Infection with Langat Flavivirus or Expression of the Envelope Protein Induces Apoptotic Cell Death

Abstract

Langat (LGT) flavivirus, derived from infectious full-length cDNA clone 636, was investigated for its apoptotic activities in mouse neuroblastoma (Neuro-2a) and simian kidney (Vero and LLC-MK(2)) cells. The hallmark of apoptosis, cleavage of cellular DNA, was observed 48 h after infection of Vero, LLC-MK(2), and Neuro-2a cells by electrophoresis analysis. Apoptosis in infected cells was also confirmed by TUNEL assay. LGT-infected Neuro-2a cells showed an increase in caspase-3-like protease (DEVDase) activity. Expression of the major envelope glycoprotein (E) alone reduced cell viability in both Vero and Neuro-2a cells, and the baculovirus P35 protein, which inhibits multiple caspases, completely blocked this effect. Cleavage of cellular DNA was observed in E gene-transfected Vero cells by TUNEL assay. Expression of E protein or caspase-9 resulted in activation of caspase-3-like proteases in Neuro-2a cells. The caspase-3-like protease specific inhibitor, Ac-DEVD-CHO peptide, partially inhibited E protein- or caspase-9-induced apoptosis in Neuro-2a cells. These observations indicate that infection of cells with LGT virus or expression of LGT virus E protein induces apoptosis through a caspase-3-like protease pathway.

Caspase 3-Dependent Cell Death of Neurons Contributes to the Pathogenesis of West Nile Virus Encephalitis

“…Flaviviruses can induce cell death directly through viral replication and the production of proapoptotic proteins (41,43,44,52,57). Notably, pancaspase and caspase 3 inhibitors reduced the WNV-mediated death of primary neurons, which is consistent with studies showing that caspase inhibitors reduced apoptosis after transgenic expression of the flavivirus NS3 and E genes (41,42,44). Caspase-dependent apoptosis of neurons in the CNS may be a general mechanism by which neuroinvasive flaviviruses induce injury.…”

Langat Flavivirus Protease NS3 Binds Caspase-8 and Induces Apoptosis

“…Recently, we reported that infection with LGT virus or expression of the envelope glycoprotein (E) induces apoptosis through a caspase-3 like protease pathway (25). Here we show that the LGT virus NS3 protein binds to caspase-8 and induces apoptosis.…”

“…However, apoptosis needs to be delayed long enough for the virus to complete its replication cycle and to produce sufficient quantities of progeny (reviewed in reference 28). Prior experiments showed that infection of Vero and Neuro-2a cells with the LGT virus resulted in release of infectious virus at 24 h in both cell lines, although LGT virus replicates to peak titers in Vero cells that are about 10-fold higher than those in Neuro-2a cells (25). These differences might be important for LGT virus infection in different cell types in vivo but may be less important for cells in culture.…”

“…Neuro-2a, Vero, and 293T cells were transfected with the NS3 plasmid, and protein expression was confirmed by immunofluorescence using LGT virus-specific hyperimmune mouse ascitic fluid. The protein was visualized 18 h after transfection in the cytoplasm of the cells and had a diffuse staining pattern as previously described for the NS3 and NS2B-NS3 proteins (25,36). Furthermore, the identical electrophoretic mobilities of the NS3 proteins expressed in both the NS3-transfected and the LGT virus-infected Neuro-2a cells were confirmed by Western blot analysis with anti-NS3 MAb (data not shown).…”

“…In inhibition experiments, caspase substrates (50 M) Z-IETD-FMK, Z-LEHD-FMK, and Z-VAD-FMK (R&D Systems, Minneapolis, Minn.) were added immediately after transfection and 24 h later to the culture medium to prevent apoptosis. At various times, cells were fixed and stained, and the percentage of apoptotic cells relative to vector control (pCI)-transfected cells, set at 0%, was calculated as described previously (24,25). Each data point represents the results of three independent experiments, and error bars indicate the standard deviations.…”