Identification Of The Interaction Between Classical Swine Fever Virus E2Protein And Porcine RACK1Protein

Classical swine fever (CSF) is a highly contagious and often lethal disease of pigs and one ofOIE-listed animal diseases, characterized by fever, extensive tissue hemorrhages andimmunosuppression. The disease is caused by classical swine fever virus (CSFV). The E2glycoproteinof CSFV is the most important antigen responsible for the induction of neutralizing antibodies and thebinding and entry of CSFV into susceptible cells.To screen the porcine proteins interacting with the CSFV E2protein, the recombinant plasmidpGBK-E2expressing the E2protein was used as a bait to screen a pGADT7-vectored porcine alveolarmacrophage expression library by yeast two-hybrid. A novel E2-interacting partner, receptor ofactivated protein kinase C1(RACK1), was identified. RACK1, containing seven WD (tryptophan andaspartate) repeat motifs, has previously been identified to specifically bind the activated form of proteinkinase C (PKC) and is assumed to anchor the kinase at membranes in the vicinity of its substrates. TheE2-RACK1interaction was validated by co-transformation of pGBK-E2and pGAD-RACK1into yeaststrain Y2H. The E2-RACK1interaction was further confirmed using GST pull-down assay and confocallaser scanning microscopy. To identify the key region within RACK1interacting with the E2protein,we constructed a series of yeast recombinant plasmids expressing truncated versions of RACK1. Weco-transformed the resulting plasmids and pGBK-E2into the yeast strain Y2H. The resultsdemonstrated that the6th to7th WD repeats of RACK1were the minimal domain interacting with theE2protein. Futhermore, we studied the effect of CSFV infection on the expression of RACK1protein.The result demonstrated that RACK1protein expression in porcine peripheral blood mononuclear cells(PBMCs) of infected pigs was remarkably downregulated after CSFV infection.In summary, this study identified porcine RACK1as a novel binding partner of the CSFV E2protein, WD6-7repeats of RACK1were defined as the minimal domain required for interaction withthe E2protein, and CSFV infection resulted in remarkabe decrease in RACK1protein expression inPBMCs.