The OCR protein of bacteriophage T7 is a small, dimeric protein which inhibits the cleavage of the phage DNA by type I restriction enzymes present in the infected host cell. I have studied the structure and the stability of OCR and analysed its binding to the <i>Eco</i>KI type I restriction-modification enzyme. OCR has an unique Tryptophan residue in position 94 which is solvent exposed and important for protein stability but not activity. The protein has a melting temperature of 72.19 °C and a molar extinction coefficient of 32095 M^-1 cm^-1. Asparagine 4 residue from one monomer is in close proximity to Asparagine 4 in the other monomer. Serine 68 residues are also at the monomer-monomer interface. Six surface exposed amino acids were substituted with cysteine and labelled with cysteine-specific fluorophores. The interaction between labelled OCR(Cys) proteins and <i>Eco</i>KI methylase revealed a huge surface area buried at the interface of the two proteins. OCR binds tightly to the R and S subunits of <i>Eco</i>KI and weakly to the M subunit of <i>Eco</i>KI. One OCR dimer binds to <i>Eco</i>KI methylase and two dimers to <i>Eco</i>KI nuclease. Both OCR-<i>Eco</i>KI methylase and OCR-<i>Eco</i>KI nuclease complexes have a K_d of about 10^{-11<i> </i>}M.

Acanthamoeba causes Granulomatous Amoebic Encephalitis (GAE) and Amoebic Keratitis (AK) in humans and in its cystic form is resistant to extreme environmental conditions. Both human pathogenic water-borne viruses and free-living protozoa share the same aquatic environment. This study set out to test the ability of both Acanthamoeba and Tetrahymena to internalise and protect enteric viruses; coxsackievirus (B3, B5), poliovirus (PV) and rotavirus (RV) following co-culture. Viral uptake was assessed by infection of cultured mammalian cells, by indirect immunofluorescence (IF), and by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that none of the free suspended viruses were internalised in Acanthamoeba or Tetrahymena. However, both coxsackievirus B3N and rotavirus Wa could be detected within Acanthamoeba by IF and confirmed by RT-PCR when the amoebae were co-cultured (fed) with virally infected mammalian cells. The co-cultured amoeba was allowed to encyst but following this procedure no viruses were detected either by cell culture or RT-PCR. In a second series of experiments, the efficacy of solar disinfection (SODIS) against viruses either alone or when co-cultured with Acanthamoeba was assessed. SODIS reduced the viral infectivity by over 3log10 after 1 h for CVB3N and over 2log10 for PV after 2 h. Repeating these experiments in the presence of riboflavin, a 6log10 reduction was observed for CVB3N after 1 h of light exposure and 6log10 after 6 h for all other viruses tested. The results suggest that Acanthamoeba does not internalise or protect viruses in suspension. However, if a virus is located with an infected mammalian cell then it may be internalised; a new potential mechanism for virus dissemination in the environment. Secondly, solar disinfection is an effective treatment method for water contaminated with viruses which is further enhanced by the addition of riboflavin. This study provides a practical example of low technology methods which could be utilised to provide safe drinking water in various circumstances.

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The last two decades have seen a major increase in the use of cryo-eleclron microscopy for virus reconstruction. Icosahedral virus reconstruction is particularly successful partly because the high symmetry of the structure can provide a guide to orient images via . for example. common lines. In this thesis. we introduce two improvements to t he icosahedral virus reconstruction methodology. Firstly. we propose a systematic probability-based orientation method to increase the accuracy of orientation. It will be shown that. relying on the statistical properties rather than the magnitudes of common-line residuals. the accuracy! can be improved substantially. Viruses such as adenovirus can be reconstructed straight- away without any model-based orientation refinement. Secondly_ we introduce a novel approach to identify icosahedral druses so that a) efficient icosahedral virus reconstruction methods can be used correctly on , viruses to achieve higher resolution" b) biologists ca better understand the structure and mechanism 0 1" ,viruses" This is done by decomposing common- line residuals into pair residuals and modelling them by normal mixture distributions. An innovative Markov chain Monte Carlo method. partition sampler. is design to efficiently estimate the number of components in the normal mixture distribution an hence help to identify the icosahedral viruses. The methodology ha'-e been tested on fixed viruses. Among these adenovirus. PRD1 and SFV are successfully identified as icosahedra viruses. and MMTV and HIV as non-icosahedral viruses.