Cloning of IBDV VP2 Gene and p62 Mutants

The IBDV VP2 gene from segment A was cloned into the pCMV-Flag-N vector (635688; Clontech, Mountain View, CA) and the pCMV-Myc-N vector (635689; Clontech, Mountain View, CA). All mutants of pCMV-Flag-VP2 and rescue plasmid T7-A were constructed by site-directed mutagenesis technology. Expression plasmid pEGFP-LC3B was constructed in our laboratory (33 (link)). The human p62 gene was amplified by PCR from total cellular RNA with gene-specific primers (5′-GCGAATTCGCATGGCGTCGCTCACC-3′ and 5′-GCTCTAGAGCTCACAACGGCGGGGG-3′) and subcloned into the pCMV-Flag-N vector and the pCMV-Myc-N vector. The mutants of Myc-p62, Myc-p62△UBA, and Myc-p62-△LIR were constructed with special primers. The primers 5′-TTGTACCCACATCTCCCCCCGCCGTTGTGA-3′ and 5-TCACAACGGCGGGGGGAGATGTGGGTACAA-3′ were used for Myc-p62△UBA. The primers 5′-GAGGAGATGATGACTCTTCAAAAGAAGT-3′ and 5′-ACTTCTTTTGAAGAGTCATCATCTCCTC-3′ were used for Myc-p62△LIR. HA-Ub, HA-K48, and HA-K63 expression plasmids were kindly gifted by Hongbin Shu (College of Life Sciences, Wuhan University). All expression plasmids were transfected into 293T cells and DF-1 cells using Lipofectamine 3000 reagent (L3000015; Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.