Metabolomic and Proteomic Profiling of Porcine Intestinal Epithelial Cells Infected with Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) infection results in severe epidemic diarrhea and the death of suckling pigs. Although new knowledge about the pathogenesis of PEDV has been improved, alterations in metabolic processes and the functional regulators involved in PEDV infection with host cells remain largely unknow. To identify cellular metabolites and proteins related to PEDV pathogenesis, we synergistically investigated the metabolome and proteome profiles of PEDV-infected porcine intestinal epithelial cells by liquid chromatography tandem mass spectrometry and isobaric tags for relative and absolute quantification techniques. We identified 522 differential metabolites in positive and negative ion modes and 295 differentially expressed proteins after PEDV infection. Pathways of cysteine and methionine metabolism, glycine, serine and threonine metabolism, and mineral absorption were significantly enriched by differential metabolites and differentially expressed proteins. The betaine-homocysteine S-methyltransferase (BHMT) was indicated as a potential regulator involved in these metabolic processes. We then knocked down the BHMT gene and observed that down-expression of BHMT obviously decreased copy numbers of PEDV and virus titers (p < 0.01). Our findings provide new insights into the metabolic and proteomic profiles in PEDV-infected host cells and contribute to our further understanding of PEDV pathogenesis.

Differential expression analysis of mRNAs, lncRNAs, and miRNAs expression profiles and construction of ceRNA networks in PEDV infection.

BACKGROUND: Porcine Epidemic Diarrhea Virus (PEDV) is a coronavirus that seriously affects the swine industry. MicroRNAs and long noncoding RNAs are two relevant non-coding RNAs (ncRNAs) class and play crucial roles in a variety of physiological processes. Increased evidence indicates a complex interaction between mRNA and ncRNA. However, our understanding of the function of ncRNA involved in host-PEDV interaction is limited. RESULTS: A total of 1,197 mRNA transcripts, 539 lncRNA transcripts, and 208 miRNA transcripts were differentially regulated at 24 h and 48 h post-infection. Gene ontology (GO) and KEGG pathway enrichment analysis showed that DE mRNAs and DE lncRNAs were mainly involved in biosynthesis, innate immunity, and lipid metabolism. Moreover, we constructed a miRNA-mRNA-pathway network using bioinformatics, including 12 DE mRNAs, 120 DE miRNAs, and 11 pathways. Finally, the target genes of DE miRNAs were screened by bioinformatics, and we constructed immune-related lncRNA-miRNA-mRNA ceRNA networks. Then, the selected DE genes were validated by qRT-PCR, which were consistent with the results from RNA-Seq data. CONCLUSIONS: This study provides the comprehensive analysis of the expression profiles of mRNAs, lncRNAs, and miRNAs during PEDV infection. We characterize the ceRNA networks which can provide new insights into the pathogenesis of PEDV.

Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication.

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.

Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.

Dynamic Dissection of the Endocytosis of Porcine Epidemic Diarrhea Coronavirus Cooperatively Mediated by Clathrin and Caveolae as Visualized by Single-Virus Tracking.

Coronaviruses (CoVs) have caused severe diseases in humans and animals. Endocytic pathways, such as clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME), play an important role for CoVs to penetrate the cell membrane barrier. In this study, a novel CoV entry manner is unraveled in which clathrin and caveolae can cooperatively mediate endocytosis of porcine epidemic diarrhea coronavirus (PEDV). Using multicolor live-cell imaging, the dynamics of the fluorescently labeled clathrin structures, caveolae structures, and PEDV were dissected. During CavME of PEDV, we found that clathrin structures can fuse with caveolae near the cell plasma membrane, and the average time of PEDV penetrating the cell membrane was within ∼3 min, exhibiting a rapid course of PEDV entry. Moreover, based on the dynamic recruitment of clathrin and caveolae structures and viral motility, the direct evidence also shows that about 20% of PEDVs can undergo an abortive entry via CME and CavME. Additionally, the dynamic trafficking of PEDV from clathrin and caveolae structures to early endosomes, and from early endosomes to late endosomes, and viral fusion were directly dissected, and PEDV fusion mainly occurred in late endosomes within ∼6.8 min after the transport of PEDV to late endosomes. Collectively, this work systematically unravels the early steps of PEDV infection, which expands our understanding of the mechanism of CoV infection.IMPORTANCE Emerging and re-emerging coronaviruses cause serious human and animal epidemics worldwide. For many enveloped viruses, including coronavirus, it is evident that breaking the plasma membrane barrier is a pivotal and complex process, which contains multiple dynamic steps. Although great efforts have been made to understand the mechanisms of coronavirus endocytic pathways, the direct real-time imaging of individual porcine epidemic diarrhea coronavirus (PEDV) internalization has not been achieved yet. In this study, we not only dissected the kinetics of PEDV entry via clathrin-mediated endocytosis and caveolae-mediated endocytosis and the kinetics of endosome trafficking and viral fusion but also found a novel productive coronavirus entry manner in which clathrin and caveolae can cooperatively mediate endocytosis of PEDV. Moreover, we uncovered the existence of PEDV abortive endocytosis. In summary, the productive PEDV entry via the cooperation between clathrin and caveolae structures and the abortive endocytosis of PEDV provide new insights into coronavirus penetrating the plasma membrane barrier.

Coronaviruses and Integrin αvß3: Does Thyroid Hormone Modify the Relationship?

BACKGROUND: Uptake of coronaviruses by target cells involves binding of the virus by cell ectoenzymes. For the etiologic agent of COVID-19 (SARS-CoV-2), a receptor has been identified as angiotensin-converting enzyme-2 (ACE2). Recently it has been suggested that plasma membrane integrins may be involved in the internalization and replication of clinically important coronaviruses. For example, integrin αvß3 is involved in the cell uptake of a model porcine enteric α-coronavirus that causes human epidemics. ACE2 modulates the intracellular signaling generated by integrins. OBJECTIVE: We propose that the cellular internalization of αvß3 applies to uptake of coronaviruses bound to the integrin, and we evaluate the possibility that clinical host T4 may contribute to target cell uptake of coronavirus and to the consequence of cell uptake of the virus. DISCUSSION AND CONCLUSIONS: The viral binding domain of the integrin is near the Arg-Gly-Asp (RGD) peptide-binding site and RGD molecules can affect virus binding. In this same locale on integrin αvß3 is the receptor for thyroid hormone analogues, particularly, L-thyroxine (T4). By binding to the integrin, T4 has been shown to modulate the affinity of the integrin for other proteins, to control internalization of αvß3 and to regulate the expression of a panel of cytokine genes, some of which are components of the 'cytokine storm' of viral infections. If T4 does influence coronavirus uptake by target cells, other thyroid hormone analogues, such as deaminated T4 and deaminated 3,5,3'-triiodo-L-thyronine (T3), are candidate agents to block the virus-relevant actions of T4 at integrin αvß3 and possibly restrict virus uptake.

Role of Porcine Aminopeptidase N and Sialic Acids in Porcine Coronavirus Infections in Primary Porcine Enterocytes.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been reported to use aminopeptidase N (APN) as a cellular receptor. Recently, the role of APN as a receptor for PEDV has been questioned. In our study, the role of APN in PEDV and TGEV infections was studied in primary porcine enterocytes. After seven days of cultivation, 89% of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to PEDV and TGEV. A significant increase of PEDV and TGEV infection was correlated with a higher expression of APN, which was indicative that APN plays an important role in porcine coronavirus infections. However, PEDV and TGEV infected both APN positive and negative enterocytes. PEDV and TGEV Miller showed a higher infectivity in APN positive cells than in APN negative cells. In contrast, TGEV Purdue replicated better in APN negative cells. These results show that an additional receptor exists, different from APN for porcine coronaviruses. Subsequently, treatment of enterocytes with neuraminidase (NA) had no effect on infection efficiency of TGEV, implying that terminal cellular sialic acids (SAs) are no receptor determinants for TGEV. Treatment of TGEV with NA significantly enhanced the infection which shows that TGEV is masked by SAs.