Enteroviral 2B Interacts with VDAC3 to Regulate Reactive Oxygen Species Generation That Is Essential to Viral Replication.

Enterovirus (EV) 71 caused episodes of outbreaks in China and Southeast Asia during the last few decades. We have previously reported that EV71 induces reactive oxygen species (ROS). However, the underlying mechanism remains elusive. Co-immunoprecipitation-proteomic analysis revealed that enteroviral 2B protein interacted with mitochondrial voltage-dependent anion channel 3 (VDAC3). Knockdown (KD) of VDAC3 expression specifically inhibited enteroviral replication. Single-round viral replication was also inhibited in KD cells, suggesting that VDAC3 plays an essential role in replication. Consistent with this, VDAC3 gene KD significantly reduced the EV71-induced mitochondrial ROS generation. Exogenous 2B expression could induce the mitochondrial ROS generation that was significantly reduced in VDAC3-KD cells or in the Mito-TEMPO-treated cells. Moreover, VDAC3 appears to be necessary for regulation of antioxidant metabolism. VDAC3 gene KD led to the enhancement of such pathways as hypotaurine/taurine synthesis in the infected cells. Taken together, these findings suggest that 2B and VDAC3 interact to enhance mitochondrial ROS generation, which promotes viral replication.

Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection.

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl α-ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

Mitomycin C modulates intracellular matrix metalloproteinase-9 expression and affects corneal fibroblast migration.

Mitomycin C (MMC) is often used to prevent postoperative corneal haze and subconjunctival fibrosis in ocular surgery. It also affects the motility and viability of the residual ocular cells, including corneal stromal cells. Extracellular matrix metalloproteinase-9 (MMP-9) contributes to the promotion of cell movement in macrophage and cancer cells, but the intracellular role of MMP-9 remained unclear. Herein, we illustrated the novel role of intracellular MMP-9 in MMC-suppressed cell migration using isolated human corneal fibroblasts (HCFs). In HCFs, MMC enhanced intracellular MMP-9 at transcriptional and protein levels. Using co-immunoprecipitation analysis, we confirmed that MMC enhanced the association between intracellular MMP-9 and inactive FAK/paxillin (PXN) complexes, i.e. PXN without phospho-tyrosine 118 (pY118) and FAK without phospho-tyrosine 397 (pY397). To verify the role of intracellular MMP-9 in migration, its gene was directly isolated from HCFs and highly expressed in HCFs by a lentivirus-based pseudovirus system with encephalomyocarditis virus (EMCV)-driven enhanced green fluorescent protein (GFP) as the MMP-9-IG-versus IG-expressing cells. Compared with the IG-expressing cells, higher intracellular MMP-9 expression in the MMP-9-IG-expressing HCFs proliferated and migrated more slowly. Phosphorylation of FAK at Y397 and PXN at both Y31 and Y118 were significantly less in the MMP-9-IG-expressing HCFs. These suggested that MMC-upregulated intracellular MMP-9 clutched inactive FAK/PXN complexes at focal adhesion sites to form a new "inactive" trimer, prohibited FAK/PXN complexes phosphorylation and retarded corneal fibroblast migration.

Mitomycin C retardation of corneal fibroblast migration via sustained dephosphorylation of paxillin at tyrosine 118.

PURPOSE: To investigate how mitomycin C (MMC) modulates corneal fibroblast migration and its molecular mechanisms in the wound healing process. METHODS: After treatment with 0 and 0.2 mg · mL(-1) MMC for 5 minutes, effect of MMC on cell migration of human corneal fibroblasts (HCFs) was examined with a cell migration assay. Both focal adhesion kinase (FAK) and paxillin (PXN) expressions in HCFs were analyzed by semiquantitative real-time PCR, immunoblotting, and immunofluorescence confocal microscopy. Using gene silencing or gene overexpression with lentiviral-based pseudovirion infection, the phosphorylation level of FAK, PXN, and mutated PXNs at tyrosine sites 31 (Y31F-EGFP) and 118 (Y118F-EGFP) were verified in HCFs. RESULTS: MMC retarded HCF migration at 1 and 2 days posttreatment (dpt). MMC reduced levels of FAK transcript and FAK protein, but increased both transcript and protein expression of PXN at 1 and 2 dpt. Furthermore, MMC upregulated FAK-pY397, which subsequently enhanced PXN-pY31 in a dose-dependent manner at 1 dpt. Concurrently, MMC downregulated PXN-pY118 at 1 dpt. However, MMC treatment resulted in dephosphorylation of FAK-pY397, PXN-pY31, and PXN-pY118 at 2 dpt. The FAK/PXN complex in MMC-treated HCFs was detected at focal adhesion sites more than at the leading edge at 1 and 2 dpt, contributing to retardation of HCF migration. Y118F-EGFP-expressing HCFs exhibited lower mobility than that of PXN-EGFP- or Y31F-EGFP-expressing HCFs. CONCLUSIONS: The sustained PXN-pY118 dephosphorylation resulted in steadfastness of an incompletely active FAK/PXN complex at focal adhesion sites and played a pivotal role in MMC-retarded HCF migration.