Virpath- Influenza, from emergence to control
Three major fields of basic research are conducting in our team:
Viral packaging and genetic reassortment.
The first field was the investigation of the mechanisms driving vRNP interactions allowing a selective packaging of the 8 gene segments in the nascent virion of influenza A (Rosa-Calatrava, Lina). This work is conducting in collaboration with Dr Roland Marquet’s lab (CNRS FRC 1589). We hypothesized that this process required RNA-RNA interactions. Our results confirmed that 5’ and 3’ extremities of specific gene segments can interact, resulting into an optimal network of interaction between the eight gene segments that allows the packaging of a full set of vRNP in each of the budding infectious particles (Fournier et al., 2012 NAR, Fournier et al., Vaccine 2012). This network can be disrupted by changing or impairing regions involved in the RNA-RNA interaction, as demonstrated by the interactions observed between H3N2 gene segments. In addition, we showed that this network of interaction is subtype specific and RNA/RNA interactions used internal nucleotide regions of H5N2 avian segments and, as a consequence, this may have some impact in the reassortment possibilities (Gavazzi et al., NAR 2013; Gavazzi et al., 2013 PNAS). Recently, we shown that genetic reassortment is strongly restricted by the sub-optimal compatibility between the vRNA packaging signals of divergent IAVs (Essere et al., 2013 PNAS). Somehow, the lab is leading this field of research in the understanding of genetic reassortment. Furthermore, our work lead to industrial applications, improving vaccine strains and yield of antigen production (FR 10/56976 and FR 2967072 – WO 2012059696).
Functional balance of HA/NA surface glycoproteins.
The second field is the long-standing research carried out by the team on the understanding of the HA and NA balance in virus fitness (Lina, Escuret). Using viruses with defective NA, or viruses with NA resistant to neuraminidase inhibitors, we carried out some investigations on the interplay between the two surface glycoproteins. First, we identified viruses lacking NA protein and the 6th gene segment. The HA of these viruses displayed low binding capacities, allowing these viruses to detach from the cell surface after budding. We also investigated viruses showing resistance to neuraminidase inhibitors. We have investigated the mechanisms for resistance to NAIs, and the possibility for viruses with combined framework mutation to maintain their fitness. This investigation on virus fitness required detailed analysis of the catalytic activity of the NA together with the affinity of HA to the sialic acids. These investigations have been carried out in clinical isolates, but also in genetically modified viruses by using Reverse Genetics, and in emerging viruses like H1N1pdm2009 and H5N1 viruses (Richard et al., Antimicrob Agents Chemother 2011; Moules et al., Virology, 2011; Richard et al., PLoS One 2012; Escuret et al., Antiviral Res, 2012; Ferraris et al., Antiviral Res 2012). Recently, with the collaboration of Dr S Gamblin and Dr J McCauley (NIMR, UK), we described a novel mutation associated to high level of resistance with detailed structural analysis of the NA catalytic pocket of A and B viruses (Escuret et al. JID, 2014). The research group (together with the NIC) is a leader in this field, as exemplified by the numerous invitations for presentation in international meetings.
Functional virus-host interactions.
The third field was the characterization of influenza-host interactions (Terrier, Rosa-Calatrava). This topic led to transcriptomic-based approaches and studies focused on functional interactions between viral proteins and cellular pathways (Josset et al. J Clin Virol, 2008; Terrier et al. Virology, 2012; Terrier et al. Virology, 2014; Dubois et al. Mbio, 2014).
The main project was focused on viral interactions with the p53 cellular pathway, known to be involved in a large panel of biological processes, including antiviral response. Transcriptional profiling studies in influenza viruses infected cells, have revealed an important deregulation of both upstream and downstream part of this pathway, including some specific miRNAs (Terrier et al. Virol J 2011; Terrier et al., J Gen Virol 2013). We have recently shown that influenza infection modulates p53 transcriptional activity and that influenza NS1 contributes to the inhibitory part of this modulation, possibly via its direct interaction with p53, ‘‘driving’’ the p53-mediated cell fate decision (Terrier et al. FEBS Letters 2013). Another level of regulation was also investigated with the study of the interplay between influenza viruses and p53 isoforms (Terrier et al. J Virol 2012). A preliminary model emerges in which the isoforms act as regulators of the p53-mediated cellular response against influenza viruses and more largely other pathogens (Terrier et al. PLoS Path 2013). Recently, our work was dedicated to the role of the main negative regulator of p53, Mdm2, during the time course of infection. Our results indicate a major role for Mdm2 during influenza infection and highlight the interest of Mdm2 antagonist molecules for optimization of viral production (Patent FR2967072 – WO 2012059696).
In parallel, we exploited transcriptomic signatures of human lung epithelial cells infected by different influenza viruses, in order to identify new inhibitors of the viral replication by targeting host cell rather viral factors. The pattern obtained was compared to a large a large database, and the screening for “reversed” signatures observed in drug-treated cells (Josset et al, PLoS One 2010). Using these in vitro & in silico approaches, we identified compounds with putative antiviral activity, and further assessed for their anti-influenza activity in vitro (Patents FR 2953410 – WO 2011069990 – EP2953410). Subsequently, we implemented two clinical trials (Lina, Rosa-Calatrava) to test in humans one of the most effective compounds in vitro. These clinical trials are the first ones that are testing an antiviral compound identified from in silico analyses of transcriptomic signatures of respiratory infections. These new approach will be implemented by new data from infected and non-infected patients, thanks to the two clinical studies that has been carried out (FLUNEXT, FLUMED) and a close partnership with Dr Guy Boivin’ lab (University of Laval, QC)