Rotaviruses are a leading cause of acute gastroenteritis in infants and young children worldwide and possess a genome of 11 double-stranded (ds) RNA segments. Early morphogenesis of RV particles and viral RNA replication occur in cytoplasmic inclusion bodies called ‘viroplasms’, of which the viral non-structural proteins NSP2 and NSP5 are essential components. Using confocal microscopy (CM), we demonstrated association of viroplasms with lipids and lipid droplet (LD)-associated proteins (perilipin A, ADRP). LD-associated proteins were found to co-localise with viroplasm-like structures (VLS) in cells co-transfected with NSP2- and NSP5- expressing plasmids in the absence of rotavirus infection, as well as viroplasms containing NSP5-EGFP. Close spatial proximity between NSP5-EGFP and perilipin A was demonstrated by Fluorescence Resonance Energy Transfer (FRET). Time course CM studies showed increasing recruitment of perilipin A to viroplasms during the progression of rotavirus infection. Separation of RV-infected cell extracts on iodixanol gradients demonstrated co-localisation of rotavirus dsRNA, NSP5 and perilipin A in low density fractions. Chemical compounds interfering with LD formation or homeostasis (isoproterenol + IBMX, triacsin C, C75) decreased the number and size of viroplasms in rotavirus-infected cells, dsRNA replication and the production of infectious progeny virus, whilst significantly protecting cells from cytopathicity caused by rotavirus infection. These data contribute to a novel understanding of the role of LDs in rotavirus replication. Using a cell line stably expressing NSP5-EGFP, the dynamics of viroplasm formation in relation to dsRNA replication and production of infectious virus was investigated.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:597592 |
| Date | January 2011 |
| Creators | Cheung, W. K. S. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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