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

Journal article

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: Maluquer de Motes, C; Cooray, S; Ren, H; Almeida, G; McGourty, K

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Public Library of Science
• Journal: PLoS pathogens
• Volume: 7
• Issue: 12
• Pages: e1002430
• Publication date: 2011-12-01
• DOI: 10.1371/journal.ppat.1002430
• EISSN: 1553-7374
• ISSN: 1553-7366
• Language: English
• Keywords: Mutation; Apoptosis; Humans; Mice; Virulence; Animals; Viral Proteins; Cell Line; Protein Binding; Protein Structure, Tertiary; Vaccinia virus; NF-kappa B