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.

痘苗病毒（Vaccinia virus, VACV）蛋白N1是一种细胞内毒力因子，隶属于一类痘苗病毒B细胞淋巴瘤-2（B-cell lymphoma, Bcl-2）样蛋白家族，该家族成员可抑制细胞凋亡或促炎转录因子的激活，例如干扰素（interferon, IFN）调节因子3（IRF-3）与核因子κB（nuclear factor-κB, NF-κB）。与该家族其他成员不同的是，N1可同时抑制细胞凋亡与NF-κB激活。为阐明N1蛋白如何介导这两种不同的功能，我们对其Bcl-2样表面凹槽及形成N1同源二聚体的界面氨基酸残基进行了诱变突变。对表面凹槽的诱变仅消除了N1的抗细胞凋亡活性，蛋白质晶体学分析显示，这些突变体仅在突变位点处与野生型N1存在差异。反之，对二聚体界面的诱变使N1转变为单体蛋白，且仅影响其对NF-κB激活的抑制作用。综上，上述实验数据表明，N1蛋白通过其表面的独立结构域分别抑制促炎信号通路与促凋亡信号通路。为明确N1蛋白的两种活性对病毒毒力的相对贡献，我们将突变型N1等位基因导入缺失N1的痘苗病毒毒株中，并通过小鼠皮内接种与鼻内接种模型分析了这些重组病毒的毒力。在两种感染模型中，携带无法抑制细胞凋亡的突变型N1的痘苗病毒，其毒力与野生型毒株无显著差异；而携带NF-κB抑制功能受损的突变型N1的痘苗病毒，则引发了与N1缺失型病毒相似的减毒感染。这一结果表明，在上述体内感染模型中，N1蛋白的抗细胞凋亡活性并非驱动病毒毒力的关键因素，同时凸显了促炎信号通路在抗病毒免疫应答中的重要作用。