IFN-beta-induced alteration of VSV protein phosphorylation in neuronal cells.
Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. VSV infection of well-known cell lines pretreated with IFN-beta results in a 10(4)-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 threefold reduction in viral transcript and protein levels was observed, despite the same 10(4)-fold reduction in released infectious virions, suggesting an assembly defect. Examination of VSV matrix (M) protein ubiquitination yielded no differences between mock- and IFN-beta-treated neuronal cells. Further analysis of potential post-translational modification events, by scintillation and two-dimensional electrophoretic methods, revealed IFN-beta-induced alterations in M protein and phosphoprotein (P) phosphorylation. Hypophosphorylated P protein was demonstrated by reduced (32)P counts, normalized by (35)S-cysteine/methionine incorporation, and by a shift in isoelectric focusing. Hypophosphorylation of VSV P protein was found to occur in neuronal cell lysates, but not within budded virions from the same IFN-beta-treated cells. In contrast, hyperphosphorylation of VSV M protein was observed in both cell lysates and viral particles from IFN-beta-treated neuronal cells. Hyperphosphorylated M protein was demonstrated by increased (32)P counts relative to (35)S-cysteine/methionine normalization, and by altered isoelectric focusing in protein populations from cell and viral lysates. Hyperphosphorylated VSV M protein was found to inhibit its association 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.
D'agostino
Paul M
PM
Biology Department, New York University, New York, New York 10003-6688, USA.
Amenta
Jessica J
JJ
Reiss
Carol Shoshkes
CS
D000818:Animals; D045744:Cell Line, Tumor / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D006801:Humans; D016899:Interferon-beta / Q000494:pharmacology*; D007739:L Cells / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D051379:Mice; D041681:NIH 3T3 Cells / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D009928:Organ Specificity; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D054875:Ubiquitination / Q000187:drug effects; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects* / Q000502:physiology; D014763:Viral Matrix Proteins / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000378:metabolism*; D019065:Virus Assembly / Q000187:drug effects* / Q000502:physiology
C094460:M protein, Vesicular stomatitis virus; C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; D015678:Viral Structural Proteins; D016899:Interferon-beta