Porcine deltacoronavirus accessory protein NS6 harnesses VPS35-mediated retrograde trafficking to facilitate efficient viral infection.

IMPORTANCE: Retrograde transport has been reported to be closely associated with normal cellular biological processes and viral replication. As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has attracted considerable attention. However, whether retrograde transport is associated with PDCoV infection remains unclear. Our present study demonstrates that retromer protein VPS35 acts as a critical host factor that is required for PDCoV infection. Mechanically, VPS35 interacts with PDCoV NS6, mediating the retrograde transport of NS6 from endosomes to the Golgi and preventing it from lysosomal degradation. Recombinant PDCoVs with an NS6 deletion display resistance to VPS35 deficiency. Our work reveals a novel evasion mechanism of PDCoV that involves the manipulation of the retrograde transport pathway by VPS35, providing new insight into the mechanism of PDCoV infection.

Unfolding of an RNA G-quadruplex motif in the negative strand genome of porcine reproductive and respiratory syndrome virus by host and viral helicases to promote viral replication.

G-quadruplex (G4) is a unique secondary structure formed by guanine-rich nucleic acid sequences. Growing studies reported that the genomes of some viruses harbor G4 structures associated with viral replication, opening up a new field to dissect viral infection. Porcine reproductive and respiratory syndrome virus (PRRSV), a representative member of Arteriviridae, is an economically significant pathogen that has devastated the swine industry worldwide for over 30 years. In this study, we identified a highly conserved G-rich sequence with parallel-type G4 structure (named PRRSV-G4) in the negative strand genome RNA of PRRSV. Pyridostatin (PDS), a well-known G4-binding ligand, stabilized the PRRSV-G4 structure and inhibited viral replication. By screening the proteins interacting with PRRSV-G4 in PRRSV-infected cells and single-molecule magnetic tweezers analysis, we found that two helicases, host DDX18 and viral nsp10, interact with and efficiently unwound the PRRSV-G4 structure, thereby facilitating viral replication. Using a PRRSV reverse genetics system, we confirmed that recombinant PRRSV with a G4-disruptive mutation exhibited resistance to PDS treatment, thereby displaying higher replication than wild-type PRRSV. Collectively, these results demonstrate that the PRRSV-G4 structure plays a crucial regulatory role in viral replication, and targeting this structure represents a promising strategy for antiviral therapies.

Exosomes mediate the antibody-resistant intercellular transmission of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes severe enteritis and lethal watery diarrhea in suckling piglets, leading to tremendous economic losses. Exosomes have been reported to participate in intercellular communication by the transportation of a variety of biological materials, including RNAs, lipids, and proteins. However, PEDV transmission routes have not yet been fully elucidated, and whether exosomes function in PEDV transmission remains unclear. In this study, we extracted and purified exosomes from PEDV-infected Vero cells using a stringent isolation method with a combination of chemical precipitation, ultracentrifugation, and incubation with CD63-labeled magnetic beads. We found that exosomes from PEDV-infected Vero cells contain viral genomic RNA and viral nucleocapsid protein. Furthermore, we demonstrated that the purified exosomes from PEDV-infected cells are capable of transmitting the virus to both PEDV-susceptible and non-susceptible cells. Importantly, exosome-mediated PEDV infection was resistant to neutralization by PEDV-specific neutralizing antibodies that potently neutralized free PEDV. Our study reveals a potential immune evasion mechanism utilized by PEDV and provides new insight into the transmission and infection of this important pathogen.

Porcine deltacoronavirus resists antibody neutralization through cell-to-cell transmission.

ABSTRACTPorcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that has been reported to infect a variety of animals and even humans. Cell-cell fusion has been identified as an alternative pathway for the cell-to-cell transmission of certain viruses, but the ability of PDCoV to exploit this transmission model, and the relevant mechanisms, have not been fully elucidated. Herein, we provide evidence that cell-to-cell transmission is the main mechanism supporting PDCoV spread in cell culture and that this efficient spread model is mediated by spike glycoprotein-driven cell-cell fusion. We found that PDCoV efficiently spread to non-susceptible cells via cell-to-cell transmission, and demonstrated that functional receptor porcine aminopeptidase N and cathepsins in endosomes are involved in the cell-to-cell transmission of PDCoV. Most importantly, compared with non-cell-to-cell infection, the cell-to-cell transmission of PDCoV was resistant to neutralizing antibodies and immune sera that potently neutralized free viruses. Taken together, our study revealed key characteristics of the cell-to-cell transmission of PDCoV and provided new insights into the mechanism of PDCoV infection.

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.

Porcine deltacoronavirus uses heparan sulfate as an attachment receptor.

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.

Porcine deltacoronavirus accessory protein NS7a possesses the functional characteristics of a viroporin.

Viroporins are virus-encoded proteins that mediate ion channel (IC) activity, playing critical roles in virus entry, replication, pathogenesis, and immune evasion. Previous studies have shown that some coronavirus accessory proteins have viroporin-like activity. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that encodes three accessory proteins, NS6, NS7, and NS7a. However, whether any of the PDCoV accessory proteins possess viroporin-like activity, and if so which, remains unknown. In this study, we analyzed the biochemical properties of the three PDCoV-encoded accessory proteins and found that NS7a could enhance the membrane permeability of both mammalian cells and Escherichia coli cells. Indirect immunofluorescence assay and co-immunoprecipitation assay results further indicated that NS7a is an integral membrane protein and can form homo-oligomers. A bioinformation analysis revealed that a putative viroporin domain (VPD) is located within amino acids 69-88 (aa69-88) of NS7a. Experiments with truncated mutants and alanine scanning mutagenesis additionally demonstrated that the amino acid residues 69FLR71 of NS7a are essential for its viroporin-like activity. Together, our findings are the first to demonstrate that PDCoV NS7a possesses viroporin-like activity and identify its key amino acid residues associated with viroporin-like activity.

Induction and modulation of the unfolded protein response during porcine deltacoronavirus infection.

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.

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

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

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.

Functions of Coronavirus Accessory Proteins: Overview of the State of the Art.

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.

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.

Porcine Deltacoronavirus Accessory Protein NS7a Antagonizes IFN-ß Production by Competing With TRAF3 and IRF3 for Binding to IKKε.

As an emerging swine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) not only causes serious diarrhea in suckling piglets but also possesses the potential for cross-species transmission, which has sparked growing interest when studying this emerging virus. We previously identified a novel accessory protein NS7a encoded by PDCoV; however, the function of NS7a was not resolved. In this study, we demonstrated that PDCoV NS7a is an interferon antagonist. Overexpression of NS7a notably inhibited Sendai virus (SeV)-induced interferon-ß (IFN-ß) production and the activation of IRF3 rather than NF-κB. NS7a also inhibited IFN-ß promoter activity induced by RIG-I, MDA5, MAVS, TBK1, and IKKε, which are key components of the RIG-I-like receptor (RLR) signaling pathway but not IRF3, the transcription factor downstream of TBK1/IKKε. Surprisingly, NS7a specifically interacts with IKKε but not with the closely related TBK1. Furthermore, NS7a interacts simultaneously with the kinase domain (KD) and the scaffold dimerization domain (SDD) of IKKε, competing with TRAF3, and IRF3 for binding to IKKε, leading to the reduction of RLR-mediated IFN-ß production. The interactions of TRAF3-IKKε and IKKε-IRF3 are also attenuated in PDCoV-infected cells. Taken together, our results demonstrate that PDCoV NS7a inhibits IFN-ß production by disrupting the association of IKKε with both TRAF3 and IRF3, revealing a new mechanism utilized by a PDCoV accessory protein to evade the host antiviral innate immune response.

Porcine deltacoronavirus (PDCoV) modulates calcium influx to favor viral replication.

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.