Nucleic Acids Research, Vol 24, Issue 22 4456-4463, Copyright © 1996 by Oxford University Press
S Gonzalez, T Zurcher and J Ortin
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.
ARTICLES
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
Centro Nacional de Biotecnologia (CSIC), Madrid, Spain.
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