Mapping the domain structure of the influenza A virus polymerase acidic protein (PA) and its interaction with the basic protein 1 (PB1) subunit.
The influenza A virus polymerase consists of three subunits (PA, PB1, and PB2) necessary for viral RNA synthesis. The heterotrimeric polymerase complex forms through PA interacting with PB1 and PB1 interacting with PB2. PA has been shown to play critical roles in the assembly, catalysis, and nuclear localization of the polymerase. To probe the structure of PA, we isolated recombinant PA from insect cells. Limited proteolysis revealed that PA contained two domains connected by a 20-residue linker (residues 257-276). Far-UV circular dichroism established that the two domains folded into a mixed alpha/beta structure when separately expressed. In vitro pull-down assays showed that neither individually nor cooperatively expressed PA domains, without the linker, could assure PA-PB1 interaction. Protease treatment of PA-PB1 complex indicated that its PA subunit was significantly more stable than free PA, suggesting that the linker is protected and it constitutes an essential component of the PA-PB1 interface.
Guu
Tom S Y
TS
Department of Biochemistry and Cell Biology, MS-140, Rice University, Houston, Texas 77005, USA.
Dong
Liping
L
Wittung-Stafshede
Pernilla
P
Tao
Yizhi J
YJ
D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D009980:Influenza A virus / Q000737:chemistry* / Q000235:genetics; D008954:Models, Biological; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D018411:Spodoptera; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C061290:PA protein, influenza viruses; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase; D010447:Peptide Hydrolases