Strategy to improve tropism and transduction efficiency of baculoviruses in mammalian cells.

AMORÓS MORALES LESLIE C.; FABRE MARÍA LAURA; PIDRE MATIAS LUIS; URE AGUSTÍN E.; CANDOLFI MARIANELA; ROMANOWSKI VÍCTOR

Buenos Aires

Congreso; LXV REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2020

SAIC

Autographa californica nucleopolyhedrovirus (AcMNPV) belongs to Baculoviridae, afamily of arthropod-specific viruses. Although AcMNPV does not replicate invertebrates, it can enter mammalian cells and express heterologous genes withmammalian promoters (gene transduction). The aim of this work was to obtainpseudotyped baculovirus to increase the transduction efficiency. To this end, wegenerated a transgenic insect cell line that constitutively expresses the G glycoproteinof vesicular stomatitis virus (VSV-G). The rationale is that baculoviruses propagatedin these cells would acquire VSV-G inserted in the envelope of budding virions.The ORF encoding VSV-G was cloned into the expression plasmid pIP-vsvG. Highfive cells (Hi5) were transfected with pIP-vsvG and selected with puromycin to obtaina Hi5-vsvG cell line. For the transduction assays, AcMNPV-dtomato was amplified inHi5 wild type (control) or Hi5-vsvG cells. Then, infection supernatants were cleared bycentrifugation and incubated with mammalian cells. Finally, viral plaques were countedby fluorescence microscopy.Initially, we verified the presence of G protein in Hi5-vsvG cells byimmunofluorescence. Then, we used the virus propagated in these cells to transducehuman liver hepatocellular cells (HepG2) and human lung adenocarcinoma cells(A549). Transfection efficiency assesed by red fluorescence showed a higherperformance of AcMVPV amplified in Hi5-vsvG, compared to the control. The sameconditions were tested in HEK 293T and in Vero cells, but no significant differences intransduction efficiency were found (p