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Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3'-end. This suggests that packaging also occurs in an ordered manner resulting in the 3'-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.

Summary. Lipid-polymer conjugates with differing hydrophobic to hydrophilic dimensions were prepared. The polymer part consisted of hydrophilic poly(2-methyl-1,3-oxazolines) ( POZO), whose chain length can be controlled by living polymerization methods. The lipid-polymer conjugates were incorporated into vesicles composed of L-a-dimyristoylphosphatidylcholine ( DMPC) or L-a-dipalmitoylphosphatidylcholine ( DPPC) in amounts of up to 25?mol%. The formation of domains within the lipid bilayer membrane is observed by atomic force microscopy (AFM), thermal analysis (DSC), and fluorescence spectroscopy. The synthetic design of the lipids allows control over the distribution of polymer chains on the vesicle surface. [ABSTRACT FROM AUTHOR]

One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes. Here we provide the strongest evidence so far of such hierarchy and determine the time constants and weights of the kinetic components of the suggested hierarchic energy landscape. To our knowledge, the idea of hierarchical folding energy barriers has never been tested over such a broad timescale. Refolding of yeast phosphoglycerate kinase was initiated from the guanidine- unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 ms to 15 mm. The strategy to build a model that describes folding of yeast phosphoglycerate kinase was to start from the simplest paradigm and modify it stepwise to the necessary minimal extent after repeated comparisons with the experiments. We made no aprion assumptions about the folding landscape. The result was a hierarchic finite level landscape model that quantitatively describes the refolding of yeast phosphoglycerate kinase from 1 ms to 15 mm. The early steps of the folding process happen in the upper region of the landscape, where the surface has a hierarchic structure. This leads to stretched kinetics in the early phase of the folding. The lower region of the energy landscape is dominated by a trap that reflects the accumulation of molten globule intermediate state. From this intermediate, the protein can reach the global energy minimum corresponding to the native state through a cross-barrier folding step. [ABSTRACT FROM AUTHOR]