Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell mRNA and functions only as the trimeric enzyme.
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 endonucleolytic cleavage of host cell mRNAs containing a cap structure to generate capped primers that are 10-14 nucleotides long which are then used to prime transcription of virus-specific mRNAs. To analyze the properties of the capped RNA-specific endonuclease associated with the influenza virus polymerase and the roles of each of the three subunits in transcription initiation, we established an in vitro assay to investigate this endonucleolytic cleavage reaction. This assay consists of an artificial RNA substrate containing a cap-0 structure at its 5' end and a partial alfalfa mosaic virus RNA 4 (AIMV RNA 4) sequence which had been shown to be cleaved by the influenza polymerase. Results showed that purified virion ribonucleoprotein complexes cleaved the RNA substrate specifically to generate a capped 14-nt RNA fragment for use as primer to initiate viral mRNA synthesis. Purified polyclonal anti-PB2 IgG inhibited the endonuclease activity, but anti-PB1 and anti-PA antibodies did not inhibit the cleavage. Partially purified trimeric polymerase expressed by recombinant baculovirus in insect cells cleaved the artificial substrate, but if one or two subunits were removed from the polymerase complex, the cleavage activity was totally lost. Our results suggest that viral PB2 protein is the endonuclease that cleaves host cell mRNA to produce the primer used to initiate transcription; however, association with the other two enzyme subunits seems to be required for this PB2 function.
Shi
L
L
Department of Microbiology and Molecular Genetics, University of California, Irvine 92715-4025, USA.
Summers
D F
DF
Peng
Q
Q
Galarz
J M
JM
D000818:Animals; D002460:Cell Line; D019281:Dimerization; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D009980:Influenza A virus / Q000201:enzymology* / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D012333:RNA, Messenger; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; D004720:Endonucleases