Exposure of eukaryotic cells to viruses will activate the host NF-kappaB transcription factor, resulting in proinflammatory and immune protein production. Vaccinia virus (VV), the prototypic orthopoxvirus, expresses products that inhibit this antiviral event. To identify novel mechanisms responsible for this effect, we made use of a VV deletion mutant (MVA) that stimulates NF-kappaBeta activation in infected 293T cells. In this virus-host system, the extents of NF-kappaBeta-regulated gene expression and nuclear translocation were reduced in the presence of either PD 98059 or U0126, two compounds capable of blocking ERK1 and ERK2 phosphorylation. A similar repression was also observed in cells that contained a dominant, nonactive form of ERK2 but not in cells where ERK1 phosphorylation was inhibited via overexpression of a dominant-negative mutant MEK1 protein. Presumably, proteins expressed from a wild-type VV that block ERK2 activity would also inhibit MVA-induced NF-kappaB activation. Indeed, the expression of one such open reading frame, M2L, supported this prediction. First, ectopic M2L expression hampered ERK2 phosphorylation induced by exposure to phorbol myristate acetate. Second, viral M2L expression via infection of cells with a recombinant MVA construct that stably expressed M2L decreased the phosphorylation of ERK2 compared to that in cells infected with the parental MVA strain. Finally, the recombinant M2L-expressing virus restored the "wild-type" NF-kappaB-inhibitory phenotype, as indicated by decreased NF-kappaB migration to infected cell nuclei and interference in transcription. Thus, in 293T cells, VV apparently utilizes its M2L protein to interfere with a step(s) that would otherwise enable ERK2 phosphorylation and the consequential activation of an NF-kappaBeta response.