BACKGROUND: Vaccinia virus, one of the best known members of poxvirus family, has a wide host range both in vivo and in vitro. The expression of Flt3 ligand (FL) by recombinant vaccinia virus (rVACV) highly influenced properties of the virus in dependence on the level of expression. RESULTS: High production of FL driven by the strong synthetic promoter decreased the growth of rVACV in macrophage cell line J774.G8 in vitro as well as its multiplication in vivo when inoculated in mice. The inhibition of replication in vivo was mirrored in low levels of antibodies against vaccinia virus (anti-VACV) which nearly approached to the negative serum level in non-infected mice. Strong FL expression changed not only the host range of the recombinant but also the basic protein contents of virions. The major proteins - H3L and D8L - which are responsible for the virus binding to the cells, and 28 K protein that serves as a virulence factor, were changed in the membrane portion of P13-E/L-FL viral particles. The core virion fraction contained multiple larger, uncleaved proteins and a higher amount of cellular proteins compared to the control virus. The overexpression of FL also resulted in its incorporation into the viral core of P13-E/L-FL IMV particles. In contrary to the equimolar ratio of glycosylated and nonglycosylated FL forms found in cells transfected with the expression plasmid, the recombinant virus incorporated mainly the smaller, nonglycosylated FL. CONCLUSIONS: It has been shown that the overexpression of the Flt3L gene in VACV results in the attenuation of the virus in vivo.
- MeSH
- buněčné linie MeSH
- exprese genu MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- myši MeSH
- replikace viru MeSH
- vakcínie genetika metabolismus virologie MeSH
- virus vakcinie genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Vaccinia virus (VV) is considered to cause lytic infection of most cells, with lysis being regarded equivalent to necrosis. Activation of caspases has not been associated with necrosis. However, we observed the activation and activity of caspases in epithelial cells HeLa G and BSC-40 lytically infected with VV. Using three different flow-cytometric approaches, we characterized the distinct stages of caspase cascade in VV-infected cells: a cleaved, activated form of caspases detected using a fluorescent pan-caspase inhibitor; caspase activity assayed by cleavage of a non-fluorescent substrate into a fluorescent product; caspase-specific cleavage of death substrates characterized by a fluorescent antibody detecting a neo-epitope in cytokeratin-18. All of these approaches yielded an increased fluorescent signal in VV-infected cells compared to mock-infected controls. Additionally, the signal was decreased by the expression of Bcl-2. The cleavage of cytokeratin-18 was confirmed by western blotting, but another key protein involved in apoptosis, PARP, was not cleaved in VV-infected lytic cells. The necrotic phenotype of the cells was confirmed by increased cell membrane permeability and/or decreased mitochondrial membrane potential. In conclusion, our data suggest that VV infection of the epithelial cells HeLa G and BSC-40 initiates the apoptotic program, however, apoptosis is not completed and switches into necrosis.
- MeSH
- aktiny genetika metabolismus MeSH
- aktivace enzymů MeSH
- apoptóza MeSH
- Cercopithecus aethiops MeSH
- cytopatogenní efekt virový MeSH
- epitelové buňky cytologie fyziologie virologie MeSH
- fenotyp MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- kaspasy genetika metabolismus MeSH
- keratin-18 genetika metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nekróza MeSH
- permeabilita buněčné membrány MeSH
- poly(ADP-ribosa)-polymerasy genetika metabolismus MeSH
- protoonkogenní proteiny c-bcl-2 genetika metabolismus MeSH
- vakcínie patofyziologie virologie MeSH
- virus vakcinie fyziologie růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- MeSH
- hepatitida B - antigeny povrchové biosyntéza genetika imunologie MeSH
- hepatitida B - antigeny biosyntéza genetika imunologie MeSH
- kuřecí embryo MeSH
- myši MeSH
- proteinové prekurzory biosyntéza imunologie MeSH
- protilátky virové biosyntéza MeSH
- rekombinantní proteiny biosyntéza genetika imunologie MeSH
- virus vakcinie fyziologie genetika MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
