Molecular Mapping of Influenza Virus RNA Polymerase by Site-Specific Antibodies

Abstract

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome, including the unique cap-I-dependent RNase activity. To map the important domains for RNA polymerization, cap-I-dependent RNase, and cap-I-binding activity, we generated site-specific antibodies against overlapping 150-amino-acid peptides that cover each entire subunit. Monospecific antibodies against each subunit inhibited RNA synthesisin vitro.Those against PB1 and PB2 inhibited the cap-I-dependent RNase activity, but those against PB2 alone slightly inhibited the cap-I-binding activity. Antibodies against the N-terminal amino acids 1–159 of PB2 that overlap the PB1-binding site on PB2 and the C-terminal amino acids 501–617 of PA that overlap the putative nucleotide-binding site and PB1-binding site on PA inhibited RNA polymerizing activity as well as monospecific antibodies. Those against the N-terminal (amino acids 1–159); the central region (amino acids 305–559) of PB2, where a part of the cap-binding domain predicted previously is localized; the N-terminal (amino acids 1–222) of PB1; and amino acids 301–517 and 601–716 of PA inhibited the cap-I-dependent RNase activity. The cap-binding domain on PB2 could be mapped in amino acids 402–559, where one of the cap-binding domains mapped previously overlapped.