Interactome between ASFV and host immune pathway proteins.

IMPORTANCE: African swine fever (ASF), caused by African swine fever virus (ASFV), has become a major crisis for the pork industry in recent years. The mechanism for ASFV pathology and the clinical symptoms difference of ASF between domestic pigs and reservoir hosts remain to be elucidated. We deciphered the comprehensive protein-protein interaction (PPI) network between ASFV and host immune pathways. The intensive PPI network contained both ASFV-host immune pathway PPI and ASFV-ASFV PPI information, providing a comprehensive ASFV-host interaction landscape. Furthermore, the ASFV-host PPI difference between domestic pigs and warthogs was explored, which will be instructive for exploring essential candidates involved in ASFV pathology. Moreover, we screened the inhibitory effect of ASFV proteins in the PPI with cGAS-STING pathway on IFN-I and NF-κB, further providing possible functions of ASFV-host PPI network in innate immune regulation.

Differences in serum metabolome profile explain individual variation in growth performance of young goats.

High growth rates and body weight are important traits of young dairy goats that can shorten generation intervals, improve animal performance, and increase economic benefits. In the present study, ninety-nine, 6-month-old, female goats were fed with the same diet and kept under the same management condition. The ten goats with highest average daily gain (ADG, HADG, 135.27 ± 4.59 g/d) and ten goats with lowest ADG (LADG, 87.74 ± 3.13 g/d) were selected to identify the key serum metabolites associated with ADG, and to investigate the relationships of serum metabolome profiles with digestive tract microbiota. The results showed that a total of 125 serum metabolites were significantly different between HADG and LADG. Of these, 43 serum metabolites were significantly higher levels in HADG, including D-ornithine, l-glutamine, L-histidine, carnosine, LysoPC (16:1(9Z)/0:0), DCTP and hydroxylysine, while, 82 serum metabolites were significantly higher levels in LADG, including P-salicylic acid and deoxycholic acid 3-glucuronide. Pathway analysis indicated that these different metabolites were mainly involved in amino acid and lipid metabolism. Furthermore, Spearman's rank correlation analysis revealed that these differential serum metabolites were correlated with ADG and ADG-related bacteria. Notably, serum hydroxylysine and L-histidine could be used as biomarkers for distinguishing HADG and LADG goats, with an accuracy of >92.0%. SIGNIFICANCE: Our study confirms that individual microbiota and metabolic differences contribute to the variations of growth rate in young goats. Some serum metabolites may be useful in improving the growth performance of young goats, which provides directions for developing further nutritional regulation in the goat industry to achieve healthy feeding and efficiency enhancement.

Effects of COVID-19 home quarantine on pregnancy outcomes of patients with gestational diabetes mellitus: a retrospective cohort study.

OBJECTIVE: This study aimed to evaluate the effects of the home quarantine on pregnancy outcomes of gestational diabetes mellitus (GDM) patients during the COVID-19 outbreak. METHODS: The complete electronic medical records of patients with GDM with home quarantine history were collected and classified into the home quarantine group from 24 February 2020 to 24 November 2020. The same period of patients with GDM without home quarantine history were included in the control group from 2018 to 2019. The pregnant outcomes of the home quarantine and control groups were systematically compared, such as neonatal weight, head circumference, body length, one-minute Apgar score, fetal macrosomia, and pre-term delivery. RESULTS: A total of 1358 patients with GDM were included in the analysis, including 484 in 2018, 468 in 2019, and 406 in 2020. Patients with GDM with home quarantine in 2020 had higher glycemic levels and adverse pregnancy outcomes than in 2018 and 2019, including higher cesarean section rates, lower Apgar scores, and higher incidence of macrosomia and umbilical cord around the neck. More importantly, the second trimester of home quarantine had brought a broader impact on pregnant women and fetuses. CONCLUSION: Home quarantine has aggravated the condition of GDM pregnant women and brought more adverse pregnancy outcomes during the COVID-19 outbreak. Therefore, we suggested governments and hospitals strengthen lifestyle guidance, glucose management, and antenatal care for patients with GDM with home quarantine during public health emergencies.

Identification, in silico selection, and mechanism study of novel antioxidant peptides derived from the rice bran protein hydrolysates.

The work aimed to assess the antioxidant ability and obtain a new antioxidant peptide from rice bran protein. Rice bran protein was hydrolyzed by Alcalase, Neutral, Pepsin, Chymotrypsin, and Trypsin, separately. Trypsin hydrolysate (T-RBPH) showed high Fe2+ chelating activity (IC50, 2.271 ± 0.007 mg/mL), DPPH and hydroxyl radical scavenging ability (IC50, 0.191 ± 0.006 and 1.038 ± 0.034 mg/mL). Moreover, T-RBPH could alleviate the H2O2-induced oxidative damage in Caco-2. The T-RBPH was purified and identified by UF, GF, FPLC, and LC-MS/MS. Finally, 9-amino acid peptide-AFDEGPWPK with low molecular weight (1045.48 Da), high antioxidant activity, good safety, and solubility was screened by in silico method and chemical oxidation determination, and its interaction with Keap1 was also demonstrated. The ORAC and DPPH radical scavenging ability of AFDEGPWPK were 44.16 ± 0.79 and 28.38 ± 0.14 µmol TE/mM. Moreover, the Molecular docking and Western blot (WB) results showed that AFDEGPWPK could enter the binding pocket in the Kelch domain and activate Keap1/Nrf2/HO-1 pathway.

Dual-responsive nanoparticles loading bevacizumab and gefitinib for molecular targeted therapy against non-small cell lung cancer.

The combination of vascular endothelial growth factor (VEGF) inhibitors and tyrosine kinase inhibitors (TKIs) is newly available for molecular targeted therapy against non-small cell lung cancer (NSCLC) in clinic. However, the therapeutic benefits remain unsatisfying due to the poor drug delivery to targets of interest. In this study, we developed bevacizumab-coated gefitinib-loaded nanoparticles (BCGN) with dual-responsive drug release for inhibiting tumor angiogenesis and phosphorylation of epidermal growth factor receptor (EGFR). Through an exogenous corona strategy, bevacizumab is easily coated on gefitinib-loaded nanoparticles via electrostatic interaction. After intravenous injection, BCGN are efficiently accumulated in NSCLC tumors as confirmed by dual-model imaging. Bevacizumab is released from BCGN upon oxidation in tumor microenvironment, whereas gefitinib is released after being internalized by tumor cells and disassembled in reduction cytoplasm. The dual-responsive release of bevacizumab and gefitinib significantly inhibits tumor growth in both A549 and HCC827 human NSCLC models. Our approach provides a promising strategy to improve combinational molecular targeted therapy of NSCLC with precisely controlled drug release.

Identification and in silico analysis of novel antioxidant peptides in broken rice protein hydrolysate and its cytoprotective effect against H2O2-induced 2BS cell model.

Broken rice is an important by-product during milling process of rice, which is rich in protein. To increase the value of by-products and search for effective antioxidants, the antioxidant peptides from broken rice protein hydrolysate were separated and identified by ultrafiltration, gel filtration chromatography, fast protein liquid chromatography, and LC-MS/MS in this study. These identified peptides were further screened using a combined in silico and in vitro method and their antioxidant mechanism was explored by Western blot and molecular docking analysis. Ninety-eight peptides were obtained after antioxidant activity-oriented isolation and four novel peptides, SGDWSDIGGR, DFGSEILPR, GEPFPSDPKKQLQ, and GEKGGIPIGIGK, with excellent solubility, safety, and antioxidant activity were synthesized. Among these, SGDWSDIGGR showed good antioxidant activities in the extracellular assay (41.57 µmol TE/g and 29.41 % in ORAC and DPPH assay, respectively.), and it possessed a protective effect against H2O2-injured oxidative stress in 2BS cells in a dose-dependent manner. Furthermore, Western blot and molecular docking results showed that SGDWSDIGGR achieves antioxidant ability by occupying the Nrf2-binding site, activating the Keap1-Nrf2 signaling pathway, and upregulating the expression of antioxidant enzymes. This study extends the rice industry chain and provides insights into the selection and mechanisms research of antioxidant peptides.

Prognosis of clear cell renal cell carcinoma patients stratified by age: A research relied on SEER database.

Objective: Clear cell renal cell carcinoma may affect patients of any age. To date, there are only a limited number of large data studies on renal clear cell carcinoma in different age groups. This study assessed CCRCC risk factors in different age groups using the Surveillance Epidemiology and End Results (SEER) database. Methods: We selected 58372 cases from the SEER database. These patients were divided into seven different age groups. Cox regression models were used to find independent risk factors for the survival of CCRCC patients. Based on independent risk factors, a nomogram was drawn with R software. Kaplan-Meier method for survival analysis and X-tile software were used to find the optimal age group for diagnosis. Results: Univariate analysis revealed that patients' age, sex, race, marital status, grade, TNM (tumor, node, metastasis) stage, surgery, WHO/ISUP grade were correlated with survival (P<0.01). Age was an independent risk factor for survival in patients with CCRCC according to multivariate Cox regression analysis (p<0.01). All-cause mortality and tumor-specific mortality increased according to the increasing age of the patients. The optimal cut-off values for age were defined as 58 and 76 years and 51 and 76 years, respectively, according to overall survival (OS) and cause-specific survival (CSS). Conclusion: There is a negative correlation between age and survival of CCRCC patients. The difference in prognosis of patients in different age groups has important implications for clinical treatment. Therefore, the diagnosis and treatment plan should be based on more detailed age grouping, which is more beneficial to improving the prognosis and survival of patients.

DEAD-box RNA helicase 21 negatively regulates cytosolic RNA-mediated innate immune signaling.

DEAD-box RNA helicase 21 (DDX21), also known as RHII/Gu, is an ATP-dependent RNA helicase. In addition to playing a vital role in regulating cellular RNA splicing, transcription, and translation, accumulated evidence has suggested that DDX21 is also involved in the regulation of innate immunity. However, whether DDX21 induces or antagonizes type I interferon (IFN-I) production has not been clear and most studies have been performed through ectopic overexpression or RNA interference-mediated knockdown. In this study, we generated DDX21 knockout cell lines and found that knockout of DDX21 enhanced Sendai virus (SeV)-induced IFN-ß production and IFN-stimulated gene (ISG) expression, suggesting that DDX21 is a negative regulator of IFN-ß. Mechanistically, DDX21 competes with retinoic acid-inducible gene I (RIG-I) for binding to double-stranded RNA (dsRNA), thereby attenuating RIG-I-mediated IFN-ß production. We also identified that the 217-784 amino acid region of DDX21 is essential for binding dsRNA and associated with its ability to antagonize IFN production. Taken together, our results clearly demonstrated that DDX21 negatively regulates IFN-ß production and functions to maintain immune homeostasis.

An intermolecular salt bridge linking substrate binding and P1 substrate specificity switch of arterivirus 3C-like proteases.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose a major threat to the equine- and swine-breeding industries throughout the world. Previously, we and others demonstrated that PRRSV 3C-like protease (3CLpro) had very high glutamic acid (Glu)-specificity at the P1 position (P1-Glu). Comparably, EAV 3CLpro exhibited recognition of both Glu and glutamine (Gln) at the P1 position. However, the underlying mechanisms of the P1 substrate specificity shift of arterivirus 3CLpro remain unclear. We systematically screened the specific amino acids in the S1 subsite of arterivirus 3CLpro using a cyclized luciferase-based biosensor and identified Gly116, His133 and Ser136 (using PRRSV 3CLpro numbering) are important for recognition of P1-Glu, whereas Ser136 is nonessential for recognition of P1-Gln. Molecular dynamics simulations and biochemical experiments highlighted that the PRRSV 3CLpro and EAV 3CLpro formed distinct S1 subsites for the P1 substrate specificity switch. Mechanistically, a specific intermolecular salt bridge between PRRSV 3CLpro and substrate P1-Glu (Lys138/P1-Glu) are invaluable for high Glu-specificity at the P1 position, and the exchange of K138T (salt bridge interruption, from PRRSV to EAV) shifted the specificity of PRRSV 3CLpro toward P1-Gln. In turn, the T139K exchange of EAV 3CLpro showed a noticeable shift in substrate specificity, such that substrates containing P1-Glu are likely to be recognized more efficiently. These findings identify an evolutionarily accessible mechanism for disrupting or reorganizing salt bridge with only a single mutation of arterivirus 3CLpro to trigger a substrate specificity switch.

Taurine Ameliorates Apoptosis via AKT Pathway in the Kidney of Diabetic Rats.

Diabetic nephropathy is one of the major diabetic complications which has become the major cause of end-stage renal disease. It has been demonstrated that apoptosis induced by hyperglycemia is a critical factor in the pathophysiology of diabetic nephropathy. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The protective effect of taurine against apoptosis in diabetic kidney deserves to be explored. In the present study, mRNA expression of cysteinyl aspartate-specific proteinase-3 (caspase-3) and caspase-9 was examined, and the activity of caspase-3 was also detected as the marker of apoptosis. The expression of Bax and Bcl-2 was detected by Western blot. In addition, the level of total Akt and phosphorylated Akt (p-Akt) was measured. We found that caspase-3 and caspase-9 mRNA expression was decreased in diabetic kidney, which was recovered by taurine treatment. The activity of caspase-3 was increased in diabetic kidney, while the increased activity was significantly attenuated after taurine treatment. We also found that the expressions of Bax and Bcl-2 were disturbed in diabetic kidney, which were reversed by taurine treatment. The decrease of the p-Akt level was also prevented by taurine treatment. These results indicated that taurine-ameliorated apoptosis in diabetic kidney may be through activating of the Akt signaling pathway.

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.

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.

DEAD-Box RNA Helicase 21 (DDX21) Positively Regulates the Replication of Porcine Reproductive and Respiratory Syndrome Virus via Multiple Mechanisms.

The porcine reproductive and respiratory syndrome virus (PRRSV) remains a persistent hazard in the global pig industry. DEAD (Glu-Asp-Ala-Glu) box helicase 21 (DDX21) is a member of the DDX family. In addition to its function of regulating cellular RNA metabolism, DDX21 also regulates innate immunity and is involved in the replication cycle of some viruses. However, the relationship between DDX21 and PRRSV has not yet been explored. Here, we found that a DDX21 overexpression promoted PRRSV replication, whereas knockdown of DDX21 reduced PRRSV proliferation. Mechanistically, DDX21 promoted PRRSV replication independently of its ATPase, RNA helicase, and foldase activities. Furthermore, overexpression of DDX21 stabilized the expressions of PRRSV nsp1α, nsp1ß, and nucleocapsid proteins, three known antagonists of interferon ß (IFN-ß). Knockdown of DDX21 activated the IFN-ß signaling pathway in PRRSV-infected cells, suggesting that the effect of DDX21 on PRRSV-encoded IFN-ß antagonists may be a driving factor for its contribution to viral proliferation. We also found that PRRSV infection enhanced DDX21 expression and promoted its nucleus-to-cytoplasm translocation. Screening PRRSV-encoded proteins showed that nsp1ß interacted with the C-terminus of DDX21 and enhanced the expression of DDX21. Taken together, these findings reveal that DDX21 plays an important role in regulating PRRSV proliferation through multiple mechanisms.

Impaired Antiviral Responses to Extracellular Double-Stranded RNA and Cytosolic DNA, but Not to Interferon-α Stimulation, in TRIM56-Deficient Cells.

The physiologic function of tripartite motif protein 56 (TRIM56), a ubiquitously expressed E3 ligase classified within the large TRIM protein family, remains elusive. Gene knockdown studies have suggested TRIM56 as a positive regulator of the type I interferon (IFN-I) antiviral response elicited via the Toll-like receptor 3 (TLR3) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathways, which detect and respond to danger signals-extracellular double-stranded (ds) RNA and cytosolic dsDNA, respectively. However, to what extent these pathways depend on TRIM56 in human cells is unclear. In addition, it is debatable whether TRIM56 plays a part in controlling the expression of IFN-stimulated genes (ISGs) resulting from IFN-I based antiviral treatment. In this study, we created HeLa-derived TRIM56 null cell lines by gene editing and used these cell models to comprehensively examine the impact of endogenous TRIM56 on innate antiviral responses. Our results showed that TRIM56 knockout severely undermined the upregulation of ISGs by extracellular dsRNA and that loss of TRIM56 weakened the response to cytosolic dsDNA. ISG induction and ISGylation following IFN-α stimulation, however, were not compromised by TRIM56 deletion. Using a vesicular stomatitis virus-based antiviral bioactivity assay, we demonstrated that IFN-α could efficiently establish an antiviral state in TRIM56 null cells, providing direct evidence that TRIM56 is not required for the general antiviral action of IFN-I. Altogether, these data ascertain the contributions of TRIM56 to TLR3- and cGAS-STING-dependent antiviral pathways in HeLa cells and add to our understanding of the roles this protein plays in innate immunity.

Evolutionary Dynamics of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by Whole-Genome Analysis.

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen in the swine industry, is a genetically highly diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus are not yet fully understood. In this study, we performed an integrated analysis of all available whole-genome sequences of type 2 PRRSV (n = 901) to reveal its evolutionary dynamics. The results showed that there were three distinct phylogenetic lineages of PRRSV in their distribution patterns. We identified that sublineage 2.7 (L2.7), associated with a NADC30 cluster, had the highest substitution rate and higher viral genetic diversity, and inter-lineage recombination is observed more frequently in L2.7 PRRSV compared to other sublineages. Most inter-lineage recombination events detected are observed between L2.7 PRRSVs (as major parents) and L3.4 (a JXA1-R-related cluster)/L3.7 (a WUH3-related cluster) PRRSVs (as minor parents). Moreover, the recombination hotspots are located in the structural protein gene ORF2 and ORF4, or in the non-structural protein gene nsp7. In addition, a GM2-related cluster, L3.2, shows inconsistent recombination modes compared to those of L2.7, suggesting that it may have undergone extensive and unique recombination in their evolutionary history. We also identified several amino acids under positive selection in GP2, GP4 and GP5, the major glycoproteins of PRRSV, showing the driving force behind adaptive evolution. Taken together, our results provide new insights into the evolutionary dynamics of PPRSV that contribute to our understanding of the critical factors involved in its evolution and guide future efforts to develop effective preventive measures against PRRSV.

Corrigendum: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms.

[This corrects the article DOI: 10.3389/fimmu.2018.01142.].

Porcine reproductive and respiratory syndrome virus infection induces endoplasmic reticulum stress, facilitates virus replication, and contributes to autophagy and apoptosis.

During viral infection, the host cell synthesizes high amounts of viral proteins, which often causes stress to the endoplasmic reticulum (ER). To manage abnormal ER stress, mammalian cells trigger a response called the unfolded protein response (UPR). Previous studies have indicated that porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has been devastating the swine industry worldwide, can induce ER stress and activate UPR, however, the activation pathways and the biological significance requires further investigation. In this study, we demonstrated that, among the three types of UPR pathways, PRRSV infection induced PERK and IRE1 pathways, but not the ATF6 pathway. Furthermore, the induction of UPR promoted PRRSV replication. We also found that PRRSV-induced UPR, particularly the PERK pathway, was involved in the induction of autophagy, a cellular degradation process that can alleviate cell stress. Besides, we also provided insights into the ER stress-mediated apoptosis in response to PRRSV infection. PRRSV infection induced the expression of the transcription factor CHOP, which activated caspase 3 and PARP led to ER stress-mediated apoptosis. Using 3-Methyladenine (3-MA) to inhibit autophagy, the increased ER stress and cell apoptosis were observed in the PRRSV infected cell. Taken together, our results revealed the associations of ER stress, autophagy, and apoptosis during PRRSV infection, helping us to further understand how PRRSV interacts with host cells.

Cross-species transmission of deltacoronavirus and the origin of porcine deltacoronavirus.

Deltacoronavirus is the last identified Coronaviridae subfamily genus. Differing from other coronavirus (CoV) genera, which mainly infect birds or mammals, deltacoronaviruses (δ-CoVs) reportedly infect both animal types. Recent studies show that a novel δ-CoV, porcine deltacoronavirus (PDCoV), can also infect calves and chickens with the potential to infect humans, raising the possibility of cross-species transmission of δ-CoVs. Here, we explored the deep phylogenetic history and cross-species transmission of δ-CoVs. Virus-host cophylogenetic analyses showed that δ-CoVs have undergone frequent host switches in birds, and sparrows may serve as the unappreciated hubs for avian to mammal transmission. Our molecular clock analyses show that PDCoV possibly originated in Southeast Asia in the 1990s and that the PDCoV cluster shares a common ancestor with Sparrow-CoV of around 1,810. Our findings contribute valuable insights into the diversification, evolution, and interspecies transmission of δ-CoVs and the origin of PDCoV, providing a model for exploring the relationships of δ-CoVs in birds and mammals.

Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease.

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.