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Biological evaluation of an L-dideoxy Bicyclic Nucleoside Analogue active against measles virus

Narayan, Rohan 2019. Biological evaluation of an L-dideoxy Bicyclic Nucleoside Analogue active against measles virus. PhD Thesis, Cardiff University.
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Measles wild-type virus (MeV) is the etiological agent of measles, a highly contagious life-threatening disease. We previously reported that a novel Lchiral dideoxy bicyclic pyrimidine nucleoside analogue (L-ddBCNA),hereby named cf2642, inhibited both MeV and vaccinia virus (VACV) infection in vitro, with a structure activity relationship indicating a host target. The aim of this project was to elucidate the mechanism of action by which cf2642 inhibits the MeV life cycle, focusing on endocytosis and particularly macropinocytosis that has recently been demonstrated to be exploited by this virus to gain cell entry. Initial cytotoxicity analysis of cf2642 in HeLa and Vero cells stably expressing the human Signalling Lymphocyte Activation Molecule (SLAM) receptor for MeV (VeroSLAM) demonstrated cell line dependent toxicity. The drug caused the generalized peripheral scattering of early and late endosomes/lysosomes in both cell lines, but did not affect traffic from the plasma membrane to lysosomes of the fluid phase/macropinocytosis probe dextran. MeV induced syncytia and autophagy in VeroSLAM cells was also inhibited by cf2642, indicating that another cellular target(s) was being affected. Given that cf2642 may have a cell-target(s), and that MeV and VACV use macropinocytosis as a mode of cell entry, the relationship between these two was tested. MeV stimulated macropinocytosis in VeroSLAM cells and this effect was abrogatedrapidly and completely by cf2642. In an attempt to 2 mimic the effects of MeV activation of SLAM receptors a non-infectious setting, a SLAM clustering model was developed using anti-SLAM antibodies to cluster these receptors. This caused a significant stimulation in macropinocytosis that was abolished by cf2642. Confocal microscopy highlighted both MeV and SLAM clustering induced effects on actin morphology that were also abrogated by cf2642. A virus entry/spread assay using MeV expressing GFP in combination with time of addition assays highlighted the inhibitory effect of the drug on virus spread and illustrated that a cellular target(s) that was important during the entire virus life cycle was targeted. Overall, the data indicates that cf2642 renders its antiviral activity by inhibiting MeV spread and possibly virus entry occurring via macropinocytosis. This is postulated to be via a general mechanism on plasma membrane and internal membranes that are critical for the virus life cycle.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Pharmacy
Date of First Compliant Deposit: 24 September 2019
Last Modified: 23 Jul 2020 02:08

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