Veratramine inhibits porcine epidemic diarrhea virus entry through macropinocytosis by suppressing PI3K/Akt pathway.

Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is available to prevent the disease. Therefore, research for novel antivirals is important. This study aimed to identify the antiviral mechanism of Veratramine (VAM), which actively inhibits PEDV replication with a 50 % inhibitory concentration (IC50) of ∼5 µM. Upon VAM treatment, both PEDV-nucleocapsid (N) protein level and virus titer decreased significantly. The time-of-addition assay results showed that VAM could inhibit PEDV replication by blocking viral entry. Importantly, VAM could inhibit PEDV-induced phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) activity and further suppress micropinocytosis, which is required for PEDV entry. In addition, PI3K inhibitor LY294002 showed anti-PEDV activity by blocking viral entry as well. Taken together, VAM possessed anti-PEDV properties against the entry stage of PEDV by inhibiting the macropinocytosis pathway by suppressing the PI3K/Akt pathway. VAM could be considered as a lead compound for the development of anti-PEDV drugs and may be used during the viral entry stage of PEDV infection.

HDAC-Specific Inhibitors Induce the Release of Porcine Epidemic Diarrhea Virus via the COPII-Coated Vesicles.

Porcine epidemic diarrhea virus (PEDV) is an alpha-coronavirus causing acute diarrhea and high mortality in neonatal suckling piglets, resulting in huge economic losses for the global swine industry. The replication, assembly and cell egression of PEDV, an enveloped RNA virus, are mediated via altered intracellular trafficking. The underlying mechanisms of PEDV secretion are poorly understood. In this study, we found that the histone deacetylase (HDAC)-specific inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), facilitate the secretion of infectious PEDV particles without interfering with its assembly. We found that PEDV N protein and its replicative intermediate dsRNA colocalize with coat protein complex II (COPII)-coated vesicles. We also showed that the colocalization of PEDV and COPII is enhanced by the HDAC-specific inhibitors. In addition, ultrastructural analysis revealed that the HDAC-specific inhibitors promote COPII-coated vesicles carrying PEDV virions and the secretion of COPII-coated vesicles. Consistently, HDAC-specific inhibitors-induced PEDV particle secretion was abolished by Sec24B knockdown, implying that the HDAC-specific inhibitors-mediated COPII-coated vesicles are required for PEDV secretion. Taken together, our findings provide initial evidence suggesting that PEDV virions can assemble in the endoplasmic reticulum (ER) and bud off from the ER in the COPII-coated vesicles. HDAC-specific inhibitors promote PEDV release by hijacking the COPII-coated vesicles.

Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus by Disrupting Virus Entry.

The porcine epidemic diarrhea virus (PEDV), belonging to the α-coronavirus, is the causative agent of porcine epidemic diarrhea (PED). Presently, protection from the existing PEDV vaccine is not effective. Therefore, anti-PEDV compounds should be studied. Berbamine (BBM), Fangchinoline (FAN), and (+)-Fangchinoline (+FAN), are types of bis-benzylisoquinoline alkaloids that are extracted from natural medicinal plants. These bis-benzylisoquinoline alkaloids have various biological activities, including antiviral, anticancer, and anti-inflammatory properties. In this study, we found that BBM, FAN, and +FAN suppressed PEDV activity with a 50% inhibitory concentration of 9.00 µM, 3.54 µM, and 4.68 µM, respectively. Furthermore, these alkaloids can decrease the PEDV-N protein levels and virus titers in vitro. The time-of-addition assay results showed that these alkaloids mainly inhibit PEDV entry. We also found that the inhibitory effects of BBM, FAN, and +FAN on PEDV rely on decreasing the activity of Cathepsin L (CTSL) and Cathepsin B (CTSB) by suppressing lysosome acidification. Taken together, these results indicated that BBM, FAN, and +FAN were effective anti-PEDV natural products that prevented PEDV entry and may be considered novel antiviral drugs.

Porcine Enteric Coronavirus PEDV Induces the ROS-ATM and Caspase7-CAD-γH2AX Signaling Pathways to Foster Its Replication.

DNA damage response (DDR) is an evolutionarily conserved mechanism by which eukaryotic cells sense DNA lesions caused by intrinsic and extrinsic stimuli, including virus infection. Although interactions between DNA viruses and DDR have been extensively studied, how RNA viruses, especially coronaviruses, regulate DDR remains unknown. A previous study showed that the porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the Coronaviridae family, induces DDR in infected cells. However, the underlying mechanism was unclear. This study showed that PEDV activates the ATM-Chk2 signaling, while inhibition of ATM or Chk2 dampens the early stage of PEDV infection. Additionally, we found that PEDV-activated ATM signaling correlates with intracellular ROS production. Interestingly, we showed that, unlike the typical γH2AX foci, PEDV infection leads to a unique γH2AX staining pattern, including phase I (nuclear ring staining), II (pan-nuclear staining), and III (co-staining with apoptotic bodies), which highly resembles the apoptosis process. Furthermore, we demonstrated that PEDV-induced H2AX phosphorylation depends on the activation of caspase-7 and caspase-activated DNAse (CAD), but not ATM-Chk2. Finally, we showed that the knockdown of H2AX attenuates PEDV replication. Taken together, we conclude that PEDV induces DDR through the ROS-ATM and caspase7-CAD-γH2AX signaling pathways to foster its early replication.

Over-expression of a glutamate dehydrogenase gene, MgGDH, from Magnaporthe grisea confers tolerance to dehydration stress in transgenic rice.

MAIN CONCLUSION: Heterologous expression of a fungal NADP(H)-GDH gene ( MgGDH ) from Magnaporthe grisea can improve dehydration stress tolerance in rice by preventing toxic accumulation of ammonium. Glutamate dehydrogenase (GDH; EC 1.4.1.2 and EC 1.4.1.4) may act as a stress-responsive enzyme in detoxification of high intracellular ammonia and production of glutamate for proline synthesis under stress conditions. In present study, a fungal NADP(H)-GDH gene (MgGDH) from Magnaporthe grisea was over-expressed in rice (Oryza sativa L. cv. 'kitaake'), and the transgenic plants showed the improvement of tolerance to dehydration stress. The kinetic analysis showed that His-TF-MgGDH preferentially utilizes ammonium to produce L-glutamate. Moreover, the affinity of His-TF-MgGDH for ammonium was dramatically higher than that of His-TF-OsGDH for ammonium. Over-expressing MgGDH transgenic rice plants showed lower water-loss rate and higher completely close stomata than the wild-type plants under dehydration stress conditions. In transgenic plants, the NADP(H)-GDH activities were markedly higher than those in wild-type plants and the amination activity was significantly higher than the deamination activity. Compared with wild-type plants, the transgenic plants accumulated much less NH4 (+) but higher amounts of glutamate, proline and soluble sugar under dehydration stress conditions. These results indicate that heterologous expression of MgGDH can prevent toxic accumulation of ammonium and in return improve dehydration stress tolerance in rice.