Auflistung nach Autor:in "Lane, Miranda Sophie"

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Reporter-based reverse genetics of murine norovirus reveals insertion-tolerant sites and a position in the capsid linked to antibody escape
(2026) Lane, Miranda Sophie
Noroviruses are members of the Caliciviridae family: positive-sense, single-stranded RNA encased within a 90 dimer protein capsid. Human norovirus (HNoV) is a major cause of gastrointestinal disease. Murine norovirus (MNV) is used as a surrogate in the study of HNoV due to it being the only norovirus currently reliably cultivatable in cell culture and possessing a reverse genetics system. Previous studies have used MNV to develop our understanding of norovirus capsid protein structure with regards to infection. Various ligands have been observed to bind the norovirus capsid including divalent cations, neutralising antibodies, bile acids, and a cellular receptor mCD300lf (MNV only). The effect of bile acids on MNV infection was previously investigated in this lab but recent research suggests dynamic conformational changes observed in the MNV capsid are due to environmental factors such as bile acid binding, pH change and divalent cation binding. This study set out to determine the effects of divalent cation binding with regards to infection. Multiple reverse genetics techniques have been developed for MNV with an RNA-based technique in a recombinant cell line developed within the last few years. This technique allows for the indirect observation of protease production via a GFP reporter system. This work compared wild‑type MNV‑1.CW1 RNA transfections with transfected MNV‑1.CW1 RNA with mutations introduced to a cDNA plasmid construct via site-directed mutagenesis. This system was used in the testing of the non‑structural (NS) polyprotein, protease cleavage sites with the aim of identifying potential sites for the insertion of a short protein-coding sequence, and to generate recombinant mutant MNVs used in the study of divalent cations. Transfection experiments of MNV‑1.CW1 RNA with an HNoV protease cleavage site identified a potential site of NS1‑2/NS3 as being suitable for a potential inserted protein, with the site receptive to a short peptide coding sequence insertion. Five recombinant MNV‑1.CW1s with mutations in the major capsid protein were generated. The generation of two divalent cation non-binding mutants (D410A and D440A) and the extensive testing of these in cell culture assays identified D440 as a crucial site of cation binding, preventing the neutralisation of the virus with a monoclonal antibody. Previous work identifying bile acid in preventing neutralisation from monoclonal antibodies was revaluated, this time controlling for divalent cations. Divalent cations were found to be essential in small concentrations – that alone could not prevent neutralisation – but assisted bile acid in neutralisation escape.