Figure 6

Hrd1 inhibition of viral replication via its E3 ligase activity. A Overexpression of Hrd1 inhibited the CDV N protein level during viral infection. Vero cells were transfected with Myc-Hrd1 or Myc-C329S mutant for 24 h, and then the transfected cells were infected with CDV 851 (MOI = 0.1) for an additional 72 h. The protein levels of cell lysates were determined by Western blot using anti-CDV N, anti-Myc, and anti-GAPDH antibodies. ImageJ was used to quantify the ratio of CDV N to GAPDH. B Overexpression of Hrd1 inhibited the N mRNA level and viral titers during viral infection. Vero cells were transfected with Myc-Hrd1 or Myc-C329S mutant for 24 h and then infected with CDV 851 for an additional 72 h. RT-qPCR was used for examination of mRNA levels of CDV N and GAPDH. The viral titer of culture supernatants were determined by the Reed–Muench method. C Knockdown of Hrd1 increased N protein level during viral infection. Vero cells were transfected with siRNA for 24 h and then infected with CDV 851 for an additional 72 h. Protein levels were determined by Western blot using anti-CDV N, anti-Hrd1, and anti-GAPDH antibodies. ImageJ was used to quantify the ratio of CDV N to GAPDH. D Blockage of the proteasome pathway restored the inhibition of viral replication by Hrd1. Vero cells were transfected with Ha-Hrd1 for 24 h and then infected with CDV 851 (MOI = 0.1) for 72 h in the presence of MG132 (20 μM). The lysates of harvested cells were subjected to Western blot analysis using anti-CDV N, anti-Ha and anti-GAPDH antibodies. Image J was used to quantify the ratio of CDV H to GAPDH. All results are shown as means ± SD from at least three separate sample preparations. *p < 0.05, **p < 0.01.