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Virus replication efficiency is influenced by two conflicting factors, kinetics of the cellular interferon (IFN) response and induction of an antiviral state versus speed of virus replication and virus-induced inhibition of the IFN response. Disablement of a virus's capacity to circumvent the IFN response enables both basic research and various practical applications. However, such IFN-sensitive viruses can be difficult to grow to high-titer in cells that produce and respond to IFN. The current default option for growing IFN-sensitive viruses is restricted to a limited selection of cell-lines (e.g. Vero cells) that have lost their ability to produce IFN. This study demonstrates that supplementing tissue-culture medium with an IFN inhibitor provides a simple, effective and flexible approach to increase the growth of IFN-sensitive viruses in a cell-line of choice. We report that IFN inhibitors targeting components of the IFN response (TBK1, IKK2, JAK1) significantly increased virus replication. More specifically, the JAK1/2 inhibitor Ruxolitinib enhances the growth of viruses that are sensitive to IFN due to (i) loss of function of the viral IFN antagonist (due to mutation or species-specific constraints) or (ii) mutations/host cell constraints that slow virus spread such that it can be controlled by the IFN response. This was demonstrated for a variety of viruses, including, viruses with disabled IFN antagonists that represent live-attenuated vaccine candidates (Respiratory Syncytial Virus (RSV), Influenza Virus), traditionally attenuated vaccine strains (Measles, Mumps) and a slow-growing wild-type virus (RSV). In conclusion, supplementing tissue culture-medium with an IFN inhibitor to increase the growth of IFN-sensitive viruses in a cell-line of choice represents an approach, which is broadly applicable to research investigating the importance of the IFN response in controlling virus infections and has utility in a number of practical applications including vaccine and oncolytic virus production, virus diagnostics and techniques to isolate newly emerging viruses.

Work in St. Andrews was supported by the Medical Research Council, UK (RER and RJR), and the Scottish Funding Council (RJR). Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins. Publisher PDF

The NS1 protein of influenza A viruses is the dedicated viral interferon (IFN)-antagonist. Viruses lacking NS1 protein expression cannot multiply in normal cells but are viable in cells deficient in their ability to produce or respond to IFN. Here we report an unbiased mutagenesis approach to identify positions in the influenza A NS1 protein that modulate the IFN response upon infection. A random library of virus ribonucleoproteins containing circa 40 000 point mutants in NS1 were transferred to infectious virus and amplified in MDCK cells unable to respond to interferon. Viruses that activated the interferon (IFN) response were subsequently selected by their ability to induce expression of green-fluorescent protein (GFP) following infection of A549 cells bearing an IFN promoter-dependent GFP gene. Using this approach we isolated individual mutant viruses that replicate to high titers in IFN-compromised cells but, compared to wild type viruses, induced higher levels of IFN in IFN-competent cells and had a reduced capacity to counteract exogenous IFN. Most of these viruses contained not previously reported NS1 mutations within either the RNA-binding domain, the effector domain or the linker region between them. These results indicate that subtle alterations in NS1 can reduce its effectiveness as an IFN antagonist without affecting the intrinsic capacity of the virus to multiply. The general approach reported here may facilitate the generation of replication-proficient, IFN-inducing virus mutants, that potentially could be developed as attenuated vaccines against a variety of viruses., This work was supported by the Spanish Ministry of Science and Innovation (Ministerio de Ciencia e Innovacion) (www.mineco.gob.es/portal/site/mineco/idi) (grant BFU2010-17540/BMC) (JO), by Fundacion Marcelino Botin (www.fundacionbotin.org/) (JO) and The Wellcome Trust (www.wellcome.ac.uk/) (grant 087751/A/08/Z) (RER). MP-C was a predoctoral fellow and awardee of a short-term travel fellowship from the Spanish Research Council (CSIC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.