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Coronaviruses are the causative agents of various mammalian diseases which have crucial economic and health-related problems and are mainly respiratory and gastrointestinal pathogens. They are positive strand RNA viruses which may require nuclear functions for replication. The nucleocapsid (N) protein of s~veral members has previously been shown to localise to the nucleus/nucleolus and the cytoplasm during infection and after transient transfection. The coronavirus N protein is a viral RNA binding protein with several functions during the virus life cycle, especially with regard to RNA replication and transcription and controlling cell signalling pathways. In order to localise to the cytoplasm and nucleus/nucleolus N protein must contain appropriate trafficking motif(s). This thesis focused on characterising the newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV) N protein. The sub-cellular localisation of the SARS-CoV N protein was determined· in virus infected and transfected cells using antibody labelling and C-terminally tagged fluorescent fusion proteins, respectively. Comparison with the avian coronavirus N protein indicated that in contrast to other coronavirus N proteins, SARS-CoV N protein localised mainly in the cytoplasm with low frequency localisation in the nucleolus. Live cell, confocal microscopy and fluorescence loss in photo-bleaching (FLIP) was used to investigate the presence of any potential trafficking signals. Based on amino acid sequence conservation with the other coronavirus N proteins the SARS-CoV N protein was divided into three regions and mutation and bioinformatic analysis was used to potential nuclear import and export motifs. This approach delineated a cryptic nucleolar localisation signal in the central portion of the protein and a novel nuclear export signal in the C-terminal part, which may be the predominant trafficking signal. These motifs may be exposed by differential phosphorylation. The N protein was expressed in vitro and experiments formally demonstrated that it was a phosphoprotein which could bind viral RNA and an RNA binding region spanned the N-terminal and central·part ofthe protein. Together, the data in this study has provided insights into the expression and sub-cellular localisation ofthe SARS Cov N protein.

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Human Immunodeficiency virus type-l (HIV-l), has a number of regulatory genes in addition to the gag, pol, and env that are common to all replication competent retrovimses. It expresses six auxilialY proteins, tat, rev, Vi/, vpu, vpx and nef. Vif (Viral Infectivity Factor) is a 23 kDa basic protein of 192 aa. Vif has been shown to be essential for the modulation of virion infectivity in nom1al host cells and is believed to function by interacting with both viral and cellular proteins. More recent studies have focused on its involvement in controlling the encapsidation of cellular proteins ~f the APOBEC3 family. However earlier work of Vif suggested an involvement in viral morphogenesis and this was the main focus of the present study. Vifhas been shown to be associated with the viral nucleocapsid and to be specifically packed into HIV patiicles, either by interaction with viral RNA and/or Gag and GagPol precursors. This study had as its primary aim definition of the molecular interactions of Vif with the Gag precursor (Pr55GAG ), the viral Protease (RR), and the antiviral cellular proteins APOBEC3G/3F. An in vivo mammalian two-hybrid assay was used to study the interactions between Vif and both Pr55GAG and the viral PR. This found that Vif interacts with Pr55GAG • To begin mapping the positions of this interaction, a series of mutations were made in both proteins. Complimenting previous studies on Vif done at Warwick, amino acid 21 was found to be cmcial for the interaction between Vif and Pr55GAG in the mammalian two-hybrid assay. Interaction between Vif and Pr55GAG was fmiher confirmed using an independent in vitro GST pull-down assay. No interactions were found between Vif and PR and between PR and Pr55GAG • The second objective of this study was to analyse the molecular interactions of HIV-1 Vif, Pr55GAG , and PR with the APOBEC3 family of cellular proteins using both an in vivo mammalian two-hybrid assay and an in vitro GST pull-down assay. In the mammalian two-hybrid assay a direct interaction between Pr55GAG and both APOBEC3G and 3F was identified. These interactions were further confirmed using the GST pull-down assay. A direct interaction between Vif and the two APOBEC proteins APOBEC3G and 3F was also seen in vitro in the GST pull-down assay; however, these interactions could not be seen in vivo using the mammalian two hybrid assay. A third are~ of work was concemed with the high level expression of Vifin a bacterial expression system and purification of the protein for structural studies. This experimental work was complemented by computer based model building using a comparative modeling method. The aim of this work being to produce an atomic level resolution model for Vifwhich could be tested against the experimental results achieved in interaction site mapping studies. Building on earlier work done at Warwick a fourth area of this study involved in vivo experiments aimed at understanding the role of HIV-1 Vif iil resistance to protease inhibitors (Adekale et al., 2005). This involved establishing the molecular reagents to allow the generation of infectious molecular clones carrying various variants of Vifand the HIV-1 protease. Plasmid constructs were generated to allow the inse11ion of different variants of the PRgene into an infectious molecular clone building on the previously available strategy which allowed similar exchanges with the Vifgene.

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Soluble and virion-associated antigens of vaccinia virus (an Orthopoxvirus), myxoma virus (a Leporipoxvirus) and molluscum contagiosum virus (an unclassified poxvirus) were studied using immunodiffusion and virus neutralisation tests. Virion-associated antigens were prepared by extraction of virus for NP. antigen (Smadel, Rivers and Hoagland 1942), or by extraction at pH 10.5 (A. antigen), followed by NP. extraction (NP.1 antigen). Technical factors greatly affected the results of immunodiffusion tests. Serologically non-specific interference with precipitin line extension was demonstrated in comparative, template immunodiffusion tests. The antigen preparations were all heterogeneous and the antigenic composition of vaccinia soluble and A. antigens varied with the virus growth system. The antigenic fractions of vaccinia NP. antigen were also identified in vaccinia NP.1 antigen and in some, but not all, preparations of vaccinia soluble and A. antigens. The soluble antigens of vaccinia and myxoma viruses contained a common antigenic fraction which was unrelated to vaccinia NP. antigen. A common fraction was also identified in vaccinia NP. and myxoma NP.1 antigens. Limited data suggested restricted cross-reactivity between molluscum antigens and vaccinia soluble and NP. antigens. The three viruses did not show unequivocal cross-reactivity in virus neutralisation tests. Normal rabbit sera reacted with certain antigens of all three viruses and with extracts of human skin. This activity was strongest in tests with myxoma antigens, and was interpreted in terms of natural antibodies. Electron microscopic studies identified at least three putative subpopulations of virions in the virus preparations. Morphological changes were not detected following extraction of virus at pH 10.5, but virions were disrupted by NP. extraction.