Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2 and PA subunits: a model for the viral RNA polymerase structure.
The domains of the PB1 subunit of the influenza virus polymerase involved in the interaction with the PB2 and PA subunits have been defined by mutational analysis of PB1 protein. The experimental approach included in vivo competition of the PB1 activity, two-hybrid interaction assays and in vitro binding to PB1-specific matrices. Mutants of the PB1 gene including N-terminal, C-terminal and internal deletions and single amino acid insertions were constructed. They were unable to support polymerase activity in a reconstituted transcription-replication system and were tested for their competition activity when expressed in excess over wild-type PB1 protein. The pattern of competition obtained suggested that the N-terminal 78 amino acids and the sequences between positions 506 and 659 in the PB1 protein are involved in the interaction with the other components of the polymerase. We identified the N-terminal region of PB1 protein as responsible for the interaction with the PA subunit by two-hybrid assays in mammalian cells. N- and C-terminal fragments of the PB1 protein were expressed as His-tagged proteins and purified on Ni2+-NTA resin. Such PB1-specific matrices were used in binding assays in vitro with metabolically labelled PB2 and PA proteins and mutants thereof. The results obtained indicated that the N-terminal and the C-terminal regions of PB1 are responsible for binding to PA and PB2 subunits, respectively. With this information and previously published results we propose a preliminary model for the architecture of the influenza virus RNA polymerase.
González
S
S
Centro Nacional de Biotecnología (CSIC), Madrid, Spain.
Zürcher
T
T
Ortín
J
J
D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D019556:COS Cells; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D009975:Orthomyxoviridae / Q000737:chemistry*; D011487:Protein Conformation; D012324:RNA-Dependent RNA Polymerase; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
C057027:PB2 protein, Influenzavirus A; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases