Elucidating the antiviral mechanism induced by type I interferons in neuroblastoma cells.

摘要

Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. VSV infection of well known cell lines, pretreated with IFNbeta, results in a 104-fold reduction in the release of infectious particles with a concomitant abrogation in viral transcript and/or protein levels. However, in cell lines of neuronal lineage only a three-fold reduction in viral transcript and proteins levels was observed, despite the same 104-fold reduction in released infectious virions, suggesting an assembly defect. Examination of VSV matrix (M) protein ubiquitination yielded no differences between mock- and IFNbeta-treated neuroblastoma cells. Further analysis of potential post-translational modification events, by scintillation and two-dimensional electrophoretic methods, revealed IFNbeta-induced alterations in M protein and phosphoprotein (P) phosphorylation. Hypophosphorylated P protein was demonstrated by reduced 32P counts, normalized by 35S-cyteine/methionine incorporation, and by a shift in isoelectric focusing. Hypophosphorylation of VSV P protein was found to occur in neuroblastoma cell lysates, but not within budded virions from the same IFNbeta-treated cells. In contrast, hyperphosphorylation of VSV M protein was observed in both cell lysates and viral particles from IFNbeta treated neuroblastoma cells. Hyper-phosphorylated M protein was demonstrated by increased 32P counts relative to 35S-cyteine/methionine normalization and by altered isoelectric focusing in protein populations from cell and virally lysates. Hyperphosphorylated VSV M protein was found to inhibit its associations with VSV nucleocapsid, suggesting a possible mechanism for type I IFN mediated misassembly through disruption of the interactions between ribonucleoprotein cores and hyperphosphorylated M protein bound to the plasma membrane inner leaflet.;In an effort to identify direct evidence for misassembly, VSV infections were tracked during an eight hour infection. In L929 fibroblast-derived cells, using immunofluorescence confocal microscopy, infection under control conditions revealed the accumulation of VSV M, P, and N proteins over time, with induced cellular morphological changes indicative of apoptosis. Observing L929 cells which had been pretreated with IFNbeta, neither detectable VSV proteins nor cytopathic effects were seen, which is consistent with type I IFN antiviral protection. However, when using the same techniques to observe VSV infections of NB41A3 cells (a neuroblastoma cell line) aside from similar viral progression in untreated controls, in IFNbeta-treated cells there was a severely attenuated VSV progression. Attenuated VSV progression was visualized through detection of VSV M, P, and N proteins in isolated cells during the first eight hours of infection, and by 18-24 hours post-infection all neuroblastomas succumb to the viral infection. Finally, upon closer inspection of IFNbeta-treated NB41A3 cells, no detectable changes in VSV protein localization were identified in comparison to untreated, virally infected neuroblastoma or non-neuronal cells.;VSV infected neuroblastoma and non-neuronal cells were then examined at the ultrastructural level. Using electron microscopy, in conjunction with immuno-gold reagents, we observed several similarities between the two cell lines either mock-treated or pre-treated with interferon-beta (IFNbeta) including: (1) identification of viroplasm-like regions containing VSV N and P proteins, and (2) stress-induced apoptosis of VSV infected cells. One difference we observed between non-neuronal and neuroblastoma cells included more numerous actively budding VSV virions across untreated L929 plasma membranes, when compared to untreated NB41A3 cells. Additionally, IFNbeta-treated VSV infected L929 cells exhibited neither cytoplasmic viral factories nor viral proteins 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 co-localized with viral N and P proteins consistent with our confocal observations. 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 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.