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Dual NDP52 Function in Persistent CSFV Infection.

ABSTRACT: Viruses have evolved many mechanisms to escape host antiviral responses. Previously, we found that classical swine fever virus (CSFV) infection induces autophagy using the autophagosome as a self-replication site, thereby evading the host immune response and promoting long-term infection. However, the underlying mechanisms used by CSFV to enter autophagosomes and the mechanism by which autophagy promotes viral replication remain unclear. We found that CSFV infection inhibited autophagy receptor nuclear dot protein 52 kDa (NDP52) expression, ubiquitination, and SUMO2-4 modification. Further analyses revealed that CSFV mediated ubiquitination and SUMOylation of NDP52 via Pten-induced kinase 1 (PINK1)-Parkin. Moreover, NDP52 inhibition also inhibited CSFV replication and the induction of mitophagy marker proteins expression. Inhibition of NDP52 reduced CD63 expression and binding to CSFV E2 protein, which has an essential role in persistent CSFV infection. As NDP52 has a close relationship with the NF-κB innate immunity pathway and plays an important role in the antiviral response, we investigated whether NDP52 inhibited CSFV replication through the release of immune factors and antivirus signals. Our results showed that inhibiting NDP52 boosted interferon and TNF release and promoted NF-κB pathway activation. In summary, we found that NDP52 inhibition not only reduces CSFV binding and entry into autophagic vesicles, but also inhibits CSFV replication by active NF-κB antiviral immune pathways. Our data reveal a novel mechanism by which NDP52, an autophagy receptor, mediates CSFV infection, and provide new avenues for the development of antiviral strategies.

SUBMITTER: Fan S

PROVIDER: S-EPMC6960106 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cellular metabolism caters to the energy and metabolite needs of cells. Although the role of the terminal metabolic enzyme LDHB (lactate dehydrogenase B) in the glycolysis pathway has been widely studied in cancer cells, its role in viral infection is relatively unknown. In this study, we found that CSFV (classical swine fever virus) infection reduces pyruvate levels while promotes lactate release in pigs and in PK-15 cells. Moreover, using a yeast two-hybrid screening system, we identified LDHB as a novel interacting partner of CSFV non-structural protein NS3. These results were confirmed via co-immunoprecipitation, glutathione S-transferase and confocal assays. Furthermore, knockdown of LDHB via interfering RNA induced mitochondrial fission and mitophagy, as detected reduced mitochondrial mass. Upon inhibition of LDHB, expression of the mitophagy proteins TOMM20 and VDAC1 decreased and the ubiquitination of MFN2, a mitochondrial fusion mediator, was promoted. In addition, a sensitive dual ﬂuorescence reporter (mito-mRFP-EGFP) was utilized to analyze the delivery of autophagosomes to lysosomes in LDHB inhibition cells. Furthermore, LDHB inhibition promoted NFKB signaling, which was regulated by mitophagy; meanwhile, infection with CSFV negated these NFKB anti-viral responses. Inhibition of LDHB also inhibited apoptosis, providing an environment conducive to persistent viral infection. Finally, we demonstrated that LDHB inhibition promoted CSFV growth via mitophagy, whereas its overexpression decreased CSFV replication. Our data revealed a novel mechanism through which LDHB, a metabolic enzyme, mediates CSFV infection, and provides new avenues for the development of anti-viral strategies.Abbreviations: 3-MA:3-methyladenine; CCCP:carbonyl cyanide 3-chlorophenylhydrazone; CCK-8:cell counting kit-8; CSFV:classical swine fever virus; DAPI:4',6-diamidino-2-phenylindole; DMSO:dimethyl sulfoxide; EGFP:enhanced green fluorescent protein; FBS:fetal bovine serum; FITC:fluorescein isothiocyanate; GST:glutathione-S-transferase; HCV:hepatitis C virus; IFN:interferon; LDH:lactate dehydrogenase; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MFN2:mitofusin 2; MOI:multiplicity of infection; NFKB:nuclear factor kappa B subunit 1; NFKBIA:nuclear factor inhibitor alpha; NS3:nonstructural protein 3; NKIRAS2:NFKB inhibitor interacting Ras like 2; PRKN:parkin E3 ubiquitin protein ligase; PBS:phosphate-buffered saline; qRT-PCR:real-time quantitative reverse transcriptase polymerase chain reaction; RELA:RELA proto-oncogene, NF-kB subunit; shRNA: short hairpin RNA; siRNA: small interfering RNA; TCID50:50% tissue culture infectious doses; TEM:transmission electron microscopy; TNF:tumor necrosis factor; TOMM20:translocase of outer mitochondrial membrane 20; VDAC1:voltage dependent anion channel 1.

Project description:BackgroundPersistent concussion symptoms (PCS) negatively affects common activities of daily living including deficits in both single and dual-task (DT) gait. DT gait deficits are present post-concussion; however, task prioritization and the effects of differing cognitive challenge remain unexplored in the PCS population.Research questionThe purpose of this study was to investigate single and dual-task gait performance in individuals with persistent concussion symptoms and to identify task priorization strategies during DT trials.MethodsFifteen adults with PCS (age: 43.9+11.7 y.o.) and 23 healthy control participants (age: 42.1+10.3 y.o.) completed five trials of single task gait followed by fifteen trials of dual task gait along a 10-m walkway. The cognitive challenges consisted of five trials each of visual stroop, verbal fluency, and working memory cognitive challenges. Groups were compared on DT cost stepping characteristics with independent samples t-test or Mann-Whitney U tests.ResultsThere were significant overall gait Dual Task Cost (DTC)difference between groups for gait speed (p = 0.009, d=0.92) and step length (p = 0.023, d=0.76). Specific to each DT challenge, PCS participants were slower during Verbal Fluency (0.98 + 0.15 m/s and 1.12 + 0.12 m/s, p = 0.008; d=1.03), Visual Stroop (1.06 + 0.19 m/s and 1.20 + 0.12 m/s, p = 0.012, d=0.88), and Working Memory (1.02 + 0.15 m/s and 1.16 + 0.14 m/s, p = 0.006, d=0.96). There were significant cognitive DTC differences between groups for WM accuracy (p = 0.008, d=0.96), but not for VS accuracy (p = 0.841, d=0.061) or VF total words (p = 0.112, d=0.56).SignificanceThe PCS participants displayed a posture-second strategy whereby gait performance generally decreased in the absence of cognitive changes. However, during the Working Memory DT, PCS participants had a mutual interference response whereby both motor and cognitive performance decreased suggesting the cognitive task plays a key role in the DT gait performance of PCS patients.

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Dual NDP52 Function in Persistent CSFV Infection.

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