It is generally recognised that novel antiviral drugs, less prone to resistance, would be a desirable alternative to current drug options in order to be able to treat potentially serious influenza infections. The viral polymerase, which performs transcription and replication of the RNA genome, is an attractive target for antiviral drugs since potent polymerase inhibitors could directly stop viral replication at an early stage. Recent structural studies on functional domains of the heterotrimeric polymerase, which comprises subunits PA, PB1 and PB2, open the way to a structure based approach to optimise inhibitors of viral replication. In particular, the unique cap-snatching mechanism of viral transcription can be inhibited by targeting either the PB2 cap-binding or PA endonuclease domains. Here we describe high resolution X-ray co-crystal structures of the 2009 pandemic H1N1 (pH1N1) PA endonuclease domain with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme. Comparison of the binding mode of the different compounds and that of a mononucleotide phosphate highlights, firstly, how different substituent groups on the basic metal binding scaffold can be orientated to bind in distinct sub-pockets within the active site cavity, and secondly, the plasticity of certain structural elements of the active site cavity, which result in induced fit binding. These results will be important in optimising the design of more potent inhibitors targeting the cap-snatching endonuclease activity of influenza virus polymerase.

We investigated the mechanism by which glycyrrhizin (GL), the main active component of licorice roots, protects cells from infection with influenza A virus (IAV). We found that GL treatment leads to a clear reduction in the number of IAV-infected human lung cells as well as a reduction in the CCID50 titer by 90%. The antiviral effect, however, was limited to one or two virus replication cycles. Analysis of different GL treatment protocols suggested that the antiviral effect of GL was limited to an early step in the virus replication cycle. A direct inhibitory action of GL on IAV particles could be excluded and GL did not interact with virus receptor binding either. The antiviral effect of GL was abolished by treatment 1h after virus infection, whereas pre-treatment and treatment during and after virus adsorption led to a reduction in the cytopathic effect, reduced viral RNA within the cells and in the cell supernatants, and reduced viral hemagglutination titers. Detailed virus uptake analyses unambiguously demonstrated reduced virus uptake in various GL-treated cells. These observations lead to the conclusion, that the antiviral activity of GL is mediated by an interaction with the cell membrane which most likely results in reduced endocytotic activity and hence reduced virus uptake. These insights might help in the design of structurally related compounds leading to potent anti-influenza therapeutics.

In this study, several influenza NS1 mutants were examined for their growth ability in interferon (IFN)-deficient Vero cells treated with human interferon alpha (IFN-alpha). Mutants with an intact RNA binding domain showed similar growth properties as the wild-type virus, whereas viruses carrying an impaired RNA binding domain were dramatically attenuated. Relying on the ability of the first half of the NS1 protein to antagonize the IFN action, we established a rescue system for the NS gene based on the transfection of one plasmid expressing recombinant NS vRNA and subsequent coinfection with an IFN sensitive helper virus followed by adding of human IFN-alpha as a selection drug. Using this method, a recombinant influenza A virus expressing green fluorescence protein (GFP) from the NS1 reading frame was rescued. To ensure the posttranslational cleavage of GFP from the N-terminal 125 amino acids (aa) of NS1 protein, a peptide sequence comprising a caspase recognition site (CRS) was inserted upstream the GFP protein. Although a rather long sequence of 275 aa was inserted into the NS1 reading frame, the rescued recombinant vector appeared to be genetically stable while passaging in Vero cells and was able to replicate in PKR knockout mice.

Influenza virus uses a unique cap-snatching mechanism characterized by hijacking and cleavage of host capped pre-mRNAs, resulting in short capped RNAs, which are used as primers for viral mRNA synthesis. The PA subunit of influenza polymerase carries the endonuclease activity that catalyzes the host mRNA cleavage reaction. Here, we show that PA is a sequence selective endonuclease with distinct preference to cleave at the 3′ end of a guanine (G) base in RNA. The G specificity is exhibited by the native influenza polymerase complex associated with viral ribonucleoprotein particles and is conferred by an intrinsic G specificity of the isolated PA endonuclease domain PA-Nter. In addition, RNA cleavage site choice by the full polymerase is also guided by cap binding to the PB2 subunit, from which RNA cleavage preferentially occurs at the 12th nt downstream of the cap. However, if a G residue is present in the region of 10–13 nucleotides from the cap, cleavage preferentially occurs at G. This is the first biochemical evidence of influenza polymerase PA showing intrinsic sequence selective endonuclease activity.