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 phosphoprotein that induces proteolytic degradation of coexpressed proteins. Point mutants 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 CLE. The human gene encodes a protein of 36 kDa (hCLE) and is expressed ubiquitously 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 experiments. Mapping of the interaction showed that hCLE interacts with PA subunit at two regions (positions 493 to 512 and 557 to 574) in the PA protein sequence. Immunofluorescence studies showed that the hCLE protein localizes in both the nucleus and the cytosol, although with a predominantly cytosolic distribution. hCLE was found associated with active, highly purified virus ribonucleoproteins reconstituted in vivo from cloned cDNAs, suggesting that PA-hCLE interaction is functionally relevant. Searches in the databases showed that hCLE has 38% sequence homology to the central region of the yeast factor Cdc68, which modulates transcription by interaction with transactivators. Similar homologies were found with the other members of the Cdc68 homologue family of transcriptional activators, including the human FACT protein.
Huarte
M
M
Campus de Cantoblanco, Centro Nacional de Biotecnología (CSIC), 28049 Madrid, Spain.
Sanz-Ezquerro
J J
JJ
Roncal
F
F
Ortín
J
J
Nieto
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001717:Birds; D019556:COS Cells; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase; D012261:Ribonucleoproteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C061290:PA protein, influenza viruses; D010750:Phosphoproteins; C435504:RTRAF protein, human; D012261:Ribonucleoproteins; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases