Negative Regulation of Interferon-β Gene Expression during Acute and Persistent Virus Infections (original) (raw)
Figure 1
The degradation of IRF3 correlates with the turn-off of IFNβ expression.
A. Virus-induced IRF3 degradation is blocked by CHX treatment. MEFs were treated with SeV and/or CHX for the indicated times. Half of the samples were lysed for total protein extracts and subjected to western blot analysis, probing for IRF3, IRF7, p65 and β-Actin. RNA was extracted from the other half of the sample. cDNAs were prepared and the expression of IFNβ and β-Actin monitored by RT-PCR. B. CHX blocks virus-induced degradation of IRF3 in human cells. Human MG63 or Namalwa cells were treated as in A, total protein was prepared and analyzed for the expression of IRF3 and β-Actin. RNase protection assays (RPA) were conducted to monitor the expression of IFNβ and γ-Actin mRNA (top panel). C. The proteosome inhibitor MG132 only partially inhibits the virus-induced degradation of IRF3. MEFs were infected with SeV in the presence or absence of 1 µM or 50 µM of MG132 for the indicated times, cells were harvested for analysis of IRF3 and β-Actin protein expression or IFNβ and β-Actin mRNA levels. D. The MAVS protein is cleaved and degraded in SeV infected MEFs. MEFs were treated the same as in A, and the level of RIG-I, MAVS, and TBK1 proteins monitored using the appropriate antibodies. E. Cleavage and degradation of MAVS revealed by another antibody. MEFs infected by SeV were lysed at different time points, and subjected to western blot analysis with another anti-mouse MAVS antibody.