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 engineered via X-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.
Macrophages are an important cell type during coronavirus infections because they “notice” the infection and respond by activating type I interferons, which then act to establish antiviral defenses and limit virus replication. In turn, coronaviruses encode proteins that mitigate the cell’s ability to detect virus replication or amplify the interferon response. Here, we evaluated the host macrophage response to two independent mutant coronaviruses: one with a reduced deubiquitinating activity (DUBmut), and the other containing an inactivated endoribonuclease (EndoUmut). We observed a rapid, robust, and focused response to the EndoUmut virus, which was characterized by enhanced expression of interferon and interferon-stimulated genes. These results indicate that coronaviruses utilize EndoU activity for preventing early activation of interferon in macrophages, thereby allowing for viral replication. In contrast, DUBmut elicited a transient interferon response and ultimately activated over 2,800 genes, including many well-known players in pro-inflammatory pathways and the unfolded protein response. These DUBmut-induced pathways are associated with development and progression of significant disease, similar to what is observed during wild type virus infection. This study demonstrates the distinct consequences of mutating different viral interferon antagonists and reveals that intact coronaviral EndoU activity substantially contributes to the ability of coronaviruses to replicate in macrophages.
Here, we report that inactivation of a coronaviral interferon antagonist, EndoU, profoundly alters the host response to viral replication in macrophages. We find that the EndoUmut virus elicits a rapid, robust, and specific antiviral response that was effective in limiting virus replication. In contrast, our data show that the WT and DUBmut viruses ultimately elicit very similar host responses that are both characteristic of an unfolded protein response and consistent with a proinflammatory profile that is associated with viral pathogenesis. The results from the DUBmut-infected macrophages indicate that the mere induction of type I IFN is not a sufficient marker for attenuation of the virus. Instead, these results suggest that the timing and the magnitude of the host antiviral response are critical for determining the outcome of infection in macrophages. Our observation that the EndoUmut virus induces an earlier and more profoundly elevated type I interferon response than even the DUBmut virus implies that there is a threshold of IFN expression that must be breached before the cell mounts an effective antiviral response.
The structure-guided approach used to generate the DUBmut virus allowed for characterization of three different classes of mutant enzymes: Class I, deficient in both DUB and de-ISGylating activity; Class II, deficient in de-ISGylating activity only; and Class III, deficient in DUB activity but competent in protease and de-ISGylating activity. We utilized three unique biochemical substrates, each with a conjugated fluorescent AMC reporter, to evoke the multi-functional activities of PLP2. Activity against the z-RLRGG-AMC peptide substrate represents the polyprotein processing activity of MHV PLP2, while UB-AMC and ISG15-AMC stimulate the deubiquitinating and deISGylating activities of the enzyme, respectively. The kinetic data for the D1772A DUBmut hydrolysis of the z-RLRGG-peptide provided indicate that the polyprotein processing ability of this mutant is likely not affected by the D1772A substitution. The deISGylating ability of the enzyme is also not affected. In contrast, the mutant enzyme’s deubiquitinating activity is significantly reduced relative to wild type, which is most likely due to a lowered binding affinity for ubiquitin as the enzyme could not be saturated with Ub-AMC as a substrate.
We were able to reproduce the enzymatic profile of the purified PLP2-D1772A mutant protein when we expressed it in cell culture. Therefore, our finding that the DUBmut virus containing the PLP2-D1772A substitution activates an elevated antiviral response in macrophages compared to the wild type virus, but that this antiviral state results in only mild attenuation of disease in mice relative to WT infection, was unexpected. Previous studies demonstrated that ubiquitin has important roles in both the activation and the attenuation of innate antiviral pathways; therefore, we anticipated a more remarkable phenotype for a DUB-mutant virus. We can imagine several possible explanations for our findings. First, it is possible that viral DUB activity has a relatively minor role in shaping pathogenesis in this system. In fact, our recent studies using SARS-CoV and SARS-related CoVs found that the papain-like protease domain/DUB is a virulence trait that varies among members of the SARS-coronavirus species. In that study, we found that replacing the SARS PLP2/DUB domain with a SARS-related PLP2/DUB domain reduced the ability of that virus to antagonize the innate immune response. Together, this previous report in conjunction with the current study support the concept that different PLP2/DUB domains may have distinct effects on antagonism of the innate immune response depending on the virus and the host cell type. Another possibility is that the DUB-mutant virus we generated may not have been sufficiently debilitated in its DUB activity to result in altered pathogenesis. We found that it was difficult to recover viable DUB-mutant viruses; indeed, this D1772A mutant was the sole viable DUB-mutant representative of our many attempts. Because an elevated interferon response was elicited from DUBmut-infected cells, this mutant virus fulfilled our criteria demonstrating the inactivation of an interferon antagonist. However, we speculate that if we are able to recover mutants that exhibit a range of DUB activity, we may be able to more fully assess the role of DUB activity as a contributor to coronaviral pathogenesis. Despite these caveats, the MHV DUB-mutant generated in this study did exhibit a reproducible phenotype of eliciting an elevated interferon response in infected macrophages that was associated with mild attenuation of pathogenesis with reduced titers in the livers and spleens of mice at day 5 post-infection. Overall, we conclude that DUB activity is indeed a virulence trait, but not a major driver of virulence for MHV. Our results support the concept that multiple viral factors likely work in concert to shape and ultimately limit the innate immune response to coronavirus replication.
The other remarkable finding from this study was the distinct transcriptional profile elicited by the EndoUmut virus during infection of bone marrow-derived macrophages compared to the profiles of wild type- and DUBmut-infected cells. We detected elevated levels of interferon and interferon-responsive genes as early as 6-9 hpi, with EndoUmut-infected cells exhibiting by far the highest expression levels of these genes. We and others found that an antiviral response to EndoUmut infection results in activation of apoptosis, which subsequently limits virus replication in cell culture and in infected mice. The current study indicates that screening MHV mutants in interferon-responsive cells may be an effective approach to identifying strains that stimulate a robust innate immune response, which may then restrict virus replication in animals. Previous studies of coronavirus-encoded interferon antagonists focused on evaluating the host transcriptional response to infection at later time points (such as 24 and 48 hpi) and in a variety of cell types or the tissues of infected animals. For example, a study comparing infection of mice with wild type SARS-CoV versus a virus containing a mutation in the interferon antagonist nsp16, termed the dNSP16 CoV, revealed that the transcriptional profile in the lungs of the dNSP16-infected mice mirrored the response to wild type virus. However, combining the dNSP16 mutation with a mutation in another interferon antagonist, ExoN, was shown to reduce disease in mice and elicit protective immunity. It would be interesting to determine if the transcriptional profile elicited by the double mutant SARS-CoV is altered compared to wild type virus, particularly at early times after infection. A study of MERS-CoV comparing the host response to wild type virus with the response to a mutant virus containing deleted accessory open reading frames (MERS-CoV dORF3-5) provides evidence of significant differences in the early transcriptional responses to infection in Calu3 2B4 cells. This study revealed that MERS-CoV dORF3-5 infection prompted earlier (7, 12, and 24 hpi) and more robust type I and type III interferon responses, and that the mutant virus was more sensitive than wild type to pre-treatment with interferon. The MERS-CoV interferon antagonist mutant virus was attenuated in mice and elicited a protective response against subsequent lethal challenge. For coronaviruses, one difficulty that arises when evaluating the role(s) of viral-mediated modulation of the host response is that multiple viral proteins, including both structural and nonstructural proteins, have been shown to antagonize the innate immune response in vitro and/or in vivo. These include: nsp16-2’O MTase, nsp14-ExoN, nsp1, nsp7, E protein, N protein, M protein, SARS-CoV-ORF6, MERS-CoV-ORF3-5, MERS-CoV-4a, and MERS-CoV-4b. Each of these interferon antagonists may play either a cell- or tissue-type specific role, or act in concert with other factors during viral replication to mitigate the innate immune response. Further studies are needed to fully understand if all or only a subset of these antagonists must be silenced to generate an effective, live-attenuated vaccine. The results presented here and from the study of the MERS-CoV dORF3-5 mutant virus indicate that inactivating interferon antagonists and screening for an early and robust antiviral transcriptional profile may represent an efficient and informative approach to evaluating live-attenuated vaccine candidate strains for existing and emerging coronaviruses.
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. 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. 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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