Antiviral innate immunity relies on the recognition of microbial structures. One such structure is viral RNA that carries a triphosphate group on its 5' terminus (PPP-RNA). By an affinity proteomics approach with PPP-RNA as the 'bait', we found that the antiviral protein IFIT1 (interferon-induced protein with tetratricopeptide repeats 1) mediated binding of a larger protein complex containing other IFIT family members. IFIT1 bound PPP-RNA with nanomolar affinity and required the arginine at position 187 in a highly charged carboxy-terminal groove of the protein. In the absence of IFIT1, the growth and pathogenicity of viruses containing PPP-RNA was much greater. In contrast, IFIT proteins were dispensable for the clearance of pathogens that did not generate PPP-RNA. On the basis of this specificity and the great abundance of IFIT proteins after infection, we propose that the IFIT complex antagonizes viruses by sequestering specific viral nucleic acids.

Viruses must enter host cells to replicate, assemble and propagate. Because of the restricted size of their genomes, viruses have had to evolve efficient ways of exploiting host cell processes to promote their own life cycles and also to escape host immune defence mechanisms. Many viral open reading frames (viORFs) with immune-modulating functions essential for productive viral growth have been identified across a range of viral classes. However, there has been no comprehensive study to identify the host factors with which these viORFs interact for a global perspective of viral perturbation strategies. Here we show that different viral perturbation patterns of the host molecular defence network can be deduced from a mass-spectrometry-based host-factor survey in a defined human cellular system by using 70 innate immune-modulating viORFs from 30 viral species. The 579 host proteins targeted by the viORFs mapped to an unexpectedly large number of signalling pathways and cellular processes, suggesting yet unknown mechanisms of antiviral immunity. We further experimentally verified the targets heterogeneous nuclear ribonucleoprotein U, phosphatidylinositol-3-OH kinase, the WNK (with-no-lysine) kinase family and USP19 (ubiquitin-specific peptidase 19) as vulnerable nodes in the host cellular defence system. Evaluation of the impact of viral immune modulators on the host molecular network revealed perturbation strategies used by individual viruses and by viral classes. Our data are also valuable for the design of broad and specific antiviral therapies.

Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner.