The coronavirus macrodomain is required to prevent PARP-mediated inhibition of virus replication and enhancement of IFN expression
(A) BMDMs were infected with WT or N1347A MHV and treated with 5 μM compound 8K or vehicle (DMSO) following a 1-hour adsorption phase. Cells were collected at 12 hpi, and IFNβ mRNA levels were determined by RT-qPCR. The data in (A) represent the combined results of three experiments; n = 9. (B) PARP14-/- or PARP14+/- littermate control BMDMs were infected with WT or N1347A virus. Cells were collected at 12 hpi, and IFNβ mRNA levels were determined by RT-qPCR. The data in (B) show one experiment representative of four independent experiments. (C) DBT cells were transfected with plasmid expressing GFP, PARP14, or a PARP14 catalytic mutant (CM). Cells were collected at 24 hours after transfection, and IFNβ mRNA levels were determined by RT-qPCR. The data in (C) show one experiment representative of two independent experiments; n = 3. (D) Six clones of CRISPR/Cas9-gRNA-mediated PARP14 KO A549 cells or gRNA-NC-transduced (irrelevant gRNA) control A549 cells were collected and analyzed by immunoblotting for PARP14 protein. (E) Schematic diagram illustrating the antiviral conditioned media assay (see Methods for details). (F,G) At 16 hpi, VSV replication on WT or IFNAR KO A549 cells was analyzed by fluorescence microscopy (F), and titers were determined by plaque assay on Vero cells (G). The images in (F) are of a single experiment representative of three independent experiments. Titers in (G) were normalized to those in cells receiving mock-treated conditioned media. The data in (G) show the combined results of three independent experiments; n = 3. x-axis labels represent VSV-infected cells. p-values represent comparisons of each poly(I:C) (pI:C)-treated PARP14 KO cell conditioned media to poly(I:C)-treated WT cell conditioned media used to pretreat WT or IFNAR KO A549 cells. Numbers above bars represent fold difference between WT and N1347A or between siPARP/siCtl or inhibitor/DMSO-treated cells infected with N1347A virus.