Deep profiling of potential substrate atlas of porcine epidemic diarrhea virus 3C-like protease.

Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.

Porcine reproductive and respiratory syndrome virus infection activates ADAM17 to induce inflammatory responses.

Porcine reproductive and respiratory syndrome (PRRS), which has posed substantial threats to the swine industry worldwide, is primarily characterized by interstitial pneumonia. A disintegrin and metalloproteinase 17 (ADAM17) is a multifunctional sheddase involved in various inflammatory diseases. Herein, our study showed that PRRS virus (PRRSV) infection elevated ADAM17 activity, as demonstrated in primary porcine alveolar macrophages (PAMs), an immortalized PAM cell line (IPAM cells), and the lung tissues of PRRSV-infected piglets. We found that PRRSV infection promoted ADAM17 translocation from the endoplasmic reticulum to the Golgi by enhancing its interaction with inactive rhomboid protein 2 (iRhom2), a newly identified ADAM17 regulator, which in turn elevated ADAM17 activity. By screening for PRRSV-encoded structural proteins, viral envelope (E) and nucleocapsid (N) proteins were identified as the predominant ADAM17 activators. E and N proteins bind with both ADAM17 and iRhom2 to form ternary protein complexes, ultimately strengthening their interactions. Additionally, we demonstrated, using an ADAM17-knockout cell line, that ADAM17 augmented the shedding of soluble TNF-α, a pivotal inflammatory mediator. We also discovered that ADAM17-mediated cleavage of porcine TNF-α occurred between Arg-78 and Ser-79. By constructing a precision mutant cell line with Arg-78-Glu/Ser-79-Glu substitution mutations in TNF-α, we further revealed that the ADAM17-mediated production of soluble TNF-α contributed to the induction of inflammatory responses by PRRSV and its E and N proteins. Taken together, our results elucidate the mechanism by which PRRSV infection activates the iRhom2/ADAM17/TNF-α axis to enhance inflammatory responses, providing valuable insights into the elucidation of PRRSV pathogenesis.

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.

Lactate-lactylation-HSPA6 axis promotes PRRSV replication by impairing IFN-ß production.

Lactate, traditionally considered a metabolic by-product, has recently been identified as a substrate for the induction of lactylation, a newly identified epigenetic modification that plays an important role in the regulation of host gene expression. Our previous study showed that lactate levels were significantly elevated in cells infected with the porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide for over 30 years. However, the role of elevated lactate in PRRSV infections remains unknown. In this study, we found that lactate was required for optimal PRRSV proliferation, and PRRSV infection increased cellular lactylation in a dose-dependent manner. Using the Cleavage Under Targets and Tagmentation (CUT&Tag) combined with RNA sequencing (RNA-seq) to screen the downstream genes regulated by lactylation in PRRSV-infected cells, we found that PRRSV-induced lactylation activated the expression of heat shock 70 kDa protein 6 (HSPA6). Follow-up experiments showed that HSPA6 is important for PRRSV proliferation by negatively modulating interferon (IFN)-ß induction. Mechanistically, HSPA6 impeded the interaction between TNF-receptor-associated factor 3 (TRAF3) and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε), thereby hindering the production of IFN-ß. Taken together, these results indicate that the activated lactate-lactylation-HSPA6 axis promotes viral growth by impairing IFN-ß induction, providing new therapeutic targets for the prevention and control of PRRSV infection. The results presented here also link lactylation to the virus life cycle, improving our understanding of epigenetic regulation in viral infection.IMPORTANCEAs a newly identified epigenetic modification, lactate-induced lactylation has received attentions because it plays important roles in gene expression and contributes to tumorigenesis and the innate immune response. Previous studies showed that many viruses upregulate cellular lactate levels; however, whether virus-elevated lactate induces lactylation and the subsequent biological significance of the modification to viral infection have not been reported. In this study, we demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection induced cellular lactylation, which, in turn, upregulated the expression of HSPA6, an IFN-negative regulator. We also dissected the mechanism by which HSPA6 negatively regulates IFN-ß production. To our knowledge, this is the first report to study virus-induced lactylation and establish the relationship between lactylation and virus infection.

Cloning, expression of porcine GSDME and identification of its site cleaved by caspase-3.

As a member of the gasdermin family, gasdermin E (GSDME) is specifically cleaved by caspase-3, resulting in pyroptosis. To date, the biological characteristics and functions of human and mouse GSDME have been extensively studied; however, little is known of porcine GSDME (pGSDME). In this study, the full-length pGSDME-FL was cloned, which encodes 495 amino acids (aa) that have closely evolutionary relationships to the homolog of camelus, aquatic mammals, cattle and goat. Moreover, pGSDME was detected at different levels of expression in 21 tissues and 5 pig-derived cell lines tested by qRT-PCR, with the highest expression levels in mesenteric lymph nodes and PK-15 cell lines. Anti-pGSDME polyclonal antibody (pAb) with good specificity was generated by expressing the truncated recombinant protein pGSDME-1-208 and immunizing the rabbits. By western blot analysis using highly specific anti-pGSDME polyclonal antibody (pAb) prepared as primary antibody, it was not only confirmed that paclitaxel and cisplatin were positive stimuli to pGSDME cleavage and caspase-3 activation, but also identified the aspartate (D268) at position 268th of pGSDME as a cleavage site of caspase-3, and the overexpressed pGSDME-1-268 possesses cytotoxicity to HEK-293T cells, indicating that pGSDME-1-268 may contain active domains and involve pGSDME-mediated pyroptosis. These results lay a foundation for further investigating the function of pGSDME, especially its role in pyroptosis and its interaction with pathogens.

PRRSV-induced inflammation in pulmonary intravascular macrophages (PIMs) and pulmonary alveolar macrophages (PAMs) contributes to endothelial barrier function injury.

Porcine reproductive and respiratory syndrome (PRRS) is a severe infectious disease currently devasting the global pig industry. PRRS is characterized by intense inflammation and severe damage to the alveolar-capillary barrier. Therefore, it is crucial to uncover the underlying mechanism by which the PRRS virus (PRRSV) induces inflammatory responses and barrier function damage. In addition to porcine alveolar macrophages (PAMs), the primary target cells of PRRSV infection in vivo, pulmonary intravascular macrophages (PIMs) are also susceptible to PRRSV infection. However, the poor isolation efficiency limits the study of PRRSV infection in PIMs. In this study, we optimized the isolation method to obtain PIMs with higher purity and yield and demonstrated that PRRSV's infection kinetics in PIMs were similar to those in PAMs. Notably, PIMs exhibited a more acute inflammation process during PRRSV infection than PAMs, as evidenced by the earlier upregulation and higher levels of pro-inflammatory cytokines, including TNF-α and IL-1ß. More acute endothelial barrier disfunction upon PRRSV infection was also observed in PIMs compared to in PAMs. Mechanistically, PRRSV-induced TNF-α and IL-1ß could cause endothelial barrier disfunction by dysregulating tight junction proteins, including claudin 1 (CLDN1), claudin 8 (CLDN8) and occludin (OCLN). Our findings revealed the crucial and novel roles of PIMs in facilitating the progression of inflammatory responses and endothelial barrier injury and provided new insights into the mechanisms of PRRSV's induction of interstitial pneumonia.

Porcine Epidemic Diarrhea Virus nsp7 Inhibits MDA5 Dephosphorylation to Antagonize Type I Interferon Production.

Porcine epidemic diarrhea virus (PEDV) is a reemerging enteropathogenic coronavirus that causes high mortality in piglets and has catastrophic effects on the global pig industry. PEDV-encoded nonstructural protein 7 (nsp7) is an important component of the viral replication and transcription complex, and a previous study reported that it inhibits poly(I:C)-induced type I interferon (IFN) production, but the mechanism by which this occurs remains unclear. Here, we demonstrated that ectopic expression of PEDV nsp7 antagonized Sendai virus (SeV)-induced interferon beta (IFN-ß) production, as well as the activation of transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB) in both HEK-293T and LLC-PK1 cells. Mechanistically, PEDV nsp7 targets melanoma differentiation-associated gene 5 (MDA5) and interacts with its caspase activation and recruitment domains (CARDs), which sequester the interactions between MDA5 and the protein phosphatase 1 (PP1) catalytic subunits (PP1α and PP1γ), thereby suppressing MDA5 S828 dephosphorylation and keeping MDA5 inactive. Furthermore, PEDV infection attenuated MDA5 multimerization and MDA5-PP1α/-γ interactions. We also tested the nsp7 orthologs of five other mammalian coronaviruses and found that all of them except severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp7 inhibited MDA5 multimerization and SeV- or MDA5-induced IFN-ß production. Collectively, these results suggest that the inhibition of MDA5 dephosphorylation and multimerization may be a common strategy employed by PEDV and some other coronaviruses to antagonize MDA5-mediated IFN production. IMPORTANCE Since late 2010, a reemerging porcine epidemic diarrhea virus variant with high pathogenesis has swept through most pig farms in many countries, resulting in significant economic losses. Coronavirus nonstructural protein 7 (nsp7), conserved within the family Coronaviridae, combines with nsp8 and nsp12 to form the viral replication and transcription complex that is indispensable for viral replication. However, the function of nsp7 in the infection and pathogenesis of coronaviruses remains largely unknown. Our present study demonstrates that PEDV nsp7 specifically competes with PP1 for binding MDA5 and impedes the PP1-mediated dephosphorylation of MDA5 at S828, thereby blocking MDA5-mediated IFN production, revealing the complex mechanism utilized by PEDV nsp7 to efficiently escape host innate immunity.

Characterization of Gut Microbiota Compositions along the Intestinal Tract in CD163/pAPN Double Knockout Piglets and Their Potential Roles in Iron Absorption.

The gut microbiota is known to play a role in regulating host metabolism, yet the mechanisms underlying this regulation are not well elucidated. Our study aimed to characterize the differences in gut microbiota compositions and their roles in iron absorption between wild-type (WT) and CD163/pAPN double-gene-knockout (DKO) weaned piglets. A total of 58 samples along the entire digestive tract were analyzed for microbial community using 16S rRNA gene sequencing. The colonic microbiota and their metabolites were determined by metagenomic sequencing and untargeted liquid chromatography-mass spectrometry (LC-MS), respectively. Our results showed that no alterations in microbial community structure and composition were observed between DKO and WT weaned piglets, with the exception of colonic microbiota. Interestingly, the DKO piglets had selectively increased the relative abundance of the Leeia genus belonging to the Neisseriaceae family and decreased the Ruminococcaceae_UCG_014 genus abundance. Functional capacity analysis showed that organic acid metabolism was enriched in the colon in DKO piglets. In addition, the DKO piglets showed increased iron levels in important tissues compared with WT piglets without any pathological changes. Pearson's correlation coefficient indicated that the specific bacteria such as Leeia and Ruminococcaceae_UCG_014 genus played a key role in host iron absorption. Moreover, the iron levels had significantly (P < 0.05) positive correlation with microbial metabolites, particularly carboxylic acids and their derivatives, which might increase iron absorption by preventing iron precipitation. Overall, this study reveals an interaction between colonic microbiota and host metabolism and has potential significance for alleviating piglet iron deficiency. IMPORTANCE Iron deficiency is a major risk factor for iron deficiency anemia, which is among the most common nutritional disorders in piglets. However, it remains unclear how the gut microbiota interacts with host iron absorption. The current report provides the first insight into iron absorption-microbiome connection in CD163/pAPN double knockout piglets. The present results showed that carboxylic acids and their derivatives contributed to the absorption of nonheme iron by preventing ferric iron precipitation.

Porcine Reproductive and Respiratory Syndrome Virus nsp1ß Stabilizes HIF-1α to Enhance Viral Replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. Severe interstitial pneumonia is the typical pathological characteristic of PRRSV-infected swine. Accumulating evidence has suggested that hypoxia-inducible factor 1α (HIF-1α) plays vital roles in the development of inflammation and the viral life cycle. However, the role and the underlying mechanism of HIF-1α in PRRSV infection remain elusive. Here, we found that PRRSV infection elevated HIF-1α expression. Furthermore, overexpression of HIF-1α increased PRRSV replication, whereas knockdown of HIF-1α inhibited PRRSV infection. Our further mechanistic analysis revealed that PRRSV-encoded nonstructural protein 1ß (nsp1ß) promoted HIF-1α transcription via its N-terminal nuclease activity and degraded the polyubiquitin chain of HIF-1α via its C-terminal deubiquitylation (DUB) enzyme activity, collectively stabilizing HIF-1α. Meanwhile, nsp1ß interacted with both HIF-1α and von Hippel-Lindau tumor suppressor (pVHL) to form a ternary complex, which may have hindered pVHL-mediated ubiquitination degradation of HIF-1α by impairing the interaction between HIF-1α and pVHL. Interestingly, pVHL also stabilized nsp1ß via K63-linked ubiquitination, forming a positive feedback loop to stabilize HIF-1α. Taken together, these results indicate that PRRSV infection stabilizes HIF-1α to facilitate viral proliferation and that viral nsp1ß plays a vital role in enhancing the expression and stabilization of HIF-1α. The regulation of HIF-1α may have great therapeutic potential for the development of novel drugs against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has devastated the swine industry worldwide for over 30 years and shows no signs of slowing down. In this study, we found that PRRSV infection elevated hypoxia-inducible factor 1α (HIF-1α) expression. In addition, overexpressed HIF-1α contributed to PRRSV replication, whereas knockdown of HIF-1α reduced PRRSV growth. The PRRSV-encoded nonstructural protein 1ß (nsp1ß) exerted a stabilizing effect on HIF-1α through its nuclease protease and papain-like cysteine protease enzymatic domains. PRRSV nsp1ß also interacted with von Hippel-Lindau tumor suppressor (pVHL) and HIF-1α, whereby nsp1ß impaired the interaction between HIF-1α and pVHL. This work deepens our understanding of the molecular mechanisms involved in PRRSV infection and provides new insights for the development of HIF-1α-based anti-PRRSV therapies.

Molecular cloning, prokaryotic expression and the anti-inflammatory activity of porcine PRDX5.

Peroxiredoxin 5 (PRDX5) is the sole member of the atypical 2-Cys subfamily of mammalian PRDXs, a family of thiol-dependent peroxidases. In addition to its antioxidant effect, PRDX5 has been implicated in modulating the inflammatory response. In this study, the full-length cDNA encoding porcine PRDX5 (pPRDX5) was cloned. Subsequently, using porcine alveolar macrophages (PAMs), the target cells of PRRSV infection in vivo, we found that the recombinant pPRDX5 protein inhibited inflammatory responses induced by tumor necrosis factor alpha (TNF-α) or porcine reproductive and respiratory syndrome virus (PRRSV), a virus causing severe interstitial pneumonia in pigs. By contrast, knockdown of endogenous pPRDX5 with specific siRNA enhanced inflammatory responses induced by TNF-α or PRRSV. We also demonstrated that the involvement of pPRDX5 in inflammation regulation depended on its peroxidase activity. Taken together, these results showed that pPRDX5 is an anti-inflammatory molecule, which may play an important immune-regulation role in the pathogenicity of PRRSV.

Norovirus 3C-Like protease antagonizes interferon-ß production by cleaving NEMO.

An epidemic owing to Norovirus (NoV) has recently been occurring worldwide. Severe cases of NoV can lead to patient death, resulting in significant public health problems. In the early stages of infection, antagonizing the production of host interferon (IFN) is an important strategy for viruses to establish infection. However, the relationship between NoV and interferon and its mechanism remains unclear. In this study, the 3C-like protease encoded by NoV was found to effectively suppress Sendai virus (SEV)-mediated IFN-ß production by cleaving the NF-κB essential modulator (NEMO). Glutamine 205 is the site of NoV3CLpro-mediated cleavage of NEMO and this cleavage suppresses the ability of NEMO to activate downstream IFN production. These findings demonstrate that NoV3CLpro-induced cleavage limits NEMO to the activation of type I IFN signaling. In summary, our findings indicate that NoV3CLpro is a new interferon antagonist, and enhances our understanding of the escape of innate immunity mediated by NoV3CLpro.

Porcine Epidemic Diarrhea Virus nsp7 Inhibits Interferon-Induced JAK-STAT Signaling through Sequestering the Interaction between KPNA1 and STAT1.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes high mortality in piglets. Interferon (IFN) responses are the primary defense mechanism against viral infection; however, viruses always evolve elaborate strategies to antagonize the antiviral action of IFN. Previous study showed that PEDV nonstructural protein 7 (nsp7), a component of the viral replicase polyprotein, can antagonize ploy(I:C)-induced type I IFN production. Here, we found that PEDV nsp7 also antagonized IFN-α-induced JAK-STAT signaling and the production of IFN-stimulated genes. PEDV nsp7 did not affect the protein and phosphorylation levels of JAK1, Tyk2, STAT1, and STAT2 or the formation of the interferon-stimulated gene factor 3 (ISGF3) complex. However, PEDV nsp7 prevented the nuclear translocation of STAT1 and STAT2. Mechanistically, PEDV nsp7 interacted with the DNA binding domain of STAT1/STAT2, which sequestered the interaction between karyopherin α1 (KPNA1) and STAT1, thereby blocking the nuclear transport of ISGF3. Collectively, these data reveal a new mechanism developed by PEDV to inhibit type I IFN signaling pathway. IMPORTANCE In recent years, an emerging porcine epidemic diarrhea virus (PEDV) variant has gained attention because of serious outbreaks of piglet diarrhea in China and the United States. Coronavirus nonstructural protein 7 (nsp7) has been proposed to act with nsp8 as part of an RNA primase to generate RNA primers for viral RNA synthesis. However, accumulating evidence indicates that coronavirus nsp7 can also antagonize type I IFN production. Our present study extends previous findings and demonstrates that PEDV nsp7 also antagonizes IFN-α-induced IFN signaling by competing with KPNA1 for binding to STAT1, thereby enriching the immune regulation function of coronavirus nsp7.

AMG487 inhibits PRRSV replication and ameliorates lung injury in pig lung xenografts by down-regulating the expression of ANXA2.

Porcine reproductive and respiratory syndrome (PRRS) is a pig disease caused by the PRRS virus (PRRSV) that is characterized with diffuse interstitial pneumonia and lung edema. High expressions of chemokine CXCL10 and its receptor CXCR3 are reported in infected porcine lungs. Since CXCR3 is a key player in host inflammatory response, it might be a therapeutic target to treat lung damage caused by PRRSV infection. The size of pigs has long hampered research into molecular mechanisms of PRRS and validating the potential pharmaceutical targets. In this study, a porcine lung xenograft model with PRRSV infection was generated in immunodeficient mice to evaluate the therapeutic effects of the CXCR3 antagonist AMG487 on PRRSV infection-induced lung injury. The porcine lung tissues developed normally two weeks after xeno-transplantation in the mouse kidney capsule. Infection of PRRSV resulted in its efficient replication in the xenografts and histological damage to the porcine lung tissue structure, with no or little effects on mouse lungs. AMG487 administration dramatically reduced the number of PRRSV genome copies and significantly alleviated the porcine lung injury. Furthermore, treatment of AMG487 in cultured porcine macrophages consistently suppressed PRRSV replication with significant downregulation of Annexin A2 (ANXA2), a cellular protein facilitating viral replication. These findings provide a suitable model for evaluating new antiviral therapies as well as a possible therapeutic option for virus infection-induced lung injury.

SARS-CoV-2 nsp5 Exhibits Stronger Catalytic Activity and Interferon Antagonism than Its SARS-CoV Ortholog.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an enormous threat to economic activity and public health worldwide. Previous studies have shown that the nonstructural protein 5 (nsp5, also called 3C-like protease) of alpha- and deltacoronaviruses cleaves Q231 of the NF-κB essential modulator (NEMO), a key kinase in the RIG-I-like receptor pathway, to inhibit type I interferon (IFN) production. In this study, we found that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleaved NEMO at multiple sites (E152, Q205, and Q231). Notably, SARS-CoV-2 nsp5 exhibited a stronger ability to cleave NEMO than SARS-CoV nsp5. Sequence and structural alignments suggested that an S/A polymorphism at position 46 of nsp5 in SARS-CoV versus SARS-CoV-2 may be responsible for this difference. Mutagenesis experiments showed that SARS-CoV-2 nsp5 (S46A) exhibited poorer cleavage of NEMO than SARS-CoV-2 nsp5 wild type (WT), while SARS-CoV nsp5 (A46S) showed enhanced NEMO cleavage compared with the WT protein. Purified recombinant SARS-CoV-2 nsp5 WT and SARS-CoV nsp5 (A46S) proteins exhibited higher hydrolysis efficiencies than SARS-CoV-2 nsp5 (S46A) and SARS-CoV nsp5 WT proteins in vitro. Furthermore, SARS-CoV-2 nsp5 exhibited stronger inhibition of Sendai virus (SEV)-induced interferon beta (IFN-ß) production than SARS-CoV-2 nsp5 (S46A), while introduction of the A46S substitution in SARS-CoV nsp5 enhanced suppression of SEV-induced IFN-ß production. Taken together, these data show that S46 is associated with the catalytic activity and IFN antagonism by SARS-CoV-2 nsp5. IMPORTANCE The nsp5-encoded 3C-like protease is the main coronavirus protease, playing a vital role in viral replication and immune evasion by cleaving viral polyproteins and host immune-related molecules. We showed that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleave the NEMO at multiple sites (E152, Q205, and Q231). This specificity differs from NEMO cleavage by alpha- and deltacoronaviruses, demonstrating the distinct substrate recognition of SARS-CoV-2 and SARS-CoV nsp5. Compared with SARS-CoV nsp5, SARS-CoV-2 nsp5 encodes S instead of A at position 46. This substitution is associated with stronger catalytic activity, enhanced cleavage of NEMO, and increased interferon antagonism of SARS-CoV-2 nsp5. These data provide new insights into the pathogenesis and transmission of SARS-CoV-2.

Porcine Deltacoronavirus Enters Porcine IPI-2I Intestinal Epithelial Cells via Macropinocytosis and Clathrin-Mediated Endocytosis Dependent on pH and Dynamin.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, small interfering RNA (siRNA) interference, specific pharmacological inhibitors, and dominant negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin, and a low-pH environment but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na+/H+ exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. IMPORTANCE An emerging enteropathogenic coronavirus, PDCoV, has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis and provide clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection in vivo. Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis, which does not require a specific receptor, and clathrin-mediated endocytosis, which requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.

Molecular characterization and functional analysis of duck CCCH-type zinc finger antiviral protein (ZAP).

This is the first study to clone duck CCCH-type zinc finger antiviral protein (duZAP) from Jingjiang duck (Anas platyrhynchos). Full-length duZAP cDNA was 2154 bp and encoded a 717-amino acid polypeptide containing four highly conserved CCCH-type finger motifs, a WWE domain and a poly (ADP-ribose) polymerase (PARP) domain. duZAP was expressed in multiple duck tissues, with the highest mRNA expression in the spleen. Overexpression of duZAP in duck embryo fibroblast cells (DEFs) led to activation of the transcription factors IRF1 and NF-κB, and induction of IFN-ß. Analysis of deletion mutants revealed that both the WWE and PARP domains of duZAP were essential for activating the IFN-ß promoter. Knockdown of duZAP in DEFs significantly reduced poly (I:C)- and duck Tembusu virus (DTMUV)-induced IFN-ß activation. Our findings further the understanding of the role of duZAP in the duck innate immune response.

ATPase and helicase activities of porcine epidemic diarrhea virus nsp13.

Porcine epidemic diarrhea virus (PEDV) is a reemerging Alphacoronavirus that causes lethal diarrhea in piglets. Coronavirus nonstructural protein 13 (nsp13) encodes helicase, which plays pivotal roles during viral replication by unwinding viral RNA. However, the biochemical characterization of PEDV nsp13 remains largely unknown. In this study, PEDV nsp13 was expressed in Escherichia coli and purified. The recombinant nsp13 possessed ATPase and helicase activities for binding and unwinding dsDNA/RNA substrates with 5'-overhangs, and Mg2+ and Mn2+ were critical for its ATPase and helicase activities. PEDV nsp13 also unwound dsDNA into ssDNA in the pH from 6.0-9.0, and used energy from all nucleoside triphosphates and deoxynucleoside triphosphates. Site-directed mutagenesis demonstrated that Lys289 (K289) of PEDV nsp13 was essential for its ATPase and helicase activities. These results provide new insights into the biochemical properties of PEDV nsp13, which is a potential target for developing 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.

Cryo-EM analysis of the HCoV-229E spike glycoprotein reveals dynamic prefusion conformational changes.

Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design.

Porcine deltacoronavirus (PDCoV) infection antagonizes interferon-λ1 production.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III interferons (IFN-λs) are the major antiviral cytokines in intestinal epithelial cells, the target cells in vivo for PDCoV. In this study, we found that PDCoV infection remarkably inhibited Sendai virus-induced IFN-λ1 production by suppressing transcription factors IRF and NF-κB in IPI-2I cells, a line of porcine intestinal mucosal epithelial cells. We also confirmed that PDCoV infection impeded the activation of IFN-λ1 promoter stimulated by RIG-I, MDA5 and MAVS, but not by TBK1 and IRF1. Although the expression levels of IRF1 and MAVS were not changed, PDCoV infection resulted in reduction of the number of peroxisomes, the platform for MAVS to activate IRF1, and subsequent type III IFN production. Taken together, our study demonstrates that PDCoV suppresses type III IFN responses to circumvent the host's antiviral immunity.