Microbial, Biochemical and Food Biotechnology

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Browsing Microbial, Biochemical and Food Biotechnology by Subject "?-oxidation"

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    The effect of oleic acid supplementation on lipid droplet production, betaoxidation and rotavirus replication
    (University of the Free State, 2022) Thobane, Tshegofatso Benedict; O'Neill, H. G.; Sander, W. J.
    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.