Characterisation of an interaction involved in viral replication : submitted in partial fulfilment of the requirements of the degree of Doctor of Philosophy, Institute of Fundamental Sciences, Massey University
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Date
2010
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Massey University
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Abstract
Human rhinoviruses (HRVs) are a major cause of illness worldwide and, as members of Picornaviridae,
are closely related to several other human and animal pathogens that exact a large
medical and economic cost on society. Viral infections in general are particularly difficult to treat,
as viruses co-opt many of the host’s own biochemical pathways, making disabling the virus
without harming the host very difficult. Carefully targeted strategies are required and detailed
structural information is useful, both to identify new drug targets, and to fully understand interactions.
One particular protein expressed by picornaviruses is 3C protease, which is responsible
for post-translational processing of the viral capsid. This protease has a cysteine as its active site
nucleophile, a functionality not found in eukaryotic proteases. The unusual active site makes
3C an attractive target for pharmaceuticals. Drugs that block the proteolytic action of 3C are
currently in clinical trials. In addition to its proteolytic activity, 3C protease also has another
function, that of an RNA binding protein. This activity has been shown to be required during
replication of the viral RNA genome. In this study, the structure of 3C protease from HRV14 is
investigated using NMR and other biophysical techniques. The structural information gained
from these studies is used, along with data on 3C protease RNA-binding activity acquired using
solution-state NMR and SAXS data, to elucidate a structure of the 3C–RNA complex. In addition,
the dynamics of the free protein and of the protein in the presence of a specific inhibitor
are investigated by solution-state NMR, and the potential role of dynamics in the function of the
protein is explored. Finally, potential allosteric interaction between the RNA-binding and proteolytic
functions of 3C is postulated, and further interactions of 3C and the 3C–RNA complex are
discussed. It is hoped that a more complete understanding of 3C and its interactions will lead to
more effective treatments for picornaviral infections in the future.
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Keywords
Human rhinoviruses (HRVs), Picornaviridae, Picornaviruses, 3C protease