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

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

Buenos Aires

Congreso; ANNUAL MEETING OF BIOSCIENCE SOCIETIES 2020; 2020

SAIC

Autographa californica nucleopolyhedrovirus (AcMNPV) belongs to Baculoviridae, a family of arthropod-specific viruses. Although AcMNPV does not replicate in vertebrates, it can enter mammalian cells and express heterologous genes with mammalian promoters (gene transduction). The aim of this work was to obtain pseudotyped baculovirus to increase the transduction efficiency. To this end, wegenerated a transgenic insect cell line that constitutively expresses the G glycoprotein of vesicular stomatitis virus (VSV-G). The rationale is that baculoviruses propagated in 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. High five cells (Hi5) were transfected with pIP-vsvG and selected with puromycin to obtain a Hi5-vsvG cell line. For the transduction assays, AcMNPV-dtomato was amplified in Hi5 wild type (control) or Hi5-vsvG cells. Then, infection supernatants were cleared by centrifugation 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 transduce human liver hepatocellular cells (HepG2) and human lung adenocarcinoma cells (A549). Transfection efficiency assesed by red fluorescence showed a higher performance of AcMVPV amplified in Hi5-vsvG, compared to the control. The same conditions were tested in HEK 293T and in Vero cells, but no significant differences in transduction efficiency were found (p