Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNA polymerase.

Abstract

Influenza virus RNA polymerase with the subunit composition of PB1-PB2-PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome. Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymerase-associated capped RNA endonuclease. To identify the RNA cap 1-binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV-crosslinking with RNA which was labelled with 32P only at the cap-1 structure. After SDS-PAGE of UV-crosslinked cores, 32P was found to be associated only with the PB2 subunit (759 amino acid residues). The labelled PB2 was subjected, together with PB2 expressed in E. coli, to limited digestion with V8 protease. Analysis of the amino terminal sequences of some isolated fragments with the crosslinked cap-1 indicated that two separate sequences within the PB2 were involved in RNA cap-1 binding, one (N-site) at the N-terminal proximal region approximately between amino acid residues 242-282 downstream from the PB1 subunit-binding site and the other (C-site) between residues 538-577 including the cap-binding motifs. Two lines of evidence support the prediction of the involvement of two separate PB2 sequences on the RNA cap-binding: (i) cross-linking of the capped RNA on to expressed and isolated PB2 fragments, each containing either the N-site or the C-site; and (ii) competition of capped RNA-binding to PB2 by both of the N- and C-terminal PB2 fragments. Taking together, we propose that two separate sequences within PB2 constitute the capped RNA-binding site of the RNA polymerase. Two separate sequences, one N-(242-282) and the other C-terminal (538-577) proximal segments of PB2 subunit, constitute the RNA cap-binding site of the influenza virus RNA polymerase.