Our study revealed an unrecognized anti-JEV pathway that p53 regulated ZDHHC1 expression to enhance IFITM3 protein stability to limit JEV infection and replication. The finding that DHHC palmitoyl transferase ZDHHC1 was regulated by p53 enriched the biological function of p53. All this results provided a theoretical basis for understanding antiviral mechanism of p53 and drug design targeting this antiviral pathway to threat virus infection.