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1.
Vet Microbiol ; 276: 109616, 2023 Jan.
Article in English | MEDLINE | ID: mdl-36495740

ABSTRACT

Porcine deltacoronavirus (PDCoV) is a newly emerging swine enteropathogenic coronavirus with extensive tissue tropism and cross-species transmission potential. Heparan sulfate (HS) is a complex polysaccharide ubiquitously expressed on cell surfaces and the extracellular matrix and acts as an attachment factor for many viruses. However, whether PDCoV uses HS as an attachment receptor is unclear. In this study, we found that treatment with heparin sodium or heparinase Ⅱ significantly inhibited PDCoV binding and infection among LLC-PK1 and IPI-2I cells. Attenuation of HS sulfuration by sodium chlorate also impeded PDCoV binding and infection. Moreover, we demonstrated that HS functioned independently of amino peptidase N (APN), a functional PDCoV receptor, in PDCoV infection. Molecular docking revealed that the S1 subunit of the PDCoV spike protein might be a putative region for HS binding. Taken together, these results firstly confirmed that HS is an attachment receptor for PDCoV infection, providing new insight into better understanding the mechanisms of PDCoV-host interactions.


Subject(s)
Coronavirus Infections , Coronavirus , Swine Diseases , Swine , Animals , Molecular Docking Simulation , Coronavirus/physiology , Coronavirus Infections/veterinary , Deltacoronavirus
2.
J Virol ; 96(24): e0162622, 2022 12 21.
Article in English | MEDLINE | ID: mdl-36453883

ABSTRACT

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To better understand host-virus genetic dependencies and find potential therapeutic targets for PDCoV, we used a porcine single-guide RNA (sgRNA) lentivirus library to screen host factors related to PDCoV infection in LLC-PK1 cells. The solute carrier family 35 member A1 (SLC35A1), a key molecule in the sialic acid (SA) synthesis pathway, was identified as a host factor required for PDCoV infection. A knockout of SLC35A1 caused decreases in the amounts of cell surface sialic acid (SA) and viral adsorption; meanwhile, trypsin promoted the use of SA in PDCoV infection. By constructing and assessing a series of recombinant PDCoV strains with the deletion or mutation of possible critical domain or amino acid residues for SA binding in the S1 N-terminal domain, we found that S T182 might be a PDCoV SA-binding site. However, the double knockout of SLC35A1 and amino peptidase N (APN) could not block PDCoV infection completely. Additionally, we found that different swine enteric coronaviruses, including transmissible gastroenteritis coronavirus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome coronavirus, are differentially dependent on SA. Overall, our study uncovered a collection of host factors that can be exploited as drug targets against PDCoV infection and deepened our understanding of the relationship between PDCoV and SA. IMPORTANCE Identifying the host factors required for replication will be helpful to uncover the pathogenesis mechanisms and develop antivirals against the emerging coronavirus porcine deltacoronavirus (PDCoV). Herein, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen, the results of which revealed that the solute carrier family 35 member A1 (SLC35A1) is a host factor required for PDCoV infection that acts by regulating cell surface sialic acid (SA). We also identified the T182 site in the N-terminal domain of PDCoV S1 subunit as being associated with the SA-binding site and found that trypsin promotes the use of cell surface SA by PDCoV. Furthermore, different swine enteric coronaviruses use SLC35A1 differently for infection. This is the first study to screen host factors required for PDCoV replication using a genome-wide CRISPR-Cas9 functional knockout, thereby providing clues for developing antiviral drugs against PDCoV infection.


Subject(s)
Coronavirus Infections , Host Microbial Interactions , Nucleotide Transport Proteins , Swine Diseases , Animals , Humans , Adsorption , Coronavirus , Coronavirus Infections/physiopathology , CRISPR-Cas Systems , N-Acetylneuraminic Acid/metabolism , Nucleotide Transport Proteins/genetics , Nucleotide Transport Proteins/metabolism , Swine , Swine Diseases/physiopathology , Trypsin , Host Microbial Interactions/genetics , Protein Domains , Binding Sites
3.
Arch Virol ; 166(3): 935-941, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33492525

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
4.
Vet Microbiol ; 247: 108785, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32768229

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 , Interferon Lambda
5.
Vet Microbiol ; 233: 21-27, 2019 Jun.
Article in English | MEDLINE | ID: mdl-31176408

ABSTRACT

Swine enteric coronavirus (CoV) is an important group of pathogens causing diarrhea in piglets. At least four kinds of swine enteric CoVs have been identified, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and the emerging HKU2-like porcine enteric alphacoronavirus (PEAV). The small intestines, particularly the jejunum and ileum, are the most common targets of these four CoVs in vivo, and co-infections by these CoVs are frequently observed in clinically infected pigs. This study was conducted to investigate the susceptibility of the porcine ileum epithelial cell line, IPI-2I, to different swine enteric CoVs. We found that IPI-2I cells are highly susceptible to TGEV, PDCoV, and PEAV, as demonstrated by cytopathic effect and virus multiplication. However, only a small number of cells could be infected by PEDV, possibly due to the heterogeneity of IPI-2I cells. A homogeneous cell line, designated IPI-FX, obtained from IPI-2I cells by sub-cloning with limited serial dilutions, was found to be highly susceptible to PEDV. Furthermore, IPI-FX cells were also highly susceptible to TGEV, PDCoV, as well as PEAV. Thus, this sub-cloned IPI-FX cell line is an ideal cell model to study the mechanisms of infection, particularly co-infections of swine enteric CoVs.


Subject(s)
Cell Culture Techniques/veterinary , Coronavirus/pathogenicity , Epithelial Cells/virology , Intestine, Small/cytology , Porcine epidemic diarrhea virus/pathogenicity , Animals , Cell Line , Diarrhea/virology , Feces/virology , Phylogeny , Porcine epidemic diarrhea virus/genetics , Swine , Swine Diseases/virology , Virus Replication
6.
Emerg Microbes Infect ; 7(1): 65, 2018 Apr 11.
Article in English | MEDLINE | ID: mdl-29636467

ABSTRACT

Porcine deltacoronavirus (PDCoV), a member of genus Deltacoronavirus, is an emerging swine enteropathogenic coronavirus (CoV). Although outstanding efforts have led to the identification of Alphacoronavirus and Betacoronavirus receptors, the receptor for Deltacoronavirus is unclear. Here, we compared the amino acid sequences of several representative CoVs. Phylogenetic analysis showed that PDCoV spike (S) protein was close to the cluster containing transmissible gastroenteritis virus (TGEV), which utilizes porcine aminopeptidase N (pAPN) as a functional receptor. Ectopic expression of pAPN in non-susceptible BHK-21 cells rendered them susceptible to PDCoV. These results indicate that pAPN may be a functional receptor for PDCoV infection. However, treatment with APN-specific antibody and inhibitors did not completely block PDCoV infection in IPI-2I porcine intestinal epithelial cells. pAPN knockout in IPI-2I cells completely blocked TGEV infection but only slightly decreased PDCoV infection. Homologous modeling of pAPN with the S1 C-terminal domain (S1-CTD) of PDCoV or TGEV showed that TGEV S1-CTD adopted ß-turns (ß1-ß2 and ß3-ß4), forming the tip of a ß-barrel, to recognize pAPN. However, only the top residues in the ß1-ß2 turn of PDCoV S1-CTD had the possibility to support an interaction with pAPN, and the ß3-ß4 turn failed to contact pAPN. We also discuss the evolution and variation of PDCoV S1-CTD based on structure information, providing clues to explain the usage of pAPN by PDCoV. Taken together, the results presented herein reveal that pAPN is likely not a critical functional receptor for PDCoV, although it is involved in PDCoV infection.


Subject(s)
CD13 Antigens/physiology , Coronavirus Infections/veterinary , Coronavirus/isolation & purification , Host-Pathogen Interactions , Receptors, Virus/metabolism , Swine Diseases/virology , Animals , Cell Line , Coronavirus/genetics , Coronavirus Infections/virology , Phylogeny , Receptors, Virus/genetics , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/genetics , Swine
7.
Oncotarget ; 8(30): 49655-49670, 2017 Jul 25.
Article in English | MEDLINE | ID: mdl-28591694

ABSTRACT

Coronaviruses (CoVs) are a huge threat to both humans and animals and have evolved elaborate mechanisms to antagonize interferons (IFNs). Nucleocapsid (N) protein is the most abundant viral protein in CoV-infected cells, and has been identified as an innate immunity antagonist in several CoVs, including mouse hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV. However, the underlying molecular mechanism(s) remain unclear. In this study, we found that MHV N protein inhibited Sendai virus and poly(I:C)-induced IFN-ß production by targeting a molecule upstream of retinoic acid-induced gene I (RIG-I) and melanoma differentiation gene 5 (MDA5). Further studies showed that both MHV and SARS-CoV N proteins directly interacted with protein activator of protein kinase R (PACT), a cellular dsRNA-binding protein that can bind to RIG-I and MDA5 to activate IFN production. The N-PACT interaction sequestered the association of PACT and RIG-I/MDA5, which in turn inhibited IFN-ß production. However, the N proteins from porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV), which are also classified in the order Nidovirales, did not interact and counteract with PACT. Taken together, our present study confirms that both MHV and SARS-CoV N proteins can perturb the function of cellular PACT to circumvent the innate antiviral response. However, this strategy does not appear to be used by all CoVs N proteins.


Subject(s)
Nucleocapsid Proteins/metabolism , Animals , Cell Line , Coronavirus Nucleocapsid Proteins , DEAD Box Protein 58/metabolism , Humans , Interferon-Induced Helicase, IFIH1/metabolism , Interferon-beta/biosynthesis , Interferon-beta/genetics , Interferon-beta/metabolism , Mice , Murine hepatitis virus/physiology , Promoter Regions, Genetic , Protein Binding , RNA, Double-Stranded/metabolism , Sendai virus/physiology , eIF-2 Kinase/metabolism
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