Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype have recently emerged from avian zoonotic reservoirs to cause fatal human disease. Adaptation of HPAI virus RNA-dependent RNA polymerase (PB1, PB2, and PA proteins) and nucleoprotein (NP) to interactions with mammalian host proteins is thought to contribute to the efficiency of viral RNA synthesis and to disease severity. While proteomics experiments have identified a number of human proteins that associate with H1N1 polymerases and/or viral ribonucleoprotein (vRNP), how these host interactions might regulate influenza virus polymerase functions and host adaptation has been largely unexplored. We took a functional genomics (RNA interference [RNAi]) approach to assess the roles of a network of human proteins interacting with influenza virus polymerase proteins in viral polymerase activity from prototype H1N1 and H5N1 viruses. A majority (18 of 31) of the cellular proteins tested, including RNA-binding (DDX17, DDX5, NPM1, and hnRNPM), stress (PARP1, DDB1, and Ku70/86), and intracellular transport proteins, were required for efficient activity of both H1N1 and H5N1 polymerases. NXP2 and NF90 antagonized both polymerases, and six more RNA-associated proteins exhibited strain-specific phenotypes. Remarkably, 12 proteins differentially regulated H5N1 polymerase according to PB2 genotype at mammalian-adaptive residue 627. Among these, DEAD box RNA helicase DDX17/p72 facilitated efficient human-adapted (627K) H5N1 virus mRNA and viral RNA (vRNA) synthesis in human cells. Likewise, the chicken DDX17 homologue was required for efficient avian (627E) H5N1 infection in chicken DF-1 fibroblasts, suggesting that this conserved virus-host interaction contributes to PB2-dependent host species specificity of influenza virus and ultimately to the outcome of human HPAI infections.

The assembly of the polymerase complex of influenza A virus from the three viral polymerase subunits PB1, PB2, and PA is required for viral RNA synthesis. We show that peptides which specifically bind to the protein-protein interaction domains in the subunits responsible for complex formation interfere with polymerase complex assembly and inhibit viral replication. Specifically, we provide evidence that a 25-amino-acid peptide corresponding to the PA-binding domain of PB1 blocks the polymerase activity of influenza A virus and inhibits viral spread. Targeting polymerase subunit interactions therefore provides a novel strategy to develop antiviral compounds against influenza A virus or other viruses.Influenza A viruses are human pathogens that are responsible for both annual epidemics and recurring devastating pandemics. The recent emergence of highly pathogenic avian influenza virus strains of the H5N1 subtype and the lethality associated with these viruses reflect the continuing threat of influenza viruses. Despite the existence of vaccines against annually recurring influenza A and B virus strains, as well as antiviral drugs targeting either the viral neuraminidase (9) or M2 proteins (14), the protection against epidemic and pandemic influenza is still incomplete. The viral RNA-dependent RNA polymerase consisting of three subunits, PA, PB1, and PB2 (the P proteins), is a potential target for the development of new anti-influenza drugs. PB1 is the central protein, containing two different domains interacting with the PB2 and the PA subunits (1,7,13,16). We hypothesized that viral RNA synthesis could be blocked by the specific inhibition of viral polymerase complex formation by using small peptides which bind to the protein-protein interaction domains responsible for hetero-oligomerization between the individual subunits. Here, we describe the characterization of a PB1-derived short peptide that fulfils these criteria.The first 25 aa of PB1 fused to GFP bind to the viral polymerase subunit PA and inhibit polymerase activity. To design an inhibitory peptide, we selected the region encompassing amino acids (aa) 1 to 25 of the PB1 polymerase subunit of influenza A virus (A/WSN/33), which has been demonstrated previously to bind the PA subunit (16, 17) and is highly conserved among influenza A virus strains (Fig. 1A). To confirm that this region indeed binds PA, we fused this sequence to the N terminus of the green fluorescent protein (GFP), transiently expressed the fusion protein (PB1 1-25 -GFP) together with hemagglutinin (HA)-tagged PA (PA-HA) in HEK293T cells, and immunoprecipitated PA-HA from cell extracts by using anti-HA antibodies. As shown in Fig. 1B, PB1 1-25 -GFP, but not Flag-tagged GFP, was specifically immunoprecipitated with PA-HA (lane 3). In a similar experiment, we also tested whether the PB1 aa 715 to 740, which are embedded in a larger PB2-binding domain (13), were sufficient for binding PB2 when fused to GFP (PB1 715-740 -GFP). However, PB1 715-740 -GFP could not be immunoprecipitated with HA-t...

The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses.