Abstract
Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.
Highlights
Cytoplasm and nucleus[12,13,14,15]
We demonstrated that the expression of Non virion (NV) or PPM1Bb negatively regulates antiviral signaling by targeting TANK-binding kinase 1 (TBK1), indicating the important role of PPM1Bb for infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) to evade innate immunity
In an effort to identify cellular partners of NV proteins, we first designed, using reverse genetics, VHSV mutants in which NV from both Novirhabdoviruses were overexpressed with an N-terminal tag allowing an easier immunopurification of the proteins
Summary
Cytoplasm and nucleus[12,13,14,15]. The recovery of NV-deletion mutants, by reverse genetics[16], demonstrated that the NV protein was dispensable but necessary for an efficient virus replication in cell culture[17,18,19,20] and essential for virus pathogenicity in rainbow trout[21,22], yellow perch[17] and Japanese olive flounder[20]. The IFN system is remarkably conserved in vertebrates and teleost fish possess functional orthologs of human retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I, MDA5 and LGP2, as well as several downstream signaling molecules, such as MAVS, TBK1 and IRF327–29 The pathogen components, such as nucleic acids and viral proteins, are recognized through specific pattern-recognition receptors (PRRs), leading to the activation of multiple signaling cascades that induce the production of interferons (IFN) and other cytokines. We report the establishment of an original system where triple flag-tagged NV proteins from IHNV and VHSV are overexpressed during the viral infection These recovered recombinant viruses have similar growth properties in vitro in cell culture and in vivo in rainbow trout compared to those of the WT virus, and they have been used to co-purify NV and its associated proteins from infected cells. We demonstrated that the expression of NV or PPM1Bb negatively regulates antiviral signaling by targeting TBK1, indicating the important role of PPM1Bb for IHNV and VHSV to evade innate immunity
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