PRRSV Viral Genes Cloning and Reporter Assay

The HA-PIAS1 and p65 genes were cloned into the pXJ41 expression vector. The nonstructural and structural genes were individually cloned in our laboratory from the FL12 infectious clone of PRRSV as the template^{80 (link)}. The coding sequences were PCR-amplified to contain ATG translation initiation and TAG termination codons. A FLAG-tag was added at the N-terminus of each gene. For membrane protein genes such as nsp2, ORF2, ORF2a, ORF5, ORF5a, and ORF6, the FLAG-tag was added to the C-terminus. The C-terminal and N-terminal deletion mutants of PIAS1 gene (pHA-PIAS1-NTD and pHA-PIAS1-CTD, respectively) were cloned into pXJ41 using the Eco RI- and Xho I-recognition sequences. The series of N gene deletion mutant are described elsewhere^{48 (link)} and were subcloned to pXJ41. The pNF-κB-luciferase reporter plasmid was purchased from Stratagene Inc (La Jolla, CA). The pRL-TK Renilla luciferase reporter plasmid was purchased from Promega (Madison, WI). The pHA-SUMO1, pHA-SUMO2, and pHA-SUMO3 plasmids were obtained from Dr. L. Flamand (Laval University, Quebec, Canada).