Influenza virus contains eight single-stranded RNA segments of negative polarity as the genome and an RNA-dependent RNA polymerase as a virion component (26). Influenza virus RNA polymerase catalyzes both transcription [the synthesis of plus-strand mRNA containing the host cell-derived cap I structure at the 5'-terminus and poly (A) tail at the 3'-terminus] and replication [the synthesis of full-length plus-strand complementary RNA (cRNA) and the cRNA-dependent synthesis of minus-strand viral RNA (vRNA)] (14,17,18,24,50). The viral RNA polymerase also catalyzes polyadenylation at the 3'-termini of mRNA in vitro (43). The viral RNA polymerase also performs templatedependent capped RNA cleavage (21, 41) and apparent proofreading of nascent RNA chains (19).The RNA polymerase purified from influenza virus consists of one part each of three subunits, PB1, PB2 and PA (15). In vitro reconstitution studies of enzymatically active RNA polymerase using individual P proteins purified either from baculovirus-infected cells (23), Pichia pastoris cells (16) or by SDS-polyacrylamide gel electrophoresis of virions (49) confirmed the subunit structure. The function of each subunit of influenza virus RNA polymerase has been genetically and biochemically characterized. For instance, PB1 subunit can be cross-linked with nucleotide substrates (3, 4, 6), and nuclear extracts containing PB1 subunit alone or cells expressing both PB1 and NP can catalyze RNA synthesis, depending on the model RNA templates (22,35,52), indicating that PB1 is involved in polymerization of RNA chains. PB1 bound with both negative and positive sense RNAs (10, 28). Cap I analog was cross-linked with PB2 in vitro (6,13,29,53), and RNA synthesized in cells without PB2 lacks the 5'-cap structure (35), suggesting that PB2 is required for cap binding and synthesis of capped mRNAs. The cap-dependent RNase active site has been mapped in PB1 recently (29). However, the information about the role of PA on viral replication remains limited. Temperature-sensitive (ts) mutations in the PA gene affected only vRNA synthesis, but not mRNA synthesis (25,30,33,45,46). Nakagawa et al. demonstrated that PA was essential for cRNA-dependent vRNA synthesis (36). We identified a unique protease activity in purified PA protein (12).The subunit binding sites of the influenza virus RNA polymerase were mapped, which demonstrated that PB1

Background: Influenza virus RNA polymerase is a multifunctional enzyme that catalyses both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood, although the enzyme is known to be required for cRNA 3 vRNA synthesis. The protease related activity of PA has been discussed ever since protease-inducing activity was demonstrated in transfection experiments.

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Influenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNAcleaving DNA enzymes from 10-23 catalytic motif to target PB2-AUG initiation codon and measured their RNA-cleaving activity in vitro. Although the RNA-cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection. ß

In Japan, the use of amantadine for treatment of influenza A virus infection was not accepted until November 1998, although it was widely used for treatment of Parkinsonism. Since then, we have monitored the emergence of amantadine-resistant viruses and isolated two viruses from patients on long-term treatment with amantadine.