Peptide-Mediated Interference with Influenza A Virus Polymerase

Abstract

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...

Cyclin T1/CDK9 Interacts with Influenza A Virus Polymerase and Facilitates Its Association with Cellular RNA Polymerase II

“…To determine the role of cyclin T1/CDK9 in replication, we asked if cyclin T1/CDK9 was involved in regulating viral transcription. An influenza A virus minireplicon assay was employed to measure viral polymerase activity (10). Using this assay, we found that overexpression of either wild-type CDK9 or the dominant negative form of CDK9 (CDK9-DN) (29) did not affect viral transcription activity, while overexpression of cyclin T1 promoted the transcription activity of vRNP significantly (Fig.…”

Single gene reassortants identify a critical role for PB1, HA, and NA in the high virulence of the 1918 pandemic influenza virus

“…Recent work where a point mutation was introduced at amino acid 66 of PB1-F2 revealed the importance of this protein in the virulence of a H5N1 virus and the 1918 pandemic virus (37). A better understanding of the contribution of polymerase proteins in virulence will aid in designing drugs that target the key intersubunit binding sites of the polymerase complex and diminish the high replication efficiency of pandemic virus strains (39). For currently circulating H5N1 influenza viruses, the PB2 polymerase subunit appears to play a greater role in the high growth phenotype and increased virulence associated with these highly pathogenic viruses in mammals (40,41).…”

The influenza virus RNA synthesis machine

“…Some of these interactions have been verified by co-crystallisation [55][56][57] and functional studies. 58 Also, some additional PB2-PA and PB1-PB2 interactions have been described in reference 40 and 59, that may play structural and/or regulatory roles in the activity of the viral polymerase.…”