Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence

Vaccinia virus (vacv) protein n1 is an intracellular virulence factor and belongs to a family of vacv b-cell lymphoma (bcl)-2-like proteins whose members inhibit apoptosis or activation of pro-inflammatory transcription factors,such as interferon (ifn) regulatory factor-3 (irf-3) and nuclear factor-κb (nf-κb). unusually,n1 inhibits both apoptosis and nf-κb activation. to understand how n1 exerts these different functions,we have mutated residues in the bcl-2-like surface groove and at the interface used to form n1 homodimers. mutagenesis of the surface groove abolished only the n1 anti-apoptotic activity and protein crystallography showed these mutants differed from wild-type n1 only at the site of mutation. conversely,mutagenesis of the dimer interface converted n1 to a monomer and affected only inhibition of nf-κb activation. collectively,these data show that n1 inhibits pro-inflammatory and pro-apoptotic signalling using independent surfaces of the protein. to determine the relative contribution of each activity to virus virulence,mutant n1 alleles were introduced into a vacv strain lacking n1 and the virulence of these viruses was analysed after intradermal and intranasal inoculation in mice. in both models,vacv containing a mutant n1 unable to inhibit apoptosis had similar virulence to wild-type virus,whereas vacv containing a mutant n1 impaired for nf-κb inhibition induced an attenuated infection similar to that of the n1-deleted virus. this indicates that anti-apoptotic activity of n1 does not drive virulence in these in vivo models,and highlights the importance of pro-inflammatory signalling in the immune response against viral infections. © 2011 maluquer de motes et al.