Inactivating Three Interferon Antagonists Attenuates Pathogenesis of an Enteric Coronavirus

ABSTRACT Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2. IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.

SARS-CoV-2 proteases cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species and the search for reservoir hosts

The genome of SARS-CoV-2 (SARS2) encodes for two viral proteases (NSP3/ papain-like protease and NSP5/ 3C-like protease or major protease) that are responsible for cleaving viral polyproteins for successful replication. NSP3 and NSP5 of SARS-CoV (SARS1) are known interferon antagonists. Here, we examined whether the protease function of SARS2 NSP3 and NSP5 target proteins involved in the host innate immune response. We designed a fluorescent based cleavage assay to rapidly screen the protease activity of NSP3 and NSP5 on a library of 71 human innate immune proteins (HIIPs), covering most pathways involved in human innate immunity. By expressing each of these HIIPs with a genetically encoded fluorophore in a cell-free system and titrating in the recombinant protease domain of NSP3 or NSP5, we could readily detect cleavage of cognate HIIPs on SDS-page gels. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type- I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of IL-6 and inflammatory response observed in COVID-19 patients. Surprisingly, both NLRP12 and TAB1 have each two distinct cleavage sites. We demonstrate that in mice, the second cleavage site of NLRP12 is absent. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for in-depth studies into the pathophysiology of COVID-19 and should facilitate the search or development of more effective animal models for severe COVID-19. Finally, we discovered that one particular species of bats, David’s Myotis, possesses the five cleavage sites found in humans for NLRP12, TAB1 and IRF3. These bats are endemic from the Hubei province in China and we discuss its potential role as reservoir for the evolution of SARS1 and SASR2.

Coronavirus Endoribonuclease and Deubiquitinating Interferon Antagonists Differentially Modulate the Host Response during Replication in Macrophages

ABSTRACT Coronaviruses (CoVs) encode multiple interferon (IFN) antagonists that modulate the host response to virus replication. Here, we evaluated the host transcriptional response to infection with murine coronaviruses encoding independent mutations in one of two different viral antagonists, the deubiquitinase (DUB) within nonstructural protein 3 or the endoribonuclease (EndoU) within nonstructural protein 15. We used transcriptomics approaches to compare the scope and kinetics of the host response to the wild-type (WT), DUBmut, and EndoUmut viruses in infected macrophages. We found that the EndoUmut virus activates a focused response that predominantly involves type I interferons and interferon-related genes, whereas the WT and DUBmut viruses more broadly stimulate upregulation of over 2,800 genes, including networks associated with activating the unfolded protein response (UPR) and the proinflammatory response 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 inactivating a dominant viral antagonist, the coronavirus endoribonuclease, dramatically alters the host response in macrophages. IMPORTANCE Macrophages are an important cell type during coronavirus infections because they “notice” the infection and respond by inducing type I interferons, which limits virus replication. In turn, coronaviruses encode proteins that mitigate the cell’s ability to signal an interferon response. Here, we evaluated the host macrophage response to two independent mutant coronaviruses, one with 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-related genes. In contrast, wild-type virus and the DUBmut virus elicited a more limited interferon response and ultimately activated over 2,800 genes, including players in the unfolded protein response and proinflammatory pathways associated with progression of significant disease. This study reveals that EndoU activity substantially contributes to the ability of coronaviruses to evade the host innate response and to replicate in macrophages.

A key region of molecular specificity orchestrates unique ephrin-B1 utilization by Cedar virus

The prototypic henipaviruses (HNV), Hendra (HeV) and Nipah (NiV), are emergent zoonotic pathogens responsible for frequent and fatal outbreaks of severe disease in domestic animals and humans. The HNV attachment glycoprotein (G) is a critical determinant of host-species and cell-type tropism. Utilization of highly conserved B-type ephrin ligands as functional entry receptors engenders HNVs with a broad permissive host range, accounts for zoonotic spillover, and is closely aligned with observed disease pathologies. Recent studies have uncovered numerous divergent clades of HNVs globally. Cedar virus (CedV), the closest relative of HeV and NiV identified to date, can establish experimental infections, yet has not been observed to cause overt disease. While the apathogenic phenotype may be attributed to a lack of P-gene derived interferon antagonists, the V and W accessory proteins, additional determinants of differential HNV pathobiology could be involved. Here, through comparative functional and structural analysis of CedV-G, we characterize molecular interactions critical to viral entry. We demonstrate that CedV possesses a unique cellular entry receptor repertoire which, in addition to functional utilization of the common HNV receptor, ephrin-B2, includes the hitherto uncharacterized interaction with ephrin-B1. Crystal structures reveal a conserved recognition mode between diverse HNV-G proteins and their distinct ephrin receptors and identify a region of molecular specificity within CedV-G that is a key determinant of ephrin selectivity. This work provides a platform for understanding the functional diversity and varied receptor tropism characteristics of HNV glycoproteins that will facilitate assessment of the pathogenic potential and transmissibility of newly discovered and uncharacterized HNVs.

V protein, the virulence factor across the family Paramyxoviridae: a review

Paramyxoviridae is a family of viruses within the order Mononegavirales and comprises 14 genera; Metaavulavirus, Orthoavulavirus, Paraavulavirus, Synodonvirus, Ferlavirus, Aquaparamyxovirus, Henipavirus, Morbillivirus, Respirovirus, Jeilongvirus, Narmovirus, Salemvirus, Pararubulavirus and Orthorubulavirus. The members within this family are negative and single-stranded RNA viruses including human and animal pathogens such as measles virus (MeV), Nipah virus (NiV), mumps virus (MuV), Sendai virus (SeV) and Newcastle disease virus (NDV). The V protein is conserved within the family and plays an essential role in viral pathogenicity. Although V proteins of many paramyxoviruses are interferon-antagonists which counteract with the host’s innate immunity, there are still differences in the mode of action of the V protein between different genera or species within the same genera. The strategies to circumvent the host interferon (IFN) pathway can be divided into three general mechanisms; degradation of signal transducers and activators of transcription (STAT) protein, inhibition of phosphorylation of the transcription factor and, inhibition of translocation of STAT proteins into the nucleus. As a result, inhibition of IFN signalling and production promotes viral replication in the host cells. This review highlights the mechanism of the paramyxoviral V protein in evading the host IFN system.