VIDA: a virus database system for the organization of animal virus genome open reading frames

VIDA is a new virus database that organizes open reading frames (ORFs) from partial and complete genomic sequences from animal viruses. Currently VIDA includes all sequences from GenBank for Herpesviridae, Coronaviridae and Arteriviridae. The ORFs are organized into homologous protein families, which are identified on the basis of sequence similarity relationships, Conserved sequence regions of potential functional importance are identified and can be retrieved as sequence alignments. We use a controlled taxonomical and functional classification for all the proteins and protein families in the database. When available, protein structures that are related to the families have also been included. The database is available for online search and sequence information retrieval at http://www.biochem.ucl.ac.uk/bsm/virus-database/ VIDA.html

Analysis of host and herpesvirus interactions using bioinformatics.

Bioinformatics methods have become central to analysing and organising the sequence data continually produced by new and existing sequencing projects. The field of bioinformatics covers both the static aspects of organising and presenting these raw data, by compiling existing knowledge into accessible databases, ontologies, and libraries; and the more dynamic aspects of knowledge discovery informatics for interpreting and mining existing data. The aim of this thesis is to utilise such methods to analyse the herpesvirus-host relationship. In Chapter 2 comparative host and herpesvirus genome analysis is used to compare the sequences of all currently sequenced herpesvirus open reading frames to the conceptually translated human genome with the aim of identifying herpesvirus-human (host) sequence homologues. Collating in one search all currently known host homologues provides the first complete assessment of herpesvirus-host homologues. This search identified 55 previously identified herpesvirus-host homologues, and 4 previously unknown herpesvirus-host homologues. The work performed in Chapter 2 highlighted the need for consistent annotation of genomes and gene products to allow greater comparative genomics. It is not feasible to manually curate large numbers of genes whose relationships to each other are not immediately clear. Therefore, Chapters 3 and 4 focus upon the use of the Gene Ontology; a resource that is made publicly available for the purpose of annotating gene products with unified vocabulary derived from a structured directed acyclic graph. The Gene Ontology was extended to allow host-pathogen interaction annotation by a) adding 187 new terms relating specifically to virus function and structure (Chapter 3), and b) using both the new and existing terms to annotate the entire Human Herpesvirus 1 genome using references from the available literature (Chapter 4). Finally, Chapter 5 examines the utility of the Gene Ontology when analysing such large-scale host and herpesvirus gene expression datasets as produced experimentally by DNA microarray studies. Using such automated annotation methods a cluster of 12 proteins were identified that increase mitochondrial function in HUVEC cells 24 hours post HCMV infection. A cluster of nine proteins that function in the MAPK pathway were also identified using the Gene Ontology that provide evidence for HCMV inhibition of the MAPK pathway

Characterisation of the doxorubicin pump of Streptomyces peucetius. The DrrA component of the DrrAB ATP-binding cassette transporter

The emergence of antibiotic resistant microorganisms is one of the most concerning problems in health welfare, and particularly, that of multi drug resistance. The active efflux of compounds from the cell is one of the key strategies used by microorganisms to avoid the noxious effects of toxic compounds and has become one of the most important mechanisms of multiple resistance, such transport processes being catalysed by an array of membrane associated proteins. ATP-Binding Cassette Transporters, that carry out an energy dependent active transport process by which substances cross the cell membrane on the hydrolysis of ATP, also display the ability to translocate a number of unrelated molecules, including antibiotics. These transporters have been identified as one of the largest and most widely distributed families of such transmembrane transport systems. Virtually ubiquitous in nature, ABC transporters have been found in the genomes of every organism from the simplest archea through to man. The fact that they contribute the main pathway for resistance to anticancer drugs in humans highlights their importance and urgent need to study them. In addition to the well known groups of antibiotics produced by members of the Streptomyces genus, that include aminoglycosides, tetracyclines, chloramphenicol, and some (3-lactams, which inhibit the protein synthesis or the proper formation of bacterial cell walls, the species Streptomyces peucetius produces the antibiotics doxorubicin and daunorubicin. These latter compounds are classified into the anthracyclines group and possess antitumoral activity, expanding the arsenal of compounds with different activity produced by Streptomyces. The manner, in which S. peucetius avoids the effect of the antitumorals it produces, is by pumping them out of the cells, and the system that it utilises is that of an ABC transporter. In this system two subunits are present, one of them, DrrA, a peripheral membrane protein that acts as the energy-transducing component, and the other, DrrB, the membrane carrier. This type of permease carries out export of antibiotics in an ATP-dependent manner. The expression of DrrA in E. coli proved to be a challenging enterprise as only low yields were obtained for DrrA at 16 °C, that were only improved when DrrA was fused to Thioredoxin. The over expression and purification of DrrA allowed a partial characterisation of the catalytic activity of DrrA fused to Thioredoxin, with traditional biochemical methods, complemented by additional characterisation assays in wild type DrrA. The characterisation was based on assays of the ATPase activity of Thio- DrrA and DrrA, which displayed a measurable catalytic activity compatible with a role in energising transmembrane transport. These proteins were shown to be cation dependent ATPases able of binding and hydrolysing ATP in a similar manner to the NBD proteins of well-characterised prokaryotic ABC transporters, also showing common features to other NBD's