Our laboratory has identified, using the yeast two-hybrid system, a cellular protein, NPI-5, which interacts with the influenza A virus nucleoprotein (NP). NPI-5, is a nuclear protein of the DEAD- box family of putative RNA helicases, and NP is a viral protein which is required for several viral functions including RNA synthesis. We are interested in NPI-5 as a potential co-factor in influenza virus replication because DEAD-box RNA helicases have been shown to participate in the replication of several viruses. In this application, experiments are proposed to determine the function in influenza virus replication of the NP:NPI-5 interaction. (1) Experiments will be performed to determine to determine whether the NP:NPI-5 interaction can be detected between purified proteins in vitro and in infected cells by co-immunofluorescence and co- immunoprecipitation. (2) A full-length NPI-5 c-DNA will be cloned, expressed in bacteria and the NPI-5 protein assayed for the biochemical activities characteristic of DEAD-box RNA helicases (NTP binding, NTPase, helicase and RNA binding activities). (3) To assess the functional significance of the NP:NPI-5 interaction for influenza virus replication, potential dominant negative NPI-5 mutants will be generated. The NP-5 mutants will be tested for their ability to affect replication of a model influenza virus RNA encoding the CAT reporter gene. If viral RNA synthesis is affected by these mutants, the precise role of NPI-5 in viral RNA synthesis will be analyzed in in vitro assays of viral transcription and replication. Additionally, cells lines stably transfected with wild- type and mutant NPI-5 will be developed and used to assess the effect of NPI-5 on viral replication.
