Matrix proteins play a central role in the assembly of enveloped pleomorphic viruses. In order to study matrix protein function during assembly, cryo-electron microscopy and tomography studies were carried out on two virus families. Studies of coronavirus virus-like particles and virions revealed that the matrix protein M forms a two-dimensional network of endodornain-Iinked homodimers at the membrane. Structural differences were detected between M on membranes with rigid positive curvature and M on flaccid membranes with variable curvature. Studies of filamentous and round influenza A virus revealed structural and functional differences in the homodimeric matrix protein Ml that were associated with differences in local membrane curvature. The conformational change happened more commonly on filamentous particles than on spherical. In both virus families matrix proteins were found to control membrane bending and rigidity through a process that appears to involve ribonucleoprotein-dependent conformational changes. Both influenza Ml and coronavirus M lost rigid membrane bending activity upon exposure to pH 5.0, demonstrating that the interactions that are proposed to lead to budding can be reversed under conditions that simulate the endosomal environment. Together, these observations help to explain how matrix proteins are able to produce sufficient force to drive the budding process through homotypic and heterotypic intramolecular interactions, and how the structural changes that drive egress can be reversed to facilitate entry.

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The integrase protein from the bacteriophage ~C31 is a member of the large serine recombinase family. Purified ~C31 integrase has been shown to only catalyse site-specific recombination between the DNA sites attB and attP in vitro. Mutagenesis of the protein enabled the identification of mutants defective in recombination and some which were 'hyperactive'. Hyperactivity was defined as the ability of a mutant integrase to recombine the products of integration (attL x attR) and reform attB and attP. The primary block on this type of recombination has been shown previously to be an inability to form a stable synapse between ~C31 integrase and attL x attR. This block was noticeably absent for the hyperactive mutant E449K and synaptic complexes were detected between attL and attR. Hyperactive mutations all localised to an area of the extended C-terminus that was predicted to form a canonical coiled-coil structure. Investigation of this coiled-~oil region as a two-helix fusion protein strongly suggested that it was capable of protein-protein interactions. Defective mutations within the N-terminal domain of ~C31 integrase were positioned close to mutations known to be important in resolvase and invertase recombination. Characterisation of the mutation V129A revealed that it was destabilising synapsis and thus inferred that the N-terminus of integrase was capable of influencing synapsis. This study has therefore identified two distinct regions of integrase that playa role in the synapsis of ~C31 integrase during recombination.

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Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. The genomes of these viruses contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The activity of the RhPV 5' IRES element has previously been demonstrated in mammalian, insect and plant translation systems. It has also been shown that the RhPV 5' IRES forms 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and elFl. It is also possible to delete large regions of the 5' IRES without affecting initiation complex formation. We have now defined the minimal sequences required for directing internal initiation in mammalian, plant and insect translation systems.

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No description available.

No description available.