Hyperacetylated microtubules assist porcine deltacoronavirus nsp8 to degrade MDA5 via SQSTM1/p62-dependent selective autophagy.

The microtubule (MT) is a highly dynamic polymer that functions in various cellular processes through MT hyperacetylation. Thus, many viruses have evolved mechanisms to hijack the MT network of the cytoskeleton to allow intracellular replication of viral genomic material. Coronavirus non-structural protein 8 (nsp8), a component of the viral replication transcriptional complex, is essential for viral survival. Here, we found that nsp8 of porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with a zoonotic potential, inhibits interferon (IFN)-ß production by targeting melanoma differentiation gene 5 (MDA5), the main pattern recognition receptor for coronaviruses in the cytoplasm. Mechanistically, PDCoV nsp8 interacted with MDA5 and induced autophagy to degrade MDA5 in wild-type cells, but not in autophagy-related (ATG)5 or ATG7 knockout cells. Further screening for autophagic degradation receptors revealed that nsp8 interacts with sequestosome 1/p62 and promotes p62-mediated selective autophagy to degrade MDA5. Importantly, PDCoV nsp8 induced hyperacetylation of MTs, which in turn triggered selective autophagic degradation of MDA5 and subsequent inhibition of IFN-ß production. Overall, our study uncovers a novel mechanism employed by PDCoV nsp8 to evade host innate immune defenses. These findings offer new insights into the interplay among viruses, IFNs, and MTs, providing a promising target to develop anti-viral drugs against PDCoV.IMPORTANCECoronavirus nsp8, a component of the viral replication transcriptional complex, is well conserved and plays a crucial role in viral replication. Exploration of the role mechanism of nsp8 is conducive to the understanding of viral pathogenesis and development of anti-viral strategies against coronavirus. Here, we found that nsp8 of PDCoV, an emerging enteropathogenic coronavirus with a zoonotic potential, is an interferon antagonist. Further studies showed that PDCoV nsp8 interacted with MDA5 and sequestosome 1/p62, promoting p62-mediated selective autophagy to degrade MDA5. We further found that PDCoV nsp8 could induce hyperacetylation of MT, therefore triggering selective autophagic degradation of MDA5 and inhibiting IFN-ß production. These findings reveal a novel immune evasion strategy used by PDCoV nsp8 and provide insights into potential therapeutic interventions.

Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3.

Porcine deltacoronavirus (PDCoV) has caused enormous economic losses to the global pig industry. However, the immune escape mechanism of PDCoV remains to be fully clarified. Transcriptomic analysis revealed a high abundance of interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) transcripts after PDCoV infection, which initially implied a correlation between IFIT3 and PDCoV. Further studies showed that PDCoV nsp5 could antagonize the host type I interferon signaling pathway by cleaving IFIT3. We demonstrated that PDCoV nsp5 cleaved porcine IFIT3 (pIFIT3) at Gln-406. Similar cleavage of endogenous IFIT3 has also been observed in PDCoV-infected cells. The pIFIT3-Q406A mutant was resistant to nsp5-mediated cleavage and exhibited a greater ability to inhibit PDCoV infection than wild-type pIFIT3. Furthermore, we found that cleavage of IFIT3 is a common characteristic of nsp5 proteins of human coronaviruses, albeit not alphacoronavirus. This finding suggests that the cleavage of IFIT3 is an important mechanism by which PDCoV nsp5 antagonizes IFN signaling. Our study provides new insights into the mechanisms by which PDCoV antagonizes the host innate immune response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a potential emerging zoonotic pathogen, and studies on the prevalence and pathogenesis of PDCoV are ongoing. The main protease (nsp5) of PDCoV provides an excellent target for antivirals due to its essential and conserved function in the viral replication cycle. Previous studies have revealed that nsp5 of PDCoV antagonizes type I interferon (IFN) production by targeting the interferon-stimulated genes. Here, we provide the first demonstration that nsp5 of PDCoV antagonizes IFN signaling by cleaving IFIT3, which affects the IFN response after PDCoV infection. Our findings reveal that PDCoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by deltacoronaviruses.

Porcine deltacoronavirus enters ST cells by clathrin-mediated endocytosis and does not require Rab5, Rab7, or Rab11.

IMPORTANCE: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus threatening pig industries worldwide. Our previous work showed that PDCoV enters porcine kidney (PK-15) cells through a caveolae-dependent pathway, but the entry mechanism for PDCoV into swine testicle (ST) cells remains unclear. Mechanisms of virus entry can be different with different virus isolates and cell types. Here, we determined that PDCoV enters ST cells via clathrin-mediated endocytosis. Additionally, we found that PDCoV entry does not require Rab5, Rab7, or Rab11. These findings provide additional understanding of the entry mechanisms of PDCoV and possible antiviral targets.

PGAM5 degrades PDCoV N protein and activates type I interferon to antagonize viral replication.

IMPORTANCE: As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication.

Chicken or Porcine Aminopeptidase N Mediates Cellular Entry of Pseudoviruses Carrying Spike Glycoprotein from the Avian Deltacoronaviruses HKU11, HKU13, and HKU17.

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Porcine Deltacoronavirus Infection Cleaves HDAC2 to Attenuate Its Antiviral Activity.

Protein acetylation plays an important role during virus infection. Thus, it is not surprising that viruses always evolve elaborate mechanisms to regulate the functions of histone deacetylases (HDACs), the essential transcriptional and epigenetic regulators for deacetylation. Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets and has the potential to infect humans. In this study, we found that PDCoV infection inhibited cellular HDAC activity. By screening the expressions of different HDAC subfamilies after PDCoV infection, we unexpectedly found that HDAC2 was cleaved. Ectopic expression of HDAC2 significantly inhibited PDCoV replication, while the reverse effects could be observed after treatment with an HDAC2 inhibitor (CAY10683) or the knockdown of HDAC2 expression by specific siRNA. Furthermore, we demonstrated that PDCoV-encoded nonstructural protein 5 (nsp5), a 3C-like protease, was responsible for HDAC2 cleavage through its protease activity. Detailed analyses showed that PDCoV nsp5 cleaved HDAC2 at glutamine 261 (Q261), and the cleaved fragments (amino acids 1 to 261 and 262 to 488) lost the ability to inhibit PDCoV replication. Interestingly, the Q261 cleavage site is highly conserved in HDAC2 homologs from other mammalian species, and the nsp5s encoded by seven tested mammalian coronaviruses also cleaved HDAC2, suggesting that cleaving HDAC2 may be a common strategy used by different mammalian coronaviruses to antagonize the antiviral role of HDAC2. IMPORTANCE As an emerging porcine enteropathogenic coronavirus that possesses the potential to infect humans, porcine deltacoronavirus (PDCoV) is receiving increasing attention. In this work, we found that PDCoV infection downregulated cellular histone deacetylase (HDAC) activity. Of particular interest, the viral 3C-like protease, encoded by the PDCoV nonstructural protein 5 (nsp5), cleaved HDAC2, and this cleavage could be observed in the context of PDCoV infection. Furthermore, the cleavage of HDAC2 appears to be a common strategy among mammalian coronaviruses, including the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to antagonize the antiviral role of HDAC2. To our knowledge, PDCoV nsp5 is the first identified viral protein that can cleave cellular HDAC2. Results from our study provide new targets to develop drugs combating coronavirus infection.

Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

Roles of Two Major Domains of the Porcine Deltacoronavirus S1 Subunit in Receptor Binding and Neutralization.

Determination of the mechanisms of interspecies transmission is of great significance for the prevention of epidemic diseases caused by emerging coronaviruses (CoVs). Recently, porcine deltacoronavirus (PDCoV) was shown to exhibit broad host cell range mediated by surface expression of aminopeptidase N (APN), and humans have been reported to be at risk of PDCoV infection. In the present study, we first demonstrated overexpression of APN orthologues from various species, including mice and felines, in the APN-deficient swine small intestine epithelial cells permitted PDCoV infection, confirming that APN broadly facilitates PDCoV cellular entry and perhaps subsequent interspecies transmission. PDCoV was able to limitedly infect mice in vivo, distributing mainly in enteric and lymphoid tissues, suggesting that mice may serve as a susceptible reservoir of PDCoV. Furthermore, elements (two glycosylation sites and four aromatic amino acids) on the surface of domain B (S1B) of the PDCoV spike glycoprotein S1 subunit were identified to be critical for cellular surface binding of APN orthologues. However, both domain A (S1A) and domain B (S1B) were able to elicit potent neutralizing antibodies against PDCoV infection. The antibodies against S1A inhibited the hemagglutination activity of PDCoV using erythrocytes from various species, which might account for the neutralizing capacity of S1A antibodies partially through a blockage of sialic acid binding. The study reveals the tremendous potential of PDCoV for interspecies transmission and the role of two major PDCoV S1 domains in receptor binding and neutralization, providing a theoretical basis for development of intervention strategies. IMPORTANCE Coronaviruses exhibit a tendency for recombination and mutation, which enables them to quickly adapt to various novel hosts. Previously, orthologues of aminopeptidase N (APN) from mammalian and avian species were found to be associated with porcine deltacoronavirus (PDCoV) cellular entry in vitro. Here, we provide in vivo evidence that mice are susceptible to PDCoV limited infection. We also show that two major domains (S1A and S1B) of the PDCoV spike glycoprotein involved in APN receptor binding can elicit neutralizing antibodies, identifying two glycosylation sites and four aromatic amino acids on the surface of the S1B domain critical for APN binding and demonstrating that the neutralization activity of S1A antibodies is partially attributed to blockage of sugar binding activity. Our findings further implicate PDCoV's great potential for interspecies transmission, and the data of receptor binding and neutralization may provide a basis for development of future intervention strategies.

Bioinformatics Analysis of Spike Proteins of Porcine Enteric Coronaviruses.

This article is aimed at analyzing the structure and function of the spike (S) proteins of porcine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) by applying bioinformatics methods. The physical and chemical properties, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, phosphorylation and glycosylation sites, epitope, functional domains, and motifs of S proteins of porcine enteric coronaviruses were predicted and analyzed through online software. The results showed that S proteins of TGEV, PEDV, SADS-CoV, and PDCoV all contained transmembrane regions and signal peptide. TGEV S protein contained 139 phosphorylation sites, 24 glycosylation sites, and 53 epitopes. PEDV S protein had 143 phosphorylation sites, 22 glycosylation sites, and 51 epitopes. SADS-CoV S protein had 109 phosphorylation sites, 20 glycosylation sites, and 43 epitopes. PDCoV S protein had 124 phosphorylation sites, 18 glycosylation sites, and 52 epitopes. Moreover, TGEV, PEDV, and PDCoV S proteins all contained two functional domains and two motifs, spike_rec_binding and corona_S2. The corona_S2 consisted of S2 subunit heptad repeat 1 (HR1) and S2 subunit heptad repeat 2 (HR2) region profiles. Additionally, SADS-CoV S protein was predicted to contain only one functional domain, the corona_S2. This analysis of the biological functions of porcine enteric coronavirus spike proteins can provide a theoretical basis for the design of antiviral drugs.

Porcine deltacoronavirus nsp10 antagonizes interferon-ß production independently of its zinc finger domains.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes serious vomiting and diarrhea in piglets. Previous work demonstrated that PDCoV infection inhibits type I interferon (IFN) production. Here, we found that ectopic expression of PDCoV nsp10 significantly inhibited Sendai virus (SeV)-induced IFN-ß production by impairing the phosphorylation and nuclear translocation of two transcription factors, IRF3 and NF-κB p65 subunit. Interestingly, experiments with truncated mutants and site-directed mutagenesis revealed that PDCoV nsp10 mutants with missing or destroyed zinc fingers (ZFs) domains also impeded SeV-induced IFN-ß production, suggesting that nsp10 does not require its ZF domains to antagonize IFN-ß production. Further work found that co-expression of nsp10 with nsp14 or nsp16, two replicative enzymes, significantly enhanced the inhibitory effects of nsp10 on IFN-ß. Taken together, our results demonstrate that PDCoV nsp10 antagonizes IFN via a ZF-independent mechanism and has a synergistic effect with nsp14 and nsp16 on inhibiting IFN-ß production.

The porcine deltacoronavirus accessory protein NS6 is expressed in vivo and incorporated into virions.

Porcine deltacoronavirus (PDCoV) is one of the emerged coronaviruses posing a significant threat to the swine industry. Previous work showed the presence of a viral accessory protein NS6 in PDCoV-infected cells. In this study, we detected the expression of the NS6 protein in small intestinal tissues of PDCoV-infected piglets. In addition, SDS-PAGE and Western blot analysis of sucrose gradient-purified virions showed the presence of a 13-kDa NS6 protein. Further evidences of the presence of NS6 in the PDCoV virions were obtained by immunogold staining of purified virions with anti-NS6 antiserum, and by immunoprecipitation of NS6 from purified virions. Finally, the anti-NS6 antibody was not able to neutralize PDCoV in cultured cells. These data establish for the first time that the accessory protein NS6 is expressed during infection in vivo and incorporated into PDCoV virions.

Subcellular localization of the porcine deltacoronavirus nucleocapsid protein.

Porcine deltacoronavirus (PDCoV) has been recently identified as an emerging enteropathogenic coronavirus that mainly infects newborn piglets and causes enteritis, diarrhea and high mortality. Although coronavirus N proteins have multifarious activities, the subcellular localization of the PDCoV N protein is still unknown. Here, we produced mouse monoclonal antibodies against the PDCoV N protein. Experiments using anti-haemagglutinin antibodies and these monoclonal antibodies revealed that the PDCoV N protein is shuttled into the nucleolus in both ectopic PDCoV N-expressing cells and PDCoV-infected cells. The results of deletion mutagenesis experiments demonstrated that the predicted nucleolar localization signal at amino acids 295-318 is critical for nucleolar localization. Cumulatively, our study yielded a monoclonal antibody against the PDCoV N protein and revealed a mechanism by which the PDCoV N protein translocated into the nucleolus. The tolls and findings from this work will facilitate further investigations on the functions of the PDCoV N protein.