Screening And Identification Of WSSV Immediate-early Protein WSV249 And WSV403 Interacting Proteins

WSSV is a rod-shaped double-stranded circular DNA virus.The virus has a strong pathogenic capacity,a wide range of transmission,and a high fatality rate.It is one of the major pathogens that endanger the shrimp aquaculture industry.In the process of virus infection,immediately early(IE)proteins of the virus play a very key regulatory role.WSV249 or WSV403 are IE proteins of WSSV.They are also E3 ubiquitylating ligase that can be activated by E2 ubiquitin-conjugating enzymes.In the early stage we screened proteins that may interact with WSV249 and WSV403 by tandem affinity purification and mass spectrometry,including the proteins striatin(STRN),Nucleoporin 50(NUP50),Serine/threonine-protein phosphatase 2A 65 k Da regulatory subunit A alpha isoform(PPP2R1A),fibroblast growth factor receptor 1 oncogene partner(FGFR1OP),karyopherin subunit alpha 6(KPNA6)that may interact with WSV403,and Sequestosome1(SQSTM1)that may interact with WSV249.In this experiment,we cloned and expressed these screened human genes and co-transfected with WSV403 or WSV249 in 293 T cells.Their interaction with WSV403 or WSV249 protein was verified by co-immunoprecipitation,confirming that WSV403 can bind to KPNA6,PPP2R1 A and STRN,and WSV249 can bind to SQSTM1.In order to verify whether the corresponding shrimp proteins homologous to these proteins interact with WSV403 or WSV249,we cloned the corresponding gene in L.vannamei and co-transfected with WSV403 or WSV249 in 293 T cells,and then we detected the interaction between the shrimp protein and the IE protein by immunofluorescence and co-immunoprecipitation.The results showed that the shrimp STRN protein(Lv-striatin-3)binds to WSV403 and co-located in the cytoplasm in a dot shape.To understand the role of Lv-stritin-3 in WSSV infection,we further analyzed the tissue distribution of Lv-stritin-3 and changes in expression level with WSSV infection by RT-PCR.Lv-stritin-3 was found to be expressed in all tested tissues,with the highest expression in brain;however,WSSV infection did not significantly affect the transcription level of Lv-stritin-3.