The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant.

Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3' splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant.

The RNA Helicase DDX6 Associates with RIG-I to Augment Induction of Antiviral Signaling.

Virus infections induce sensitive antiviral responses within the host cell. The RNA helicase retinoic acid-inducible gene I (RIG-I) is a key sensor of influenza virus RNA that induces the expression of antiviral type I interferons. Recent evidence suggests a complex pattern of RIG-I regulation involving multiple interactions and cellular sites. In an approach employing affinity purification and quantitative mass spectrometry, we identified proteins with increased binding to RIG-I in response to influenza B virus infection. Among them was the RIG-I related RNA helicase DEAD box helicase 6 (DDX6), a known component of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules like P-bodies and stress granules (SGs). RIG-I and DDX6 both localized to the cytosol and were detected in virus-induced SGs. Coimmunoprecipitation assays detected a basal level of complexes harboring RIG-I and DDX6 that increased after infection. Functionally, DDX6 augmented RIG-I mediated induction of interferon (IFN)-ß expression. Notably, DDX6 was found to bind viral RNA capable to stimulate RIG-I. These findings imply a novel function for DDX6 as an RNA co-sensor and signaling enhancer for RIG-I.

Quantitative Proteomic Approach Identifies Vpr Binding Protein as Novel Host Factor Supporting Influenza A Virus Infections in Human Cells.

Influenza A virus (IAV) infections are a major cause for respiratory disease in humans, which affects all age groups and contributes substantially to global morbidity and mortality. IAV have a large natural host reservoir in avian species. However, many avian IAV strains lack adaptation to other hosts and hardly propagate in humans. While seasonal or pandemic IAV strains replicate efficiently in permissive human cells, many avian IAV cause abortive nonproductive infections in these hosts despite successful cell entry. However, the precise reasons for these differential outcomes are poorly defined. We hypothesized that the distinct course of an IAV infection with a given virus strain is determined by the differential interplay between specific host and viral factors. By using Spike-in SILAC mass spectrometry-based quantitative proteomics we characterized sets of cellular factors whose abundance is specifically up- or downregulated in the course of permissive versus nonpermissive IAV infection, respectively. This approach allowed for the definition and quantitative comparison of about 3500 proteins in human lung epithelial cells in response to seasonal or low-pathogenic avian H3N2 IAV. Many identified proteins were similarly regulated by both virus strains, but also 16 candidates with distinct changes in permissive versus nonpermissive infection were found. RNAi-mediated knockdown of these differentially regulated host factors identified Vpr binding protein (VprBP) as proviral host factor because its downregulation inhibited efficient propagation of seasonal IAV whereas overexpression increased viral replication of both seasonal and avian IAV. These results not only show that there are similar differences in the overall changes during permissive and nonpermissive influenza virus infections, but also provide a basis to evaluate VprBP as novel anti-IAV drug target.

Disruption of Src homology 3-binding motif within non-structural protein 1 of influenza B virus unexpectedly enhances viral replication in human cells.

The influenza virus non-structural protein 1 (NS1) is a multifunctional virulence factor that plays a crucial role during infection by blocking the innate antiviral immune response of infected cells. In contrast to the well-studied NS1 protein of influenza A virus, knowledge about structure and functions of the influenza B virus homologue B/NS1, which shares less than 25 % sequence identity, is still limited. Here, we report on a reverse genetic analysis to study the role of a highly conserved class II Src homology 3 domain-binding motif matching the consensus PxxPx(K/R) that we identified at positions 122-127 of the B/NS1 protein. Surprisingly, glycine substitutions in the Src homology 3 domain-binding motif increased virus replication up to three orders of magnitude in human lung cells. Enhanced mutant virus propagation was accompanied by increased gene expression and apoptosis induction linking this motif to the control of programmed cell death. A MS-based interactome study revealed that the glycine substitutions facilitate binding of B/NS1 to heat shock protein 90-beta (HSP90ß). Moreover, recruitment of the viral polymerase basic protein 2 to the B/NS1-HSP90ß complex was observed. Pharmacological inhibition of HSP90 reduced mutant virus propagation suggesting that the mutation-induced involvement of HSP90ß enhanced viral replication. This study not only functionally characterizes a conserved motif within the B/NS1 protein, but also illustrates a rare example in which mutation of a highly conserved sequence within a viral protein does not result in high fitness costs, but rather increases viral replication via recruitment of a host factor.

Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication.

UNLABELLED: During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE: Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.