
 Fig . 6. p38 mediates BPXV mRNA translation by regulating p38–MNK1–eIF4 E  signaling axis. (A/B)  MNK1 and eIF4E inhibition suppresses BPXV replication.  Vero cells, in triplicates, were infected with BPXV at MOI of 0.1 in the presence of 1 μg/mL of CGP57380 (MNK1 inhibitor), 2 μg/mL 4EGI-1 (eIF4 E  inhibitor) or equivalent volumes of DMSO. At 48 hpi, infectious virus particles released in the infected cell culture supernatants were quantified by plaque assay. Reduction in viral yields in the presence of CGP57380 ( A ) and 4EGI-1 ( B ) is shown. ( C ) p38 inhibition blocks binding of the viral mRNA with eIF4E (CHIP assay): Vero cells were infected with BPXV at MOI of 5. At 6 hpi, the cells were treated with SB239063, CGP57380 (MNK1 inhibitor) or DMSO. At 16 hpi, cell lysates were prepared as per the procedure described for CHIP assay (materials and method section). The clarified cell lysates were incubated with α-eIF4 E  (reactive antibody), α-ERK (nonreactive antibody) or equivalent volume of IP buffer (Beads control), followed by incubation with Protein A Sepharose slurry. The beads were then washed five times in IP buffer. To reverse the cross-linking, the complexes were then incubated with Proteinase K. Finally, the reaction mixtures were centrifuged and the supernatant was subjected to cDNA preparation and quantitation of BPXV RNA ( M  gene) by qRT-PCR. Error bars indicate SD. Pair-wise statistical comparisons were performed using Student's t-test (** =  P  < 0.01). Values are means ± SD and representative of the result of at least 3 independent experiments.