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1.
Comput Struct Biotechnol J ; 20: 3409-3421, 2022.
Article in English | MEDLINE | ID: covidwho-1926353

ABSTRACT

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.

2.
Vet Microbiol ; 271: 109494, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1886124

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that has the potential for cross-species infection. Many viruses have been reported to induce endoplasmic reticulum stress (ERS) and activate the unfolded protein response (UPR). To date, little is known about whether and, if so, how the UPR is activated by PDCoV infection. Here, we investigated the activation state of UPR pathways and their effects on viral replication during PDCoV infection. We found that PDCoV infection induced ERS and activated all three known UPR pathways (inositol-requiring enzyme 1 [IRE1], activating transcription factor 6 [ATF6], and PKR-like ER kinase [PERK]), as demonstrated by IRE1-mediated XBP1 mRNA cleavage and increased mRNA expression of XBP1s, ATF4, CHOP, GADD34, GRP78, and GRP94, as well as phosphorylated eIF2α expression. Through pharmacologic treatment, RNA interference, and overexpression experiments, we confirmed the negative role of the PERK-eIF2α pathway and the positive regulatory role of the ATF6 pathway, but found no obvious effect of IRE1 pathway, on PDCoV replication. Taken together, our results characterize, for the first time, the state of the ERS response during PDCoV infection and identify the PERK and ATF6 pathways as potential antiviral targets.


Subject(s)
Protein Serine-Threonine Kinases , Unfolded Protein Response , Animals , Deltacoronavirus , Endoplasmic Reticulum Stress , Eukaryotic Initiation Factor-2/metabolism , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/metabolism , Swine , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism
3.
J Virol ; 96(9): e0040022, 2022 05 11.
Article in English | MEDLINE | ID: covidwho-1807320

ABSTRACT

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.


Subject(s)
Janus Kinase 1 , Porcine epidemic diarrhea virus , STAT1 Transcription Factor , Signal Transduction , Viral Nonstructural Proteins , alpha Karyopherins , Animals , Cell Line , Interferons/metabolism , Janus Kinase 1/metabolism , Porcine epidemic diarrhea virus/genetics , STAT1 Transcription Factor/metabolism , Swine , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , alpha Karyopherins/metabolism
4.
Virology ; 571: 12-20, 2022 06.
Article in English | MEDLINE | ID: covidwho-1799672

ABSTRACT

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.


Subject(s)
Norovirus , Peptide Hydrolases , Antiviral Agents , Cysteine Endopeptidases , Humans , Interferon-beta/genetics , Interferons/genetics , Norovirus/genetics
5.
J Virol ; 96(8): e0003722, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1779311

ABSTRACT

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.


Subject(s)
Coronavirus 3C Proteases , Interferon Type I , SARS Virus , SARS-CoV-2 , Antiviral Agents , COVID-19/immunology , COVID-19/virology , Coronavirus 3C Proteases/metabolism , Humans , Immune Evasion/genetics , Interferon Type I/antagonists & inhibitors , Interferon Type I/metabolism , SARS Virus/enzymology , SARS Virus/genetics , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Virus Replication/genetics
7.
Viruses ; 13(10)2021 10 04.
Article in English | MEDLINE | ID: covidwho-1463827

ABSTRACT

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.


Subject(s)
Coronavirus Infections/veterinary , Deltacoronavirus/genetics , Deltacoronavirus/metabolism , Genes, Reporter/genetics , Luciferases/genetics , A549 Cells , Animals , Cell Line , Chlorocebus aethiops , Chromosomes, Artificial, Bacterial/genetics , Coronavirus Infections/pathology , Deltacoronavirus/growth & development , Dogs , Genome, Viral/genetics , Humans , Luciferases/biosynthesis , Madin Darby Canine Kidney Cells , Nanostructures , Swine , Swine Diseases/virology , Vero Cells , Virus Replication/genetics
8.
J Virol ; 95(24): e0134521, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1441856

ABSTRACT

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.


Subject(s)
Coronavirus Infections/virology , Deltacoronavirus/physiology , Dynamins/metabolism , Endocytosis , Epithelial Cells/virology , Animals , Cell Line , Cell Survival , Clathrin/metabolism , Coronavirus/genetics , Hydrogen-Ion Concentration , Ileum/virology , Kidney/virology , Phosphatidylinositol 3-Kinases/metabolism , Pinocytosis , RNA, Small Interfering/metabolism , Swine , Swine Diseases/virology , Virus Internalization , rab5 GTP-Binding Proteins/metabolism
9.
J Virol ; 94(20)2020 09 29.
Article in English | MEDLINE | ID: covidwho-1271852

ABSTRACT

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.


Subject(s)
Coronavirus 3C Proteases/metabolism , Protein Processing, Post-Translational , Proteolysis , Torovirus Infections/embryology , Torovirus/enzymology , Animals , Coronavirus 3C Proteases/genetics , HEK293 Cells , Humans , Substrate Specificity , Swine , Torovirus/genetics , Torovirus Infections/genetics
10.
Viruses ; 13(6)2021 06 13.
Article in English | MEDLINE | ID: covidwho-1270126

ABSTRACT

Coronavirus accessory proteins are a unique set of proteins whose genes are interspersed among or within the genes encoding structural proteins. Different coronavirus genera, or even different species within the same coronavirus genus, encode varying amounts of accessory proteins, leading to genus- or species-specificity. Though accessory proteins are dispensable for the replication of coronavirus in vitro, they play important roles in regulating innate immunity, viral proliferation, and pathogenicity. The function of accessory proteins on virus infection and pathogenesis is an area of particular interest. In this review, we summarize the current knowledge on accessory proteins of several representative coronaviruses that infect humans or animals, including the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with an emphasis on their roles in interaction between virus and host, mainly involving stress response, innate immunity, autophagy, and apoptosis. The cross-talking among these pathways is also discussed.


Subject(s)
Immunity, Innate , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Viral Regulatory and Accessory Proteins/metabolism , COVID-19/immunology , COVID-19/virology , Host-Pathogen Interactions , Humans , Immune Evasion , Open Reading Frames , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Regulatory and Accessory Proteins/genetics , Virus Replication
11.
Arch Virol ; 166(3): 935-941, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1045234

ABSTRACT

Enteric coronaviruses (CoVs) are major pathogens that cause diarrhea in piglets. To date, four porcine enteric CoVs have been identified: transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and HKU2-like porcine enteric alphacoronavirus (PEAV). In this study, we investigated the replicative capacity of these four enteric CoVs in LLC-PK1 cells, a porcine kidney cell line. The results showed that LLC-PK1 cells are susceptible to all four enteric CoVs, particularly to TGEV and PDCoV infections, indicating that LLC-PK1 cells can be applied to porcine enteric CoV research in vitro, particularly for coinfection studies.


Subject(s)
Deltacoronavirus/growth & development , Gastroenteritis, Transmissible, of Swine/virology , Porcine epidemic diarrhea virus/growth & development , Transmissible gastroenteritis virus/growth & development , Virus Replication/physiology , Animals , Cell Line , Chlorocebus aethiops , Disease Susceptibility , Fluorescent Antibody Technique, Indirect , Intestine, Small/virology , LLC-PK1 Cells , Swine , Swine Diseases/virology , Vero Cells
12.
Virus Res ; 295: 198306, 2021 04 02.
Article in English | MEDLINE | ID: covidwho-1031553

ABSTRACT

Cholesterol 25-hydroxylase (CH25 H) is a key enzyme regulating cholesterol metabolism and also acts as a broad antiviral host restriction factor. Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that can cause vomiting, diarrhea, dehydration and even death in newborn piglets. In this study, we found that PDCoV infection significantly upregulated the expression of CH25H in IPI-FX cells, a cell line of porcine ileum epithelium. Overexpression of CH25H inhibited PDCoV replication, whereas CH25H silencing using RNA interference promoted PDCoV infection. Treatment with 25-hydroxycholesterol (25HC), the catalysate of cholesterol via CH25H, inhibited PDCoV proliferation by impairing viral invasion of IPI-FX cells. Furthermore, a mutant CH25H (CH25H-M) lacking hydroxylase activity also inhibited PDCoV infection to a lesser extent. Taken together, our data suggest that CH25H acts as a host restriction factor to inhibit the proliferation of PDCoV but this inhibitory effect is not completely dependent on its enzymatic activity.


Subject(s)
Coronavirus Infections/prevention & control , Deltacoronavirus , Steroid Hydroxylases/physiology , Virus Internalization , Animals , Cells, Cultured , Coronavirus Infections/enzymology , Steroid Hydroxylases/antagonists & inhibitors , Swine , Virus Replication
13.
J Virol Methods ; 276: 113772, 2020 02.
Article in English | MEDLINE | ID: covidwho-829838

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-ΔORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-ΔORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.


Subject(s)
CRISPR-Cas Systems , Coronavirus Infections/virology , Porcine epidemic diarrhea virus/genetics , Swine Diseases/virology , Animals , Coronavirus Infections/prevention & control , Genome, Viral , Recombination, Genetic , Swine , Viral Vaccines/genetics
14.
Vet Microbiol ; 247: 108785, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-827867

ABSTRACT

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.


Subject(s)
Coronavirus Infections/veterinary , Epithelial Cells/immunology , Epithelial Cells/virology , Host-Pathogen Interactions/immunology , Interferons/antagonists & inhibitors , Animals , Cell Line , Coronavirus , Coronavirus Infections/immunology , Coronavirus Infections/virology , Interferon Regulatory Factor-1/antagonists & inhibitors , Interferon Regulatory Factor-1/immunology , Interferons/immunology , Intestines/cytology , Intestines/virology , Kidney/cytology , Kidney/virology , NF-kappa B/antagonists & inhibitors , NF-kappa B/immunology , Sendai virus/immunology , Signal Transduction/immunology , Swine/virology , Swine Diseases/immunology , Swine Diseases/virology
15.
Antiviral Res ; 173: 104651, 2020 01.
Article in English | MEDLINE | ID: covidwho-824493

ABSTRACT

Emerging coronaviruses (CoVs) primarily cause severe gastroenteric or respiratory diseases in humans and animals, and no approved therapeutics are currently available. Here, A9, a receptor tyrosine kinase inhibitor (RTKI) of the tyrphostin class, is identified as a robust inhibitor of transmissible gastroenteritis virus (TGEV) infection in cell-based assays. Moreover, A9 exhibited potent antiviral activity against the replication of various CoVs, including murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV). We further performed a comparative phosphoproteomic analysis to investigate the mechanism of action of A9 against TGEV infection in vitro. We specifically identified p38 and JNK1, which are the downstream molecules of receptor tyrosine kinases (RTKs) required for efficient TGEV replication, as A9 targets through plaque assays, qRT-PCR and Western blotting assays. p38 and JNK1 inhibitors and RNA interference further showed that the inhibitory activity of A9 against TGEV infection was mainly mediated by the p38 mitogen-activated protein kinase (MAPK) signaling pathway. All these findings indicated that the RTKI A9 directly inhibits TGEV replication and that its inhibitory activity against TGEV replication mainly occurs by targeting p38, which provides vital clues to the design of novel drugs against CoVs.


Subject(s)
Antiviral Agents/pharmacology , Host-Pathogen Interactions , MAP Kinase Signaling System/drug effects , Protein Kinase Inhibitors/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Transmissible gastroenteritis virus/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Cats , Cell Line , Cells, Cultured , Chlorocebus aethiops , Chromatography, Liquid , Gastroenteritis, Transmissible, of Swine/drug therapy , Gastroenteritis, Transmissible, of Swine/metabolism , Gastroenteritis, Transmissible, of Swine/virology , High-Throughput Screening Assays , Life Cycle Stages , Phosphoproteins/metabolism , Protein Kinase Inhibitors/chemistry , Proteomics/methods , Small Molecule Libraries , Swine , Tandem Mass Spectrometry , Vero Cells
16.
Virology ; 539: 38-48, 2020 01 02.
Article in English | MEDLINE | ID: covidwho-822398

ABSTRACT

Ionic calcium (Ca2+) is a versatile intracellular second messenger that plays important roles in cellular physiological and pathological processes. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that causes serious vomiting and diarrhea in suckling piglets. In this study, the role of Ca2+ to PDCoV infection was investigated. PDCoV infection was found to upregulate intracellular Ca2+ concentrations of IPI-2I cells. Chelating extracellular Ca2+ by EGTA inhibited PDCoV replication, and this inhibitory effect was overcome by replenishment with CaCl2. Treatment with Ca2+ channel blockers, particularly the L-type Ca2+ channel blocker diltiazem hydrochloride, inhibited PDCoV infection significantly. Mechanistically, diltiazem hydrochloride reduces PDCoV infection by inhibiting the replication step of the viral replication cycle. Additionally, knockdown of CACNA1S, the L-type Ca2+ voltage-gated channel subunit, inhibited PDCoV replication. The combined results demonstrate that PDCoV modulates calcium influx to favor its replication.


Subject(s)
Calcium/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Coronavirus/physiology , Swine Diseases/metabolism , Swine Diseases/virology , Virus Replication , Animals , Calcium Signaling , Swine , Swine, Miniature
17.
ACS Appl Bio Mater ; 3(8): 4809-4819, 2020 08 17.
Article in English | MEDLINE | ID: covidwho-833523

ABSTRACT

Despite the good biocompatibility and antibacterial activity of zinc sulfide nanoparticles (ZnS NPs), whether they possess antiviral activity is still unclear. Here, GSH-modified ZnS NPs (GSH-ZnS NPs) were synthesized and their significant antiviral activity was demonstrated using the Arteriviridae family RNA virus, porcine reproductive and respiratory syndrome virus (PRRSV), as a model. Mechanistically, GSH-ZnS NPs were shown to reduce PRRSV-induced ROS production to prevent PRRSV multiplication, with no activating effect on the interferon (IFN) signal pathway, the first defense line against virus infection. Furthermore, isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis of GSH-ZnS NP-treated cells revealed the involvement of numerous crucial proteins in virus proliferation, with vitronectin (VTN) being confirmed as an efficient PRRSV antagonist here. Furthermore, GSH-ZnS NPs were found to have potent antiviral effects on the Herpesviridae family DNA virus, pseudorabies virus (PRV), the Coronaviridae family positive-sense RNA virus, porcine epidemic diarrhea virus (PEDV), and the Rhabdoviridae family negative-stranded RNA virus, vesicular stomatitis virus (VSV), indicating their broad-spectrum antiviral activity against viruses from different families with various genome types. Overall, GSH-ZnS NP is a prospective candidate for the development of antiviral nanomaterials and may serve as a model for investigation of potential host restriction factors in combination with proteomics.


Subject(s)
Antiviral Agents/pharmacology , Glutathione/chemistry , Nanoparticles/chemistry , Sulfides/chemistry , Viruses/drug effects , Zinc Compounds/chemistry , Animals , Cell Line , Chlorocebus aethiops , Microbial Sensitivity Tests , Reactive Oxygen Species/metabolism , Viruses/classification
18.
RSC Adv ; 10(24): 14161-14169, 2020 Apr 06.
Article in English | MEDLINE | ID: covidwho-833522

ABSTRACT

Heparan sulfate (HS) is a kind of cellular adhesion receptor that mediates the attachment and internalization of virus infection. Herein, to mimic the cell surface receptor, mercaptoethane sulfonate (MES), an analogue of HS, was used as the surface modifier to synthesize bovine serum albumin (BSA)-coated tellurium nanoparticles (Te/BSA NPs) with a unique triangular star shape (Te/BSA nanostars). Using porcine reproductive and respiratory syndrome virus (PRRSV), which utilizes HS as a cellular receptor, as a model of arterivirus, we found that Te/BSA nanostars suppressed virus infection mainly by inhibiting the virus internalization process. Interestingly, Te/BSA nanostars exhibited much higher antiviral activity than the spherical Te/BSA NPs (Te/BSA nanospheres), the Te/BSA NPs were synthesized with GSH as a substitute of MES, suggesting that both MES modification and the novel shapes of Te/BSA NPs enhance their antiviral activity. Finally, the antiviral effect of Te/BSA nanostars on porcine epidemic diarrhea virus (PEDV), a model of coronavirus, was also demonstrated, indicating the potential broad-spectrum antiviral property of Te/BSA nanostars.

19.
Elife ; 92020 09 02.
Article in English | MEDLINE | ID: covidwho-740561

ABSTRACT

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.


Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses ­ the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus ­ cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.


Subject(s)
CD13 Antigens/deficiency , Coronavirus Infections/prevention & control , Coronavirus/pathogenicity , Gastroenteritis, Transmissible, of Swine/prevention & control , Porcine Reproductive and Respiratory Syndrome/prevention & control , Porcine respiratory and reproductive syndrome virus/pathogenicity , Receptors, Cell Surface/deficiency , Transmissible gastroenteritis virus/pathogenicity , Animals , Animals, Genetically Modified , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/immunology , Body Composition , CD13 Antigens/genetics , CD13 Antigens/immunology , Coronavirus/immunology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Disease Susceptibility , Gastroenteritis, Transmissible, of Swine/genetics , Gastroenteritis, Transmissible, of Swine/immunology , Gastroenteritis, Transmissible, of Swine/virology , Gene Knockdown Techniques , Host Microbial Interactions , Meat-Packing Industry , Phenotype , Porcine Reproductive and Respiratory Syndrome/genetics , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/immunology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Sus scrofa/genetics , Swine , Transmissible gastroenteritis virus/immunology , Weight Gain
20.
J Virol ; 94(15)2020 07 16.
Article in English | MEDLINE | ID: covidwho-382053

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The nonstructural protein nsp5, also called 3C-like protease, is responsible for processing viral polyprotein precursors in coronavirus (CoV) replication. Previous studies have shown that PDCoV nsp5 cleaves the NF-κB essential modulator and the signal transducer and activator of transcription 2 to disrupt interferon (IFN) production and signaling, respectively. Whether PDCoV nsp5 also cleaves IFN-stimulated genes (ISGs), IFN-induced antiviral effector molecules, remains unclear. In this study, we screened 14 classical ISGs and found that PDCoV nsp5 cleaved the porcine mRNA-decapping enzyme 1a (pDCP1A) through its protease activity. Similar cleavage of endogenous pDCP1A was also observed in PDCoV-infected cells. PDCoV nsp5 cleaved pDCP1A at glutamine 343 (Q343), and the cleaved pDCP1A fragments, pDCP1A1-343 and pDCP1A344-580, were unable to inhibit PDCoV infection. Mutant pDCP1A-Q343A, which resists nsp5-mediated cleavage, exhibited a stronger ability to inhibit PDCoV infection than wild-type pDCP1A. Interestingly, the Q343 cleavage site is highly conserved in DCP1A homologs from other mammalian species. Further analyses demonstrated that nsp5 encoded by seven tested CoVs that can infect human or pig also cleaved pDCP1A and human DCP1A, suggesting that DCP1A may be the common target for cleavage by nsp5 of mammalian CoVs.IMPORTANCE Interferon (IFN)-stimulated gene (ISG) induction through IFN signaling is important to create an antiviral state and usually directly inhibits virus infection. The present study first demonstrated that PDCoV nsp5 can cleave mRNA-decapping enzyme 1a (DCP1A) to attenuate its antiviral activity. Furthermore, cleaving DCP1A is a common characteristic of nsp5 proteins from different coronaviruses (CoVs), which represents a common immune evasion mechanism of CoVs. Previous evidence showed that CoV nsp5 cleaves the NF-κB essential modulator and signal transducer and activator of transcription 2. Taken together, CoV nsp5 is a potent IFN antagonist because it can simultaneously target different aspects of the host IFN system, including IFN production and signaling and effector molecules.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Coronavirus/metabolism , Cysteine Endopeptidases/metabolism , Endoribonucleases/metabolism , Trans-Activators/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Coronavirus 3C Proteases , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Cysteine Endopeptidases/chemistry , Exoribonucleases/metabolism , HEK293 Cells , Host-Pathogen Interactions , Humans , Immune Evasion , Interferons/metabolism , STAT2 Transcription Factor/metabolism , Signal Transduction , Swine , Swine Diseases/virology
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