Final Report Summary - FLU-POLII (Association between influenza virus RNA polymerase and the transcriptional machinery of the host cell) The general aim of this project was to understand how interactions of the influenza virus polymerase with cellular factors regulate its function at the molecular level. The project addressed two specific aims and their corresponding specific objectives:Aim 1: Characterize the interaction between the viral RNA polymerase and Pol II.Objective 1.1. Compare the in vitro binding of purified RdRp heterotrimer and vRNP to synthetic CTD mimic peptides.Objective 1.2.a. Map the Pol II interaction binding domain of the viral polymerase.Objective 1.2.b. Identify factors mediating the interaction between the viral polymerase and Pol II.(Objectives 1.2.a. and 1.2.b. were mutually exclusive)Objective 1.3. Analyze the transcriptional and replicative activity of viral polymerase mutants that do not interact with Pol II.Aim 2: Conduct a genome-wide search for associations of influenza virus RNA polymerase.Objective 2.1. Develop a method to analyze interactions between influenza virus proteins and DNA on a genome-wide scale.Objective 2.2. Identify sites in the human genome targeted by the viral RNAThe major achievements expected from this project were to: (1) elucidate whether the interaction between the viral RNA polymerase and cellular Pol II is direct or indirect (i.e. mediated by host factors), (2) map the binding domain of the viral polymerase to Pol II (or to the mediating host factor(s)), and (3) identify sites in the human genome targeted by the viral polymerase. To this end, an assay was developed to compare the in vitro binding of purified RdRp heterotrimer and vRNP to synthetic CTD mimic peptides. Peptide pulldowns were carried out either with purified viral polymerase or infected cell lysates. In order to map the binding domain, bioinformatic predictions were used for rational design of mutants predicted to be disabled in binding to Pol II CTD. The transcriptional and replicative activity of those mutants was analysed by primer extension assay. Crystal trials were also set up to attempt co-crystallisation of the viral polymerase with synthetic peptides mimicking the CTD of Pol II. BIACORE experiments were carried out to determine the affinity of the interaction between the viral polymerase and Pol II CTD.Genome-wide experiments have continued beyond the time-frame of this project and have opened a new research line in the host laboratory. Those results have not yet been published and therefore are not discussed in this section for confidentiality.The main results of the project show that fully assembled viral ribonucleoproteins, as well as RNA-free polymerases, bind to the CTD of the serine-5 phosphorylated form of Pol II, which is engaged in transcription initiation. Results also show that the binding is direct and occurs via the viral polymerase. Moreover, the binding is conserved in influenza C viruses, which are evolutionarily distant. This suggests that the interaction with Pol II is important for influenza. Although mapping the binding domain of the viral polymerase was not achieved in this project, tools have been developed towards that objective. Eventually, mapping the binding domain of the viral polymerase to Pol II will uncover targets for development of novel antivirals. This will have a profound socioeconomic impact, because measures to combat influenza are currently scarce. Circulating strains do not always match the manufactured vaccines, and development of resistance to the few available antivirals is a matter of concern. Therefore, identifying new targets for antiviral development is of utmost importance.In addition to showing the global effects of influenza infection on cellular transcription, genome-wide analysis has also provided data on specific genes that are targeted upon infection, which will contribute to shed light into gene expression regulation upon influenza virus infection.
