Bishop, Kate Nanette; (2001) The interactions of the viral restriction gene, Fv1, and its target. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Fvl gene controls the sensitivity of murine cells to the retrovirus Murine Leukemia Virus (MLV). This inhibition of viral replication is called restriction. Restriction is specific, with different alleles of Fvl having different activities against individual subclasses of MLV. Fvl has homology with certain murine endogenous retroviruses, and has a putative Major Homology Region (MHR) which is a domain found in the capsid protein of most exogenous retroviruses. The aim of my PhD project was to further investigate the mechanism of Fvl restriction. This thesis initially describes the development of a fast, transient assay to test Fvl function using two colour FACS analysis. Using this assay, the regions of Fvl necessary for restriction of MLV were investigated. First, stop codons were introduced into Fvl to demonstrate that Fvl functions as protein rather than RNA. Then, mix-and-match mutants were generated by shuffling the major alleles of the gene about the three positions at which they differ. Residues 358 and/or 399 were also mutated to alanine for each mutant, to study specificity further. A variety of mutations were then introduced into the Fvl gene, producing both N- and C-terminus deletions, internal deletions throughout the coding region, and point mutations in the putative MHR domain of Fvl. These Fvl derivatives were all tested for activity. A significant fraction of the Fvl protein was not required for restriction, however retention of an intact MHR and domains toward the N- and C-termini was essential. Binding specificity appeared to be a combinatorial property of a number of residues within the C-terminal portion of Fvl. Certain mutations were also found to affect the localisation pattern of the protein. Finally, this thesis includes studies to determine the effect of mutations on the solubility of Fvl. Bacterially-expressed full length Fvl is highly insoluble, complicating future biochemical and structure analysis of the Fvl-MLV interaction. All Fvl mutants tested were as insoluble as wild-type, but initial attempts to purify Fvl protein using a baculovirus expression system look promising.

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