Theoretical studies of viral capsid proteins.

Recent results in structural biology and increases in computer power have prompted initial theoretical studies on capsids of nonenveloped icosahedral viruses. The macromolecular assembly of 60 to 180 protein copies into a protein shell results in a structure of considerable size for molecular dynamics simulations. Nonetheless, progress has been made in examining these capsid assemblies from molecular dynamics calculations and kinetic models. The goals of these studies are to understand capsid function and structural properties, including quarternary structural stability, effects of antiviral compounds that bind the capsid and the self-assembly process. The insight that can be gained from the detailed information provided by simulations is demonstrated in studies of human rhinovirus; an entropic basis for the antiviral activity of hydrophobic compounds, predicted from calculated compressibility values, has been corroborated by experimental measurements on poliovirus.

Molecular dynamics investigation of the effect of an antiviral compound on human rhinovirus.

The factors that influence the enhanced stability observed experimentally of human rhinovirus 14 (HRV14) upon binding a hydrophobic antiviral drug have been investigated by molecular dynamics. Simulations centered about the HRV14 drug-binding pocket allow the reliable assessment of differences in capsid protein motions of HRV14 and drug-bound HRV14. We propose that the experimentally observed stabilization of the ligated virus arises from higher entropy, rather than enthalpy. Time-averaged interaction energies between the viral protein and molecules occupying the pocket are less favorable in the presence of the drug, consistent with the proposal that the observed stability arises from entropic effects. Interaction energies characterizing subunit-subunit contacts within one viral protomer are found to be substantially stronger than those between two protomers. Such distinction in subunit interaction would have clear implications on assembly and disassembly. Drug binding is found to affect large-scale, collective properties, while leaving local atomic properties unperturbed. Specifically, the simulations reveal a weakening of long-range correlations in atomic motions upon drug binding. On the other hand, neither the fast time scale RMS fluctuations of individual atomic positions nor the fluctuation build-up curves from the capsid beta-sandwich forming the drug-binding pocket show a consistent distinction between the drug-bound and drug-free viral simulations. Collectively, the detailed description available from the simulations provides an understanding of the experimental observations on the drug-induced changes in thermal stability and protease sensitivity reported for picornaviruses. The predicted significance of binding entropy can be explored experimentally and should be considered in the design of new antiviral compounds.

Influence of an antiviral compound on the temperature dependence of viral protein flexibility and packing: a molecular dynamics study.

The antiviral activity of compounds that bind an internal pocket of picornaviruses is due in part to stabilization of the protein capsid and inhibition of the uncoating process required for virus replication. Information on the basis for this structural stabilization of the virus capsid is important to elucidate the mechanism of antiviral action and provide insights into the disassembly process. It has been proposed that this stabilization is entropically based, since binding the nonpolar antiviral compound increases the compressibility, and thus the conformational flexibility, of the virus. Such a proposal predicts a difference in the temperature dependence of the atomic positional fluctuations for free virus and drug-bound virus; nonpolar interactions are weaker and less directional, and would give rise to greater conformational disorder at low temperature. Further, the transition that has been observed in globular proteins to a state resembling a frozen liquid, in which the protein is considered "trapped" in potential energy wells, is predicted to occur at lower temperature when the antiviral compound is bound. Results described here from computer simulations of rhinovirus over a range in temperature show these predicted changes in conformational disorder and the temperature of the transition in mobility. In addition to providing independent support for the above proposal for antiviral activity, these results indicate that the mobility transition of a protein can be controlled by the binding of an appropriate ligand, an effect not previously reported.

A novel basis of capsid stabilization by antiviral compounds.

Picornaviruses are inactivated by a family of hydrophobic drugs that bind at an internal site in the viral capsid and inhibit viral uncoating. A basis for the capsid stabilization previously unrecognized is revealed by molecular dynamics simulations of the antiviral drug WIN52084s bound to a hydrophobic pocket of solvated human rhinovirus 14. Isothermal compressibilities of the complex and human rhinovirus 14 without the antiviral drug calculated from density fluctuations show that the presence of WIN52084s increases the compressibility of the viral capsid near the antiviral drug. This counterintuitive result is understandable on the basis of the empirical evidence of thermal melting temperatures and protein-folding entropies of globular proteins. Based on this evidence, we propose that a larger compressibility from drug binding confers greater thermal stability to capsid proteins by increasing the conformational entropy of capsids, thereby diminishing the entropy gain with uncoating. We suggest that compressibility is fundamental to the structural integrity of viral capsids and that examination of compressibility and antiviral activity will provide insights into the disassembly process.

Tissue-specific differences in accumulation of stress proteins in Mytilus edulis exposed to a range of copper concentrations.

This study examines the expression and accumulation of two major stress proteins, stress70 and chaperonin60 (cpn60), in the gill and mantle of blue mussels, Mytilus edulis, which were exposed to a range of Cu concentrations for 7 days. Scope-for-growth (SFG), mortality, and Cu accumulation in gill and mantle tissue were also measured to monitor the physiological effects of Cu exposure in the organisms. In general Cu accumulated to a greater extent in gill relative to mantle tissue. A reduction of SFG index and increased mortality was also observed at the two highest Cu concentrations. We found no significant differences between the two tissues in the expression of cpn60 and stress70 for mussels exposed to Cu ranging from 0 to 10 micrograms/liter Cu (cpn60) and 0 to 32 micrograms/liter Cu (stress70) in sea-water. However, differences in the stress response were observed between the gill and the mantle tissue of mussels exposed to higher Cu concentrations. Chaperonin concentrations were greater than an order of magnitude higher in the gill than in the mantle for these mussels. Further, although the accumulation of stress70 was similar between the two tissues, two additional proteins reacted with antibody to stress70 in gill, but not mantle tissue, of mussels exposed to 100 micrograms/litter Cu. This study suggests that the physiological processes involved in contaminant uptake, distribution, and detoxification may affect the tissue-level expression of the stress response in multicellular organisms. Further, the intensity of the stress response and relative concentrations of chaperonin and stress70 among tissues may help identify tissues which are the most vulnerable to damage caused by a particular environmental stressor.

Hexachlorobenzene in selected marine samples: an environmental perspective.

Hexachlorobenzene (HCB) has been designated by the US Environmental Protection Agency as a chemical of interest. Re-examination of previously analysed gas chromatograms and archived samples revealed peaks that co-eluted with the authentic HCB standard. Levels of HCB were less than 1 ng/g in all samples re-examined, and were at least three to four orders of magnitude lower than those of poly-chlorinated biphenyls, two orders of magnitude lower than those of pyrene and one to two orders of magnitude lower than levels of either phenanthrene or benzo[a]-pyrene found in the same mussel samples. Chromatograms of sea water (dissolved and particulate phases) and sediments also revealed very low levels of HCB. Because gas chromatography-mass spectrometry results indicated that levels of HCB were below the level of detection it was not possible to verify its presence. The environmental data on HCB provided by the US Mussel Watch programme demonstrate that HCB levels in mussels are minor compared with other organic compounds such as polychlorinated biphenyls and polycyclic aromatic hydrocarbons (phenanthrene, pyrene and benzo[a]pyrene). Mussels were transplanted to areas affected by industrial activity and by disposal of sewage sludge and dredged materials, and in relatively clean reference sites. Transplanted mussels were used to monitor the marine food chain, as surrogates for other biota in the same areas and as sentinel organisms to monitor localized differences in biologically available chemical compounds. The degree of environmental threat posed by HCB in the marine environment, as indicated by the US Mussel Watch data, is relatively low. It is therefore recommended that a decision be made not to pursue an expanded HCB monitoring programme in the marine environment solely to collect more HCB data.