The effect of oleic acid supplementation on lipid droplet production, betaoxidation and rotavirus replication
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Date
2022
Authors
Thobane, Tshegofatso Benedict
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
Rotavirus (RV) is the most common cause of severe acute gastroenteritis in infants and young
children globally. Rotavirus infections induce cytoplasmic inclusion bodies called viroplasms
serving as a site of RV genome replication and assembly. Viroplasms recruit lipid droplets (LDs),
which play major roles in energy homeostasis and is a site for lipid storage. The successful
formation of viroplasm-LD complexes is essential for RV propagation. The extracellular supply of
oleic acid (OA, C18:1) to host cells induce LD biogenesis and modulate cellular fatty acid (FA)
metabolism. Rotavirus modulates the host cell lipidome and influence cell LD biogenesis and FA
metabolism by β-oxidation for successful viral propagation. Previous studies have shown that
chemical fragmentation of LDs by treatment with 3-isobutyl-1-methylxanthine (IBMX) and
isoproterenol (ISP) to be deleterious for RV infection. In addition, the chemical modulation of FA
β-oxidation by etomoxir (ETO) treatment has been shown to affect the propagation of other
viruses including hepatitis C virus (HCV). In this study, we sought to investigate (1) the effect of
OA supplementation on LDs during RV infection, (2) the effect of OA supplementation on FA β-
oxidation during RV infection and lastly, (3) the effect of LD and FA β-oxidation modulation on RV
infection. Briefly, HEK293 cells were supplemented with OA in the presence and absence of IBMX and ISP as well as ETO treatment during RV infection (MOI=5) for 6 h post infection (p.i). Control cells
were not treated with OA in the presence and absence of RV infection in which they were also
maintained in parallel with the test samples. At the end of each timepoint p.i, LDs where isolated
for triacylglyceride (TAG) quantification (LD quantification) while the test samples treated with the
same conditions were analyzed for changes in oxygen consumption rate (OCR) (FA β-oxidation
quantification) in HEK293 cells. For viral replication kinetics, HEK293 cells were again
supplemented with OA and treated with IBMX, ISP and ETO during RV infection as previously
describe for 0 h, 2 h, 6 h, 12 h and 16 h p.i, respectively. At the end of each time point, RV was
harvested and quantified using tissue culture infection doses 50 (TCID₅₀). Our study shows that supplementation of HEK293 cells with OA increase the overall TAG content
in isolated LDs, while the treatment of HEK293 cells with IBMX and ISP reduce TAG content of
isolated LDs. We further show an increase in total TAG content of isolated LDs when HEK293
cells are treated with ETO. Rotavirus infection increased the total TAG content of isolated LDs.
The supplementation of HEK293 cells with OA increased HEK293 cell OCR, while the treatment
with IBMX, ISP and ETO reduced the overall host cell OCR. Treatment of OA-supplemented HEK293 cells with IBMX, ISP and ETO slightly increased the overall OCR of the treated HEK293
cells compared to the OA unsupplemented HEK293 treated with the chemical inhibitors. It is also
important to note that RV infection reduced the overall OCR of HEK293 cells. Analysis of viral
kinetics showed that OA supplementation increases RV replication over time, while the treatment
of HEK293 cells with IBMX, ISP and ETO in the presence or absence of OA supplementation
reduce the overall replication of RV over time. The OA supplementation of RV infected HEK293
cells with IBMX and ISP rescues viral propagation over time, which was not observed when
HEK293 cells were treated with ETO. The data thus show that OA supplementation increases total TAG content in isolated LDs as well
as the overall host OCR while promoting RV infection over time. Rotavirus infection promotes
enrichment of LDs by increasing the overall TAG content of isolated LDs while reducing the overall
OCR of host cells. It was shown that the chemical modulation of both LD biogenesis and FA β-
oxidation is deleterious to RV propagation over time.
Description
Dissertation (M.Sc.(Microbial and Biochemical))--University of the Free State, 2022
Keywords
Rotavirus (RV), Viroplasms, Lipid droplets, ?-oxidation, Energy homeostasis, Fatty
acids (FA), Triacylglycerides (TAG), Oleic acid, 3-isobutyl-1-methylxanthine (IBMX), Isoproterenol (ISP), Etomoxir (ETO), Oxygen consumption rate (OCR), Tissue culture infection doses 50 (TCID50)