Abstract
BackgroundVaccinia 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.ResultsHigh 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.ConclusionsIt has been shown that the overexpression of the Flt3L gene in VACV results in the attenuation of the virus in vivo.
Highlights
Vaccinia virus, one of the best known members of poxvirus family, has a wide host range both in vivo and in vitro
Integration of soluble isoform of FL (sFL) in virions is associated with aberrant protein composition of viral particles As the P13-E/L-Flt3 ligand (FL) virions were distinct in structure from P13-H5-FL virus particles, we looked for other differences in the protein composition
It could mean that the inhibition of virus multiplication is not mediated by high level of extrinsic FL. This fact has been supported by the failure to find any Flt3 (CD135) molecules on the membrane of J774.G8 cells and by experimental addition of extrinsic Flt3L to J774.G8 cells that had no effect on the multiplication of the control P13-preS2S-βgal or P13-E7 viruses at any step of infection.The inhibition of multiplication of P13-E/LFL in macrophages might be reversible since we showed the dependence of β-galactosidase production by P13βgal-E/L-FL on the growing activity of cell cultures
Summary
One of the best known members of poxvirus family, has a wide host range both in vivo and in vitro. The expression of Flt ligand (FL) by recombinant vaccinia virus (rVACV) highly influenced properties of the virus in dependence on the level of expression. Vaccinia virus (VACV) is the best-studied member of the Orthopoxvirus genus of the poxvirus family. It has a wide host range and is able to infect cells of many different origins. As VACV highly stimulates both the innate and adaptive arms of the immune system, it was used as the vaccine for eradication of smallpox and recently, the virus has been used as a live recombinant vaccine for the induction of protective immune response against many pathogens in experimental animals. The resultant recombinant virus (rVACV) usually expresses foreign genes without remarkable impact on viral infectivity.
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