Cells exposed to intact, noninfectious influenza virus were shown to be recognized by class I-restricted anti-influenza cytotoxic T lymphocytes (CTLs). Both internal and external proteins derived from virions were processed by cells for CTL recognition. Sensitization required the inactivation of viral neuraminidase activity and could be inhibited by preventing fusion of viral and cellular membranes. These findings are important in designing vaccines to elicit CTL responses, since they demonstrate that cells can process intact, exogenous proteins for recognition by CTLs and suggest that such processing depends on introduction of exogenous proteins into the cytoplasm.
We studied influenza virus M1 protein by generating HeLa and MDCK cell lines that express M1 genetically fused to green fluorescent protein (GFP). GFP-M1 was incorporated into virions produced by influenza virus infected MDCK cells expressing the fusion protein indicating that the fusion protein is at least partially functional. Following infection of either HeLa or MDCK cells with influenza A virus (but not influenza B virus), GFP-M1 redistributes from its cytosolic/nuclear location and accumulates in nuclear dots. Immunofluorescence revealed that the nuclear dots represent nuclear dot 10 (ND10) structures. The colocalization of authentic M1, as well as NS1 and NS2 protein, with ND10 was confirmed by immunofluorescence following in situ isolation of ND10. These findings demonstrate a previously unappreciated involvement of influenza virus with ND10, a structure involved in cellular responses to immune cytokines as well as the replication of a rapidly increasing list of viruses.
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