Rudge, Helen Janet; (2003) Characterisation of murine gammaherpesvirus-68 alakaline nuclease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Gammaherpesviruses are associated with a number of serious diseases of man. Unfortunately, these human viruses are intractable in laboratory models but infections of mice with murine gammaherpesvirus-68 (MHV-68) can be used to study natural infections of these viruses. Open reading frame (ORF) 37 of MHV-68 is predicted to encode an alkaline nuclease. Alkaline nucleases have been found in all herpesviruses sequenced to date, suggesting that the enzyme plays an important role in the lifecycle of the virus. The aim of this study was to characterise the putative alkaline nuclease encoded by MHV-68. This was approached by using a variety of methods to examine ORF 37 from the RNA to the functional protein level. ORF 37 mRNA was found to be expressed as early as 2 hours (h) post-infection (p.i.) and forms part of an overlapping family of mRNAs as seen in herpes simplex virus (HSV). Anti-alkaline nuclease monoclonal antibodies were produced and characterised. Alkaline nuclease was found to be produced by 6h p.i. and was localised in the nucleus of MHV-68-infected NIH-3T3 cells. MHV-68 bacterial artificial chromosome (BAC) alkaline nuclease mutant and revertant viruses were characterised in vitro. Alkaline nuclease mutations caused a growth deficiency phenotype which was confirmed by complementation with the alkaline nuclease gene in trans. An alkaline nuclease activity assay was optimised and used to purify and characterise the MHV-68 enzyme. The nuclease was partially purified from infected cells. It was then expressed in Escherichia coli, both with and without a 6xHis-tag, and large quantities of highly purified protein were obtained. ORF 37 was found to be an active alkaline nuclease with similar biochemical properties to other studied alkaline nucleases. The secondary structure of the protein was studied with dynamic light scattering and circular dichroism and attempts were made to crystallise the protein. This study has advanced research in the areas of both alkaline nuclease and MHV-68 by characterising a previously unstudied protein. A large amount of pure protein has been produced and monoclonal antibodies generated against this protein are the first monoclonal antibodies directed against a specific MHV-68 protein. This study also presents one of the first characterisations of a gammaherpesvirus with a mutated alkaline nuclease gene.

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