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. 2019 Jun 28;93(14):e00081-19. doi: 10.1128/JVI.00081-19

FIG 1.

FIG 1

Genetically dissecting the role of RelA in regulating CHPV propagation. (A) A current model for the cellular defense to RNA virus infections. In general, viral sensing activates the RelA NF-κB heterodimer, which in collaboration with IRF3 induces the expression of type I interferons, including IFN-β. Autocrine and paracrine signals of IFN-β through the cognate IFNAR restrict viral multiplication and produce an antiviral state in neighboring cells. Despite the relevance of CHPV to human health, immune signaling pathways activated by CHPV per se have not been characterized. (B) Primary MEFs derived from WT and Rela−/− mice were infected with CHPV at an MOI of 2; culture supernatants were collected at the indicated time points, and progeny virus yield was measured using a plaque assay. Data represent means of three biological replicates ± SEM. (C) Rela−/− MEFs immortalized using the NIH 3T3 protocol were transduced with retrovirus expressing RelA from a transgene (RelA-Tg). Subsequently, cells were infected with CHPV, and the progeny virus yield was measured. Data represent means of three biological replicates ± SEM. (D) RT-qPCR revealing the relative abundance of viral genomic RNA as well as N and P mRNAs in WT and Rela−/− primary MEFs infected with CHPV an MOI of 2 for the indicated times. The abundance of viral RNAs was normalized to that of beta-actin mRNA. Data represent means of four biological replicates ± SEM. The inset shows the relative level of genome RNA subsequent to viral absorption in WT and RelA-deficient cells. (E) WT and Rela−/− MEFs were infected with CHPV at an MOI of 5 and harvested at the indicated times postinfection, and whole-cell extracts were subjected to immunoblot analyses using antibodies against the indicated viral proteins. GAPDH served as a loading control. The panel on the right shows a densitometric analysis of the relative abundances of N, P, and M proteins quantified from three independent experiments. (F) Effect of shRNA-mediated knockdown of RelA in THP-1 cells on the progeny CHPV titer. Data represent means of three experimental repeats ± SEM. The immunoblot on the left reveals the effect of shRNA-mediated knockdown of RelA expressions. NS, nonsilencing shRNA. Statistical significance was determined using two-tailed Student's t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.