Purification and molecular structure of RNA polymerase from influenza virus A/PR8.
The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.
Honda
A
A
Department of Molecular Genetics, National Institute of Genetics, Shizuoka.
Mukaigawa
J
J
Yokoiyama
A
A
Kato
A
A
Ueda
S
S
Nagata
K
K
Krystal
M
M
Nayak
D P
DP
Ishihama
A
A
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002499:Centrifugation, Density Gradient; D002586:Cesium; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D066298:In Vitro Techniques; D009980:Influenza A virus / Q000201:enzymology*; D012367:RNA, Viral / Q000302:isolation & purification; D011817:Rabbits; D012261:Ribonucleoproteins / Q000302:isolation & purification*
D000906:Antibodies; D012367:RNA, Viral; D012261:Ribonucleoproteins; D002586:Cesium; D012321:DNA-Directed RNA Polymerases