Department of Biology, New York University, New York, 10003-6688, USA.
DNA Cell Biol. 2010 Mar;29(3):103-20. doi: 10.1089/dna.2009.0963.
Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. Pretreatment of sensitive cultured cells with IFNbeta results in a 10(4)-fold reduction in the release of infectious VSV particles. However, differences exist between the mechanisms of reduced infectious particle titers in cell lines of neuroblastoma and nonneuronal lineage. In L929-fibroblast-derived cells, using immunofluorescence confocal microscopy, infection under control conditions reveals the accumulation of VSV matrix, phosphoprotein (P), and nucleocapsid (N) proteins over time, with induced cellular morphological changes indicative of cytopathic effects (CPEs). Upon observing L929 cells that had been pretreated with IFNbeta, neither detectable VSV proteins nor CPEs were seen, consistent with type I IFN antiviral protection. When using the same techniques to observe VSV infections of NB41A3 cells, a neuroblastoma cell line, aside from similar viral progression in the untreated control cells, IFNbeta-treated cells illustrated a severely attenuated VSV infection. Attenuated VSV progression was observed through detection of VSV matrix, P, and N proteins in isolated cells during the first 8 h of infection. However, by 18-24 h postinfection all neuroblastomas had succumbed to the viral infection. Finally, upon closer inspection of IFNbeta-treated NB41A3 cells, no detectable changes in VSV protein localization were identified compared with untreated, virally infected neuroblastomas. Next, to extend our study to test our hypothesis that virion assembly is compromised within type I IFN-treated neuroblastoma cells, we employed electron microscopy to examine our experimental conditions at the ultrastructural level. Using VSV-specific antibodies in conjunction with immuno-gold reagents, we observed several similarities between the two cell lines, such as identification of viroplasmic regions containing VSV N and P proteins and signs of stress-induced CPEs of VSV-infected cells, which had either been mock-treated or pretreated with interferon-beta (IFNbeta). One difference we observed between nonneuronal and neuroblastoma cells was more numerous actively budding VSV virions across untreated L929 plasma membranes compared with untreated NB41A3 cells. Additionally, IFNbeta-treated, VSV-infected L929 cells exhibited neither cytoplasmic viroplasm nor viral protein expression. In contrast, IFNbeta-treated, VSV-infected NB41A3 cells showed evidence of VSV infection at a very low frequency as well as small-scale viroplasmic regions that colocalized with viral N and P proteins. Finally, we observed that VSV viral particles harvested from untreated VSV-infected L929 and NB41A3 cells were statistically similar in size and shape. A portion of VSV virions from IFNbeta-treated, virally infected NB41A3 cells were similar in size and shape to virus from both untreated cell types. However, among the sampling of virions, pleomorphic viral particles that were identified from IFNbeta-treated, VSV-infected NB41A3 cells were different enough to suggest a misassembly mechanism as part of the IFNbeta antiviral state in neuroblastoma cells.
水疱性口炎病毒（VSV）的复制对干扰素（IFN）诱导的抗病毒反应非常敏感。用 IFNβ预处理敏感的培养细胞会导致感染性 VSV 颗粒的释放减少 104 倍。然而，神经母细胞瘤和非神经元谱系细胞系中降低感染性颗粒滴度的机制存在差异。在 L929 成纤维细胞衍生的细胞中，通过免疫荧光共聚焦显微镜观察，在对照条件下的感染会随着时间的推移积累 VSV 基质、磷酸蛋白（P）和核衣壳（N）蛋白，同时发生诱导的细胞形态变化表明细胞病变效应（CPE）。在用 IFNβ预处理的 L929 细胞中观察到，既没有检测到 VSV 蛋白，也没有观察到 CPE，这与 I 型 IFN 的抗病毒保护作用一致。当使用相同的技术观察 NB41A3 细胞（一种神经母细胞瘤细胞系）的 VSV 感染时，除了未经处理的对照细胞中的类似病毒进展外，IFNβ处理的细胞显示出严重减弱的 VSV 感染。通过在感染的前 8 小时内检测分离细胞中的 VSV 基质、P 和 N 蛋白，观察到减弱的 VSV 进展。然而，在感染后 18-24 小时，所有神经母细胞瘤都已屈服于病毒感染。最后，通过仔细检查 IFNβ处理的 NB41A3 细胞，与未经处理、感染病毒的神经母细胞瘤相比，未发现 VSV 蛋白定位的可检测变化。接下来，为了扩展我们的研究以检验我们的假设，即 I 型 IFN 处理的神经母细胞瘤细胞中的病毒装配受到损害，我们采用电子显微镜在超微结构水平上检查我们的实验条件。使用 VSV 特异性抗体结合免疫金试剂，我们观察到两种细胞系之间存在一些相似之处，例如鉴定含有 VSV N 和 P 蛋白的 viroplasm 区域以及 VSV 感染细胞的应激诱导 CPE 的迹象，这些细胞要么未经处理，要么用干扰素-β（IFNβ）预处理。我们在非神经元和神经母细胞瘤细胞之间观察到的一个区别是，未经处理的 L929 质膜上比未经处理的 NB41A3 细胞有更多的活跃出芽 VSV 病毒粒子。此外，IFNβ处理的、感染 VSV 的 L929 细胞既没有细胞质 viroplasm 也没有病毒蛋白表达。相比之下，IFNβ处理的、感染 VSV 的 NB41A3 细胞显示出 VSV 感染的证据频率非常低，以及与病毒 N 和 P 蛋白共定位的小规模 viroplasm 区域。最后，我们观察到从未经处理的 VSV 感染的 L929 和 NB41A3 细胞中收获的 VSV 病毒粒子在大小和形状上统计学上相似。来自 IFNβ处理的、感染病毒的 NB41A3 细胞的一部分 VSV 病毒粒子在大小和形状上与两种未经处理的细胞类型的病毒相似。然而，在病毒粒子的取样中，从 IFNβ处理的、感染 VSV 的 NB41A3 细胞中鉴定出的多形病毒粒子足以表明在神经母细胞瘤细胞中作为 IFNβ抗病毒状态的一部分存在错误装配机制。
