Skip to main content
. 2019 Nov 26;93(24):e01499-19. doi: 10.1128/JVI.01499-19

FIG 12.

FIG 12

CARD11 inhibits viral polymerase activity. (A) Establishment of NDV MG systems. The LaSota strain MG system was constructed under the T7 promoter with a firefly luciferase reporter (MG-Fluc). For the 3-plasmid system, the BHK-21 cells were cotransfected with pCAGGS-T7, pCMV-NP-P-L, and MG-Fluc at a ratio of 2:1:1. For the 5-plasmid system, the BHK-21 cells were cotransfected with pCAGGS-T7, MG-Fluc, pcDNA3-HA-NP, pcDNA3-HA-P, and pCAGGS-L at a ratio of 5:5:2:2:1 or without the three support plasmids, pCAGGS-L, and pCAGGS-T7. (B) CARD11 dose-dependently inhibits viral RNA polymerase activity using the 3-plasmid system. The BHK-21 cells were cotransfected with the 3-plasmid system and different doses of pcDNA3-2×Flag-CARD11. β-Tubulin was used as a protein loading control. (C to E) Viral RNA polymerase activity with different doses of P, NP, or L and CARD11 (1 μg) using the 3-plasmid system. The BHK-21 cells were cotransfected with the 3-plasmid system, pcDNA3-2×Flag (1 μg) or pcDNA3-2×Flag-CARD11 (1 μg), and additional P, NP, or L constructs (pcDNA3-HA-NP, pcDNA3-HA-P, or pCAGGS-L). (F) Inhibition of viral RNA polymerase activity by the CARD11 CC1 domain using a 3-plasmid system. The BHK-21 cells were cotransfected with the 3-plasmid system and truncated CARD11 constructs (1 μg) as well as with negative-control plasmids. β-Tubulin was used as a protein loading control. (G) The viral polymerase activity of P truncations and CARD11 (1 μg) using the 5-plasmid system. The BHK-21 cells were cotransfected with pCAGGS-T7, MG-Fluc, pcDNA3-HA-NP, pcDNA3-HA-P or pcDNA3-HA-P ΔX or pcDNA3-HA-P ΔPMD, and pCAGGS-L at a ratio of 5:5:2:2:1 or with pcDNA3-2×Flag-CARD11 (1 μg). In addition, the 5-plasmid system without the P was detected as previously described. All the cells above were cotransfected with pRL-SV40-N expressing Renilla luciferase as a normalizing standard and were lysed at 36 h posttransfection. All the representative data, shown as the means ± SDs (n = 3), were analyzed by two-tailed Student's t tests. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.