Influenza A virus alters structural and biochemical functions of the neutrophil cytoskeleton

Influenza A virus (IAV) has previously been shown to alter chemotactic, oxidative, and secretory functions of polymorphonuclear leukocytes (PMNL). Because of the role of cytoskeletal proteins in these processes, studies were carried out to determine if IAV altered the PMNL cytoskeleton. PMNL were incubated with buffer of IAV, stimulated with f-met-leu-phe (FMLP), fixed and stained with NBD-phallacidin (NBD-Ph) and studied by flow cytometry. Mean F-actin fluorescence was increased 18% in virus treated cells pre-FMLP stimulation and 13% 5 and 10 min post-FMLP (P less than .03); no significant difference in F-actin fluorescence was noted in virus treated PMNL 15-30 s post-FMLP compared to control cells. PMNL exposed to the same conditions were solubilized and actin content was determined following SDS-PAGE of triton insoluble precipitates. Increased actin was recovered from virus treated compared to buffer treated cells before and after FMLP stimulation in the 8,000g precipitates (P less than .001). Immunofluorescent microscopy studies of F-actin distribution were done in PMNL stained with NBD-Ph following FMLP stimulation for 10 min. These studies showed an increased lamellipod F-actin/uropod F-actin ratio in PMNL pre-incubated with IAV compared to controls (4.6 vs. 1.0; P less than .025). Phosphorylation of specific cytoskeletal proteins was examined after immunoprecipitation. IAV alone altered phosphorylation of both vimentin and vinculin, and in stimulated PMNL virus led to decreased phosphorylation of vimentin and vinculin. These data show distributional and biochemical effects of IAV on PMNL cytoskeletal proteins, indicating additional targets for IAV interference in the PMNL signal-transduction-function process.