Protein-RNA Interactions and Virus Stability as Probed by the Dynamics of Tryptophan Side Chains

Abstract

The correlation between dynamics and stability of icosahedral viruses was studied by steady-state and timeresolved fluorescence approaches. We compared the environment and dynamics of tryptophan side chains of empty capsids and ribonucleoprotein particles of two icosahedral viruses from the comovirus group: cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV). We found a great difference between tryptophan fluorescence emission spectra of the ribonucleoprotein particles and the empty capsids of BPMV. For CPMV, timeresolved fluorescence revealed differences in the tryptophan environments of the capsid protein. The excited-state lifetimes of tryptophan residues were significantly modified by the presence of RNA in the capsid. More than half of the emission of the tryptophans in the ribonucleoprotein particles of CPMV originates from a single exponential decay that can be explained by a similar, nonpolar environment in the local structure of most of the tryptophans, even though they are physically located in different regions of the x-ray structure. CPMV particles without RNA lost this discrete component of emission. Anisotropy decay measurements demonstrated that tryptophans rotate faster in empty particles when compared with the ribonucleoprotein particles. The increased structural breathing facilitates the denaturation of the empty particles. Our studies bring new insights into the intricate interactions between protein and RNA where part of the missing structural information on the nucleic acid molecule is compensated for by the dynamics.Virus assembly is a puzzle when viewed from the perspectives of thermodynamics and kinetics. Virus assembly touches questions related to protein folding, protein-protein interactions, and protein-nucleic acid interactions. As previously shown for several viruses, these questions are quite linked in many bacteria, plant, and animal viruses (1-5). Although much has been learned about the structure of many macromolecular assemblies, in most cases the direct correlation with function is not possible because of the lack of information about the dynamics of the system (4). In solution, icosahedral viruses can be viewed as molecular "quasicrystals," and they are good models for understanding the linkage among protein folding, protein-nucleic acid interactions, and macromolecular assembly.Comoviridae is a family of icosahedral plant viruses characterized by a divided genome. This genome consists in two single-stranded, positive-sense RNA molecules, which are encapsidated into distinct particles. The capsids contain equimolar amounts of a large (L) and a small (S) proteins. 1 The two RNA molecules are referred to as RNA 1 and RNA 2, with ϳ6.0 and 3.5 kb, respectively (6). Isolation of comovirus from infected plants results in a mixture of the two ribonucleoprotein particles, as well as empty shells. Thus, three different particles can be separated by gradient ultracentrifugation from preparations of these viruses: the top (no RNA), the middle (containing RNA 2), and the bottom (c...

Electrospray ion mobility spectrometry of intact viruses

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Adenovirus Virion Stability and the Viral Genome: Size Matters

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Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

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