Recombinant-baculovirus-expressed PB2 subunit of the influenza A virus RNA polymerase binds cap groups as an isolated subunit.
The influenza A virus RNA-dependent RNA polymerase catalyzes several reactions in transcription and replication of the genome RNA. The first step in viral mRNA synthesis is the recognition of the 5' end cap structure of host cell hnRNA and the cleavage of the RNA substrate between 10 and 14 nucleotides from the 5' end to generate capped primers for initiation of transcription of virus-specific mRNAs. This report describes the use of an in vitro UV crosslinking and protein renaturation assay to identify the polymerase subunits which interact with the 5' end cap structure of an artificial RNA substrate. Our results showed, for the first time, that purified polymerase subunit PB2 expressed by recombinant baculovirus in insect cells possessed cap-binding activity by itself after renaturation by Escherichia coli thioredoxin, whereas cleavage of the artificial capped substrate required the holoenzyme expressed in insect cells triply-infected with baculovirus containing all three polypeptide components, PB1, PB2, and PA. Purified polyclonal anti-PB2 IgG inhibited the binding activity; anti-PB1 and anti-PA IgGs did not.
Shi
L
L
Department of Microbiology and Molecular Genetics, University of California, Irvine 92717-4025, USA.
Galarza
J M
JM
Summers
D F
DF
D000818:Animals; D003432:Cross-Linking Reagents; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G; D009980:Influenza A virus / Q000201:enzymology*; D012315:RNA Caps / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; C057027:PB2 protein, Influenzavirus A; D012315:RNA Caps; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase