Molecular mapping of influenza virus RNA polymerase by site-specific antibodies

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is inv olved in both transcription and replication of the RNA genome, including th e unique cap-I-dependent RNase activity. To map the important domains for R NA polymerization, cap-I-dependent RNase, and cap-I-binding activity, we ge nerated site-specific antibodies against overlapping 150-amino-acid peptide s that cover each entire subunit. Monospecific antibodies against each subu nit inhibited RNA synthesis in vitro. Those against PB1 and PB2 inhibited t he cap-I-dependent RNase activity, but those against P82 alone slightly inh ibited 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-termi nal 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 1-15 9); the central region (amino acids 305-559) of PB2, where a part of the ca p-binding domain predicted previously is localized; the N-terminal (amino a cids 1-222) of PBI; 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 map ped in amino acids 402-559, where one of the cap-binding domains mapped pre viously overlapped. (C) 1999 Academic Press.