We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme B^P (GzmB^P/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmB^W/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmB^P and GzmB^W activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmB^P or GzmB^W. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmB^W, and so, would persist in infected cells targeted by CTLs from GzmB^P/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmB^P, and would persist in cells targeted by CTLs of GzmB^W/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.

我们调查了表达不同促凋亡丝氨酸蛋白酶颗粒酶B的不同同种型对小鼠巨细胞病毒感染的小鼠存活结果显着不同的分子基础。粒酶B ^P（GzmB ^{P / P}）纯合子的C57BL / 6小鼠产生可有效杀死受感染细胞的细胞毒性T淋巴细胞，而近交等位基因（GzmB ^{W / W}）的同基因C57BL / 6小鼠则无法杀死MCMV感染的细胞。并死于不受控制的肝细胞感染和急性肝衰竭。我们发现了GzmB ^P和GzmB ^W的细微差异激活细胞死亡信号-两种同种异体形式都主要直接激活前胱天蛋白酶，而裂解的促细胞凋亡出价则很差。因此，同种异体都不会启动线粒体外膜通透性，也不会被Bcl-2，Bcl-XL或MCMV蛋白M38.5 / M41.1的共表达所阻断，后者通过螯合Bak / Bax共同稳定线粒体。值得注意的是，对来自MCMV感染的原代小鼠胚胎成纤维细胞的蛋白质进行的质谱分析确定了九种病毒蛋白质（M18，M25，M28，M45，M80，M98，M102，M155，M164）中的13个切割位点，这些切割位点被切割了20倍以上通过GzmB ^P或GzmB ^W有效地。值得注意的是，GzmB ^W更有效地将M18，M28，M45，M80，M98，M102和M164裂解20-> 100倍，因此，将持续存在于GzmB ^{P / P}小鼠的CTL靶向的感染细胞中。相反，M155被GzmB ^P切割的效率更高> 100倍，并会在GzmB ^{W / W}小鼠CTL靶向的细胞中持续存在。M25被两种蛋白酶有效地切割，但是在不同的位点。我们得出的结论是，对MCMV的不同易感性不是由偏向的内源性细胞死亡途径引起的，而是由迄今尚未表征的最终抑制粒酶B诱导的细胞死亡的MCMV内在途径引起的。