Molecular and cellular biology of human enteroviruses : cell signalling, recombination and replication

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Enteroviruses are small, positive-stranded, RNA viruses that generally cause acute infections in humans. The prototype enterovirus, poliovirus ? the aetiological agent of paralytic poliomyelitis ? has been extensively studied over the last two decades and is a model for our understanding of related human pathogens, and the cellular processes these viruses subvert during cell infection. The replication cycle of the virus is initiated by receptor-mediated entry, occurs on membrane-bound cytoplasmic vesicles, and leads to cytolysis and release of progeny virions within eight hours. This proposal seeks support for advanced studies of three aspects of the biology of human enteroviruses ? the role of cell signalling in virus entry, the characterisation of cis-acting replication elements (CREs) and the molecular for intraspecific recombination.

Many non-poliovirus enteroviruses use decay accelerating factor (DAF) as a receptor to bind and enter cells. DAF is known to transduce signals to the cell, and we have demonstrated that inhibiting this cell signalling blocks infection at an early stage. We hypothesise that signalling is required for virus entry to an endocytic vesicle and propose to test this using classical virologic approaches, confocal microscopy, live cell imaging and biochemical analysis. These studies will provide insights into a little understood aspect of the life cycle of enteroviruses.

We have previously identified a novel CRE in the enterovirus genome and present bioinformatic and experimental data to suggest poliovirus and related species C enteroviruses (but not species A, B or D) have two additional, previously unsuspected, CREs of undefined function. We will use a range of reverse genetic and biochemical approaches, including in vivo studies in transgenic mice, to determine the role of these regions in virus replication. As well as providing important information on enterovirus replication, these studies may highlight features of replication mechanisms shared by other ? less tractable to analyse ? positive strand RNA viruses.

Enteroviruses recombine frequently, generating novel combinations of structural and non-structural proteins. In preliminary studies we demonstrate an engineered recombinant between poliovirus and a coxsackie A virus is blocked at a late stage in the life cycle e.g. particle morphogenesis or encapsidation. We propose to engineer or select a range of recombinants to identify the requirements for viability. Since recombination is a major evolutionary mechanism these studies will define genomic regions that may interact, thereby contributing to our understanding of enterovirus evolution and cis-acting functions within the genome.