Coronaviruses encode multiple interferon antagonists that modulate the host response to virus replication. Here, we evaluated pathogenesis and host transcription in response to infection with murine coronaviruses encoding independent mutations in two different viral antagonists: the deubiquitinase (DUB) within nonstructural protein 3 and the endoribonuclease (EndoU) within nonstructural protein 15. The virus with reduced ability to deubiquitinate proteins, herein termed the DUBmut virus, was engineeredviaX-ray structure-guided mutagenesis and activates an earlier interferon response than the wild type virus. However, the replication kinetics of DUBmut in cultured cells are similar to wild type virus and pathogenesis in mice is also similar to what was observed during infection with wild type virus. On the other hand, we previously reported that an EndoUmut virus containing an inactivated endoribonuclease activity elicited rapid and robust activation of type I interferon, which limited virus replication and pathogenesis. Here, using a transcriptomics approach, we compared the scope and kinetics of the host response to the wild type, DUBmut, and EndoUmut viruses in infected macrophages. We found that the EndoUmut virus activates a focused response, predominantly involving type I interferons and a subset of interferon-responsive genes, within 12 hours after infection. In contrast, the wild type and DUBmut viruses stimulate upregulation of over 2,800 genes, including activation of unfolded protein response (UPR) pathways and a proinflammatory profile associated with viral pathogenesis. This study highlights the role of viral interferon antagonists in shaping the kinetics and magnitude of the host response during virus infection and demonstrates that inactivation of a dominant viral antagonist, the coronavirus endoribonuclease, dramatically alters the host response in macrophages and the disease process.
Our results demonstrating upregulation of the unfolded protein response (UPR) in response to wild type and DUBmut coronavirus replication confirm and extend the work of earlier studies that documented activation of the ER sensors PERK, IRE1, and ATF-6 during coronavirus infection [32,34,35]. Heavy utilization of the endoplasmic reticulum for generating coronavirus replication complexes and of the ER-Golgi intermediate compartment for assembling virus particles places a substantial load on the host translational machinery during infection. Host sensors IRE1, ATF-6, and PERK are situated in the ER to sense and respond to such overload by prompting upregulated expression of genes encoding ER chaperones, amino acid transporters, and activators of lipid biosynthesis. Ironically, many of these proteins ultimately facilitate virus replication and assembly. Notably, it has been demonstrated that UPR pathways that promote apoptosis are blocked during coronavirus replication [32, 34]. The ability of viruses to modulate the UPR has important implications for the innate immune response to such viruses because the UPR has been shown to attenuate antiviral defenses by way of degrading the type I interferon receptor . To our knowledge, the results presented here provide the first transcriptomic evidence of UPR activation in coronavirus-infected macrophages, underlining an important role for UPR pathways in the coronavirus life cycle. Our observation that EndoUmut-infected macrophages exhibit significantly lower expression of several genes involved in UPR pathways compared with wild type- and DUBmut-infected cells is consistent with the reduced levels of virus replication detected in EndoUmut-infected macrophages.
The notion of inactivating viral interferon antagonists as a means of generating live-attenuated vaccines is supported by recent reports of screening for inactivation of influenza A virus-encoded interferon antagonists , as well as studies that revealed that the classic vaccine strain of yellow fever virus encodes an interferon antagonist in the NS5 protein . For coronaviruses, it is not yet clear if disabling a single interferon antagonist, such as the highly conserved EndoU, will be sufficient to attenuate viruses that infect different cell types in different species. Promisingly, our studies of a coronavirus that causes lethal disease in piglets, porcine epidemic diarrhea virus (PEDV), revealed that inactivation of EndoU activity is associated with attenuated disease . However, additional work is needed to evaluate potential reversion of EndoU mutant viruses and to determine if inactivating multiple interferon antagonists is an effective approach for generating safe and effective live-attenuated coronavirus vaccines.
Reference & source information: https://www.biorxiv.org/
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