title: Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence.
abstract: 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.
authors.affiliation: Department of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Carlos
authors.initials: C
authors.lastname: Maluquer de Motes
authors.forename: Samantha
authors.initials: S
authors.lastname: Cooray
authors.forename: Hongwei
authors.initials: H
authors.lastname: Ren
authors.forename: Gabriel M F
authors.initials: GM
authors.lastname: Almeida
authors.forename: Kieran
authors.initials: K
authors.lastname: McGourty
authors.forename: Mohammad W
authors.initials: MW
authors.lastname: Bahar
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Grimes
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Graham
authors.forename: Geoffrey L
authors.initials: GL
authors.lastname: Smith
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014774:Virulence
chemical_list: C464479:N1L protein, Vaccinia virus; D016328:NF-kappa B; D014764:Viral Proteins