PA subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators

The PA subunit of the influenza virus polymerase complex is a phosphoprotei n that induces proteolytic degradation of coexpressed proteins. Point mutan ts with reduced proteolysis induction reconstitute viral ribonucleoproteins defective in replication but not in transcriptional activity. To look for cellular factors that could associate with PA protein, we have carried out a yeast two-hybrid screen. Using a human kidney cDNA library, we identified two different interacting clones. One of them was identified as the human homologue of a previously described cDNA clone from Gallus gallus called CL E. The human gene encodes a protein of 36 kDa (hCLE) and is expressed ubiqu itously in all human organs tested. The interaction of PA and hCLE was also observed with purified proteins in vitro by using pull-down and pep-spot e xperiments. Mapping of the interaction showed that hCLE interacts with PA s ubunit at two regions (positions 493 to 512 and 557 to 574) in the PA prote in sequence. Immunofluorescence studies showed that the hCLE protein locali zes in both the nucleus and the cytosol, although with a predominantly cyto solic distribution. hCLE was found associated with active, highly purified virus ribonucleoproteins reconstituted in vivo from cloned cDNAs, suggestin g that PA-hCLE interaction is functionally relevant. Searches in the databa ses showed that hCLE has 38% sequence homology to the central region of the yeast factor Cdc68, which modulates transcription by interaction with tran sactivators. Similar homologies were found with the other members of the Cd c68 homologue family of transcriptional activators, including the human FAC T protein.