Analysis of the interaction of influenza virus polymerase complex with human cell factors.
The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. Here we present the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. The virus polymerase was expressed by cotransfection of the polymerase subunits cDNAs, one of which fused to the tandem-affinity purification (TAP) tag. The intracellular complexes were purified by the TAP approach, which involves IgG-Sepharose and calmodulin-agarose chromatography, under very mild conditions. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of untagged polymerase used as a control. Several influenza polymerase-associated proteins were identified by MALDI-MS and their presence in purified polymerase-containing complexes were verified by Western blot. Their relevance for influenza infection was established by colocalisation with virus ribonucleoproteins in human infected cells. Most of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification and nucleo-cytoplasmic export, but some were cytosolic proteins involved in translation and transport. The interactions recognised in this proteomic approach suggest that the influenza polymerase might be involved in steps of the infection cycle other than RNA replication and transcription.
Jorba
Núria
N
Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, Madrid, Spain.
Juarez
Silvia
S
Torreira
Eva
E
Gastaminza
Pablo
P
Zamarreño
Noelia
N
Albar
Juan Pablo
JP
Ortín
Juan
J
D053487:DEAD-box RNA Helicases / Q000378:metabolism; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M / Q000378:metabolism; D006801:Humans; D009975:Orthomyxoviridae / Q000378:metabolism*; D011485:Protein Binding; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014764:Viral Proteins / Q000378:metabolism*
D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; C119332:DDX3X protein, human; C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases