ARFGAP1 binds to classical swine fever virus NS5A protein and enhances CSFV replication in PK-15 cells

Zhang, Liang; Jin, Mingxing; Song, Mengzhao; Liu, Shanchuan; Wang, Tao; Guo, Kangkang; Zhang, Yanming

doi:10.1016/j.vetmic.2021.109034

Cited by 7 publications

(3 citation statements)

References 34 publications

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“…Autophagy is induced and participates in CSFV replication, but the mechanism is not clearly understood. We analyzed whether the NS5A protein plays a significant role in inducing autophagy in vitro in the PK-15 cell line, a common model cell line for studying CSFV infection (Grummer et al, 2006;Zhang et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

et al. 2021

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Although classic swine fever virus (CSFV) infection has been reported to induce autophagy, the specific induced mechanism remains unrevealed. Nonstructural protein 5A (NS5A) of CSFV is a multiphosphorylated protein with multiple functions to regulate viral replication and the host cell immune responses. Herein, we demonstrated that CSFV NS5A could induce cellular autophagy and promote viral replication. In the current study, we showed that NS5A expression significantly increased the levels of autophagy-related genes (ATGs), including light chain 3 (LC3), ATG5, and Beclin 1; conversely, degradation of P62/sequestosome 1 (SQSTM1) was observed by Western blotting. The number of autophagy-like vesicles was also obviously increased in NS5A-expressing cells, as analyzed by transmission electron microscopy (TEM). Furthermore, we observed the co-localization of the NS5A and LC3 proteins by confocal immunofluorescence analysis. Direct binding of NS5A to the autophagy-related LC3 protein was confirmed by coimmunoprecipitation in vivo and by a GST pulldown assay in vitro. Through segmentation and point mutation research on the NS5A protein, we found that the N-terminal region and the phosphorylation of amino acids 81 and 92 of the NS5A protein were essential for inducing autophagy. Finally, we demonstrated that the LC3 protein had a positive effect on CSFV replication. These findings emphasize a previously unascertained interaction relationship between NS5A and LC3 in the autophagy process. Furthermore, our research revealed a new role of CSFV NS5A, particularly its N-terminal amino acids serine 81 and serine 92, as a critical regulator of CSFV-induced autophagy and have significance for extending our understanding of the CSFV-autophagy interplay.

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Section: Discussionmentioning

confidence: 99%

Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

et al. 2021

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“…The lentiviruses with RPLP1 knockdown or overexpression were produced as previously described [ 28 ]. Briefly, HEK-293 T cells within logarithmic growth phase were co-transfected with indicated constructs (CMV-RPLP1 or RPLP1 shRNA) and three assistant vectors (pVSV-G, pRev and pGag/Pol).…”

Section: Methodsmentioning

confidence: 99%

RPLP1, an NS4B-interacting protein, enhances production of CSFV through promoting translation of viral genome

et al. 2022

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Classical swine fever virus (CSFV), the etiological agent of classical swine fever (CSF), causes serious financial losses to the pig industry. Using yeast two-hybrid screening, we have previously identified ribosomal protein RPLP1 as a potential binding partner of CSFV NS4B. In this study, the interaction between host RPLP1 and CSFV NS4B was further characterized by co-immunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and confocal microscopy. In addition, lentivirus-mediated shRNA knockdown of RPLP1 drastically attenuated CSFV growth, while stable overexpression of RPLP1 markedly enhanced CSFV production. Moreover, cellular RPLP1 expression was found to be significantly up-regulated along with CSFV infection. Dual-luciferase reporter assay showed that depletion of RPLP1 had no effects on the activity of CSFV internal ribosome entry site (IRES). In the first life cycle of CSFV, further studies revealed that RPLP1 depletion did not influence the intracellular viral RNA abundance but diminished the intracellular and extracellular progeny virus titers as well as the viral E2 protein expression, which indicates that RPLP1 is crucial for CSFV genome translation. In summary, this study demonstrated that RPLP1 interacts with CSFV NS4B and enhances virus production via promoting translation of viral genome.

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“…The IPEC-J2 cells were obtained from College of Veterinary Medicine at Northwest A&F University and maintained in high glucose Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco) and penicillinstreptomycin solution (Sigma-Aldrich, St Louis, MO, USA). 16 The cells were incubated in a 5% CO 2 atmosphere of at 37 °C. 17 After growing to 80-90% confluence, the cultured cells were digested with 0.25% trypsin-ethylenediaminetetraacetic acid for subsequent passage or protein extractions.…”

Section: Cell and Bacterial Strain Culturementioning

confidence: 99%

Tannic acid alleviates ETEC K88‐induced intestinal damage through regulating the p62‐keap1‐Nrf2 and TLR4‐NF‐κB‐NLRP3 pathway in IPEC‐J2 cells

Liu,

Guo

2024

J Sci Food Agric

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BackgroundTannic acid (TA), a naturally occurring polyphenol, has shown diverse potential in preventing intestinal damage in piglet diarrhea induced by ETEC K88. However, the protective effect of TA on ETEC k88 infection‐induced post‐weaning diarrhea and its potential mechanism has not been well elucidated. Therefore, an animal trial was carried out to investigate the effects of dietary supplementation with TA on the intestinal diarrhea of weaned piglets challenged with ETEC K88. In addition, porcine intestinal epithelial cells were used as an in vitro model to explore the mechanism through which TA alleviates intestinal oxidative damage and inflammation.ResultsThe results indicated that TA supplementation (2 g·kg‐1 and 4 g·kg‐1) reduced diarrhea rate, enzyme activity (DAO and MAD) and serum inflammatory cytokines concentration (TNF‐α, IL‐1β) (P < 0.05) compared to the IG group in vivo. In vitro, TA treatment effectively alleviated ETEC‐induced cytotoxicity, increased the expression of ZO‐1, occludin and claudin‐1 at both mRNA and protein levels. Moreover, TA pre‐treatment increased the activity of antioxidant enzymes (such as T‐SOD) and decreased serum cytokine levels (TNF‐α, IL‐1β). Furthermore, TA increased cellular antioxidant capacity by activating the Nrf2 signaling pathway and decreased inflammatory response by down‐regulating the expression of TLR4, MyD88, NF‐kB and NLRP3.ConclusionThe study showed that TA reduced the diarrhea rate of weaned piglets by restoring the intestinal mucosal mechanical barrier function, alleviating oxidative stress and inflammation. The underlying mechanismwas achieved by modulating p62‐keap1‐Nrf2 and TLR4‐NF‐κB‐NLRP3 pathway.This article is protected by copyright. All rights reserved.

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ARFGAP1 binds to classical swine fever virus NS5A protein and enhances CSFV replication in PK-15 cells

Cited by 7 publications

References 34 publications

Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

RPLP1, an NS4B-interacting protein, enhances production of CSFV through promoting translation of viral genome

Tannic acid alleviates ETEC K88‐induced intestinal damage through regulating the p62‐keap1‐Nrf2 and TLR4‐NF‐κB‐NLRP3 pathway in IPEC‐J2 cells

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