Hommel-Berrey G A, Bochan M R, Montel A H, Goebel W S, Froelich C J, Brahmi Z
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
Cell Immunol. 1997 Aug 25;180(1):1-9. doi: 10.1006/cimm.1997.1173.
Cytotoxic cells provide a crucial defense against DNA and RNA viral infections. Here we describe an in vitro model to study the fate of vesicular stomatitis virus (VSV) RNA in cells undergoing apoptosis. Using the [3H]uridine release assay, we show that human LAK cells induce the degradation of RNA in infected U937 cells in addition to inhibiting the production of infectious virions. LAK cell-mediated RNA degradation was blocked by the serine protease inhibitor, 3,4-dichloroisocoumarin. Purified human granzyme B but not inactivated granzyme B, granzyme A, or perforin rapidly induced degradation of RNA in VSV-infected U937 cells in a dose- and time-dependent manner without lysing the cells and suppressed viral production. Northern analysis of RNA extracted from infected cells with a VSV full-length cDNA probe confirmed that levels of viral transcripts were reduced by treatment with granzyme B. Nevertheless, the amount of host beta-actin mRNA was also reduced in infected cells, suggesting that treatment with granzyme B induced apoptosis. Consistent with this notion, infected cells exposed to granzyme B rapidly developed DNA strand breakage. Taken together, the data suggest that granzyme B in the absence of perforin reduced VSV production by activating a mechanism that degraded viral transcripts in infected U937 cells.
细胞毒性细胞为抵抗DNA和RNA病毒感染提供了关键防御。在此，我们描述了一种体外模型，用于研究水疱性口炎病毒（VSV）RNA在经历凋亡的细胞中的命运。使用[3H]尿苷释放试验，我们发现人LAK细胞除了抑制感染性病毒粒子的产生外，还能诱导受感染的U937细胞中RNA的降解。丝氨酸蛋白酶抑制剂3,4-二氯异香豆素可阻断LAK细胞介导的RNA降解。纯化的人颗粒酶B而非失活的颗粒酶B、颗粒酶A或穿孔素能以剂量和时间依赖性方式迅速诱导VSV感染的U937细胞中RNA的降解，且不会裂解细胞，并抑制病毒产生。用VSV全长cDNA探针分析从受感染细胞中提取的RNA的Northern印迹证实，颗粒酶B处理可降低病毒转录本水平。然而，受感染细胞中宿主β-肌动蛋白mRNA的量也减少了，这表明颗粒酶B处理诱导了细胞凋亡。与此观点一致，暴露于颗粒酶B的受感染细胞迅速出现DNA链断裂。综上所述，数据表明在没有穿孔素的情况下，颗粒酶B通过激活一种机制来降低VSV的产生，该机制可降解受感染的U937细胞中的病毒转录本。
