Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m 7 GpppNm) on RNAs and in initiating viral RNA transcription

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Abstract
Purified influenza viral cores catalyze the entire process of viral RNA transcription, which includes the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m7GpppNm) structures to generate capped primers 10-13 nucleotides long, the initiation of transcription via the incorporation of a guanosine residue onto the primers, and elongation of the viral mRNAs [Plotch, S. J., Bouloy, M., Ulmanen, L & Krug, R. M. (1980) Cell 23, 847-858]. To identify which viral core protein (nucleocapsid protein, P1, P2, or P3) recognizes the cap 1 structure on the RNA primer, we irradiated (UV) endonuclease reactions carried out by viral cores in the absence of ribonucleoside triphosphates, with a primer RNA labeled in its cap 1 structure with 32P. The labeled cap was crosslinked to a protein that had a mobility similar to that of the P3 protein, the smaller of the two basic P proteins, in both one- and two-dimensional gel electrophoresis. This strongly suggests that this crosslinked protein is the viral P3 protein. Competition experiments with unlabeled RNAs containing or lacking a cap 1 structure established that this protein recognizes the cap 1 structure on RNAs. This protein remained associated with the cap throughout the transcription reaction, even after the viral mRNA molecules were elongated. To identify the viral core protein that catalyzes the initiation of transcription via the incorporation of a guanosine residue onto primer fragments, we irradiated transcription reactions carried out by viral cores in the presence of [α-32P]GTP as the only ribonucleoside triphosphate with an unlabeled primer RNA. A labeled guanosine residue was crosslinked to a protein that had a mobility similar to that of the P1 protein, the larger of the two basic P proteins, in both one-and two-dimensional gel electrophoresis. The transcription reaction conditions required to bring this protein in close association with a labeled guanosine residue so that crosslinking could occur indicated that this association most likely occurred coincident with the guanosine residue's being incorporated onto the primer. These results suggest that the viral P1 protein catalyzes this incorporation and hence initiates transcription.