Preliminary Studies On Regulation Of JAK-STAT3 Signaling Pathway By Varicella-zoster Virus Immediate-early Protein 62

Varicella-zoster virus(VZV)causes chickenpox(varicella)and shingles(herpes zoster,HZ).Chickenpox is caused by primary infection with VZV,which can establish long-term latency in the host ganglia.In older and immunocompromised hosts,the virus can be reactivated to cause shingles,which may impact the quality of life.Currently,the VZV-induced diseases are lack of any specific treatment.Recent advances in bacterial artificial chromosome(BAC)technology and humanized SCID mouse model have greatly facilitated studies on functions of VZV genes and their products,virus-host interactions in VZV pathogenesis.A previous study showed that VZV infection can trigger STAT3 phosphorylation in human embryonic lung fibroblasts(HELF)in vitro and in human skin xenografts in SCID mice in vivo;moreover,small-molecule inhibitors of STAT3 phosphorylation restrict VZV replication in vitro,and also impair the VZV infection of skin xenografts in vivo.These results indicated that activation of STAT3 is required for the VZV replication.However,the mechnaism of activation and regulation of the JAK-STAT3 signaling pathway in the host cell by VZV remain obscure.The exploration of these questions could improve our understanding of VZV pathogenesis.In this study,phosphorylation of STAT3 was firstly verified in VZV-rOka infected MRC-5 and HFF-1 cells.Then,by inhibiting viral protein synthesis and UV-induced DNA damage,it was confirmed that the phosphorylation of STAT3 was caused by the expression of VZV viral proteins.Next,to identify which VZV protein could activate STAT3,part of VZV proteins have been screened using a STAT3 luciferase reporter assay in 293T cells;the results showed that VZV immediate-early protein 62(IE62)can activate STAT3.Subsequently,phosphorylated STAT3 were detected by western blotting,and the mRNA levels of downstream genes of the STAT3 pathway were shown to be upregulated by RT-PCR when IE62 was expressed alone after transient transfection of 293T cells.These results suggested that IE62 can activate the whole STAT3 signaling pathway.Furthermore,by JAKs knockdown using siRNA,it was demonstrated that JAK1 kinase was required for the IE62-mediated activation of STAT3 in a cell-based luciferase assay.On the other hand,it was shown that IE62-mediated of STAT3 can be inhibited by co-expression of VZV ORF66 kinase and mutation of the nuclear localization signal of IE62.It was,therefore,presumed that nuclear localization of IE62 was associated with its function in STAT3 activation.In addition,using high-throughput transcript sequencing(RNA-seq),differences in expression of 256 genes,seven of which showed dramatic and highly significant differences,were observed between IE62-expressing 293T cells and mock-transfected control ones.Finally,it was found that STAT1 and STAT2 can also be activated by IE62 expression using an ISRE luciferase reporter assay.However,whether the mechanism of IE62-mediated activation of STAT1 and STAT2 is same as that of STAT3 should be further studied in the future.In conclusion,this research confirmed the phosphorylation of STAT3 in VZV-infected MRC-5 and HFF-1 cells in vitro.IE62 was verified to be able to trigger STAT3 activation after screening part of VZV proteins.Further studies showed that IE62-mediated activation of STAT3 signaling pathway was JAK-1-dependent,and the activation of STAT3 may be related to the nuclear localization of IE62.Finally,effects of IE62 expression on cellular genome expression was analyzed.In addition,STAT1 and STAT2 were also found to be activated by IE62.Overall,the present study provided a prelimilary evidence for the activation and regulation of JAK-STAT3 signaling pathway by VZV IE62,and laid the foundation for a better understanding of VZV pathogenesis in future.