Isolation and characterisation of extracellular vesicles in baculovirus infection of insect cells

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an insect-specific virus. Its infection can be studied in Spodoptera frugiperda. It has a double-stranded DNA genome and a biphasic lifecycle, producing budded virus (BV) to infect neighbouring cells and occlusion derived virus to infect new insect hosts. The antiviral response of insects relies mainly on apoptosis and RNA interference (RNAi). In Drosophila melanogaster, following Sindbis virus infection, an RNAi signal was reportedly spread via extracellular vesicles (EVs). Extracellular vesicles incorporate proteins and nucleic acids of the cell they originate from. Proteins involved in their biogenesis are frequently used as a marker for these vesicles. When infected with a virus, the cell-derived cargo of EVs can change, and viral proteins or nucleic acids may be incorporated. Upon reaching a recipient cell the EV cargo can exert a biological function. Small EVs (sEVs) derived from infected cells can exert a pro- or antiviral response in uninfected recipient cells, as observed for herpes simplex virus 1, HIV-1 and Sindbis virus, among others. These studies have focused on viruses capable of infecting mammals. To examine if the modulation of the antiviral response via sEVs is a phenomenon more broadly exploited by viruses, the role of sEVs in AcMNPV infection of insect cells was studied here. In order to study this, sEVs had to be separated from BV. Using size exclusion chromatography combined with density gradient ultracentrifugation, the vast majority of BV was removed. However, full separation was required for functional studies. Therefore, the AcΔp6.9 bacmid was used, which does not produce new BV. Preliminary experiments showed no significant effect of sEVs from AcΔp6.9-transfected Sf9 cells on BV production, and no transfer of an RNAi signal was observed. The protein content of sEVs from both mock- and AcΔp6.9-transfected Sf9 cells was then analysed. Homologs of sEV protein markers TSG101 and syntenin-1 were identified. In sEVs from AcΔp6.9-transfected cells proteins from the mitochondrion, proteasome complex, ribosome, and involved in ubiquitination were enriched compared to sEVs from mock-transfected cells. These were presumably passively enriched, due to an increase in cellular abundance of the same proteins. Further research on the RNA in sEVs from AcΔp6.9-transfected cells, as well as in vivo studies on their role in infection, could provide more insight on the biological significance of these vesicles.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2023

Author dc:creator

• Van Es, Lex Joan Claas

Contributors dc:contributor

• Van Es, Lex J. C.
• King, Linda A.
• Possee, Robert D.

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/GDJ3-8648

OAI identifier oai:identifier

tle:5a546a8b-8f10-424f-89bf-1ca4a47c4ed5:d6bd9758-527a-46cd-bfe2-c433766e8fca:1