Figure 2

ARV σA protein enhances the proteasome activity and expression level of PSMB6. (A) Vero cell lysates from ARV-infected, σA-transfected, and PSMB6- depleted cells with ARV at an MOI of 10 for 24 hours were used to quantify relative proteasome activity. (B) Examination of the PSMB6 levels. Vero cells were infected with ARV at an MOI of 10 at indicated time points, followed by Western blot assay with an anti-PSMB6 antibody. (C) Analysis of mRNA levels of PSMB6 and other subunits by real-time RT-PCR.The data reveal that PSMB6 is transcriptionally upregulated by σA. (D) To confirm whether PSMB6 mediates ribosomal protein ubiquitin-proteasome-mediated degradation and inhibits Akt phosphorylation at S473, Vero cells were transfected with plasmids overexpressing PSMB6 followed by Western blot assay with the indicated antibodies (E) In the presence of MG132, the decrease in the levels of p-Akt (S473) in ARV-infected vero cells could be reversed in ARV-infected cells. (F) The levels of PSMB6, Rpl26, Rpl27, and p-Akt (S473) were examined in ARV-infected cells co-transfected with an shRNA against σA. (G) To confirm whether both PSMB6 and MDM2 mediate ribosomal proteins degradation, knockdown of either PSMB6 or MDM2 with shRNAs was performed followed by Western blot analysis with the indicated antibodies. (H) Vero cells were transfected with pcDNA3.1-p17 or co-transfected with pcDNA3.1-p17 and pcDNA3.1-σA plasmids for 24 hours, respectively, followed by Western blot assays with the indicated antibodies. (I) Individual 24-well plates of Vero cells were transfected with a PSMB6 shRNA for 24 hours, followed by ARV infection at an MOI of 5 for 24 hours. The ARV-infected cell supernatant was collected at 24 hpi for determining the virus titer. All data shown represent the mean ± SD calculated from three independent experiments. (J) A model depicting the cooperation between p17 and σA proteins of ARV to trigger ribosomal protein degradation is shown. The protein levels were normalized to those for β-actin. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The activation and inactivation folds indicated below each lane were normalized against against values for the mock control or at 0 h. The uncropped blots with molecular weights are shown in Figs S5 and S6.