Bair C H, Chung C S, Vasilevskaya I A, Chang W
Institute of Molecular Biology, Academia Sinica, Nankang, Taiwan, Republic of China.
J Virol. 1996 Jul;70(7):4655-66. doi: 10.1128/JVI.70.7.4655-4666.1996.
The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.
中国仓鼠卵巢（CHO）细胞系对痘苗病毒不敏感，据报道病毒中间基因的翻译被阻断（A. Ramsey-Ewing和B. Moss，《病毒学》206:984 - 993，1995）。然而，细胞很容易被痘苗病毒杀死。通过逆转录病毒插入诱变分离出了一种抗痘苗病毒的CHO突变体VV5 - 4。亲本CHO细胞在感染痘苗病毒后，会在2至3天内死亡，而VV5 - 4细胞能优先在这种细胞毒性作用下存活。VV5 - 4的存活表型是部分性的，且与所用的感染复数呈负相关。此外，病毒感染在VV5 - 4中未能阻断宿主蛋白合成。VV5 - 4被用于研究病毒生命周期进展与细胞命运的关系。我们发现，在亲本CHO细胞中，痘苗病毒依次经历病毒早期基因表达、脱壳、病毒DNA复制以及中间和晚期启动子的表达。相比之下，我们在VV5 - 4细胞中仅检测到早期基因表达和脱壳，而病毒DNA复制似乎被阻断。与病毒基因表达的级联调控模型一致，我们在VV5 - 4细胞中几乎检测不到中间和晚期基因表达。由于已知痘苗病毒具有细胞溶解性，因此该突变体的分离证明了一种影响病毒生命周期进展并导致不同细胞命运的细胞微环境新模式。这个过程似乎是由一种普遍机制介导的，因为VV5 - 4对肖普纤维瘤病毒和黏液瘤病毒的杀伤也具有抗性。另一方面，VV5 - 4对牛痘病毒的杀伤仍然敏感。为了研究VV5 - 4存活的机制，我们研究了是否涉及细胞凋亡。在用痘苗病毒感染的CHO和VV5 - 4细胞中均观察到了DNA梯状条带以及用Hoechst 33258对凋亡细胞核进行的染色。我们得出结论，阻断病毒DNA复制并使VV5 - 4存活的细胞途径与细胞凋亡无关。该突变体还提供了证据，表明痘苗病毒感染后诱导细胞凋亡的信号发生在病毒DNA复制之前。
