Virus Maturation Targets the Protein Capsid to Concerted Disassembly and Unfolding

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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...

Section: Discussionmentioning

confidence: 83%

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

Poian

Johnson

Silva

2002

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“…Recently we found that the maturation/ cleavage of flock house virus capsid converts the coat protein into a metastable conformation, whereas a cleavage-defective mutant particle coat protein reversibly reassembles into particles. The mature capsid is more sensitive to pressure than the cleavage-defective, immature particle (42).…”

Section: Fig 3 Pressure Effects On Sindbis and Influenza Virusesmentioning

confidence: 99%

“…This change makes it sensitive to both pH and pressure. Cavities have been implicated in protein metastability in several systems (32,(41)(42)(43). The influenza virus system has the unique property of having a water-penetrated cavity in the precursor protein, which eliminates water on proteolytic cleavage, originating the pH and pressure sensitivity.…”

Section: Fig 3 Pressure Effects On Sindbis and Influenza Virusesmentioning

confidence: 99%

Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

Gaspar¹,

Silva²,

Gomes³

et al. 2002

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Enveloped animal viruses must undergo membrane fusion to deliver their genome into the host cell. We demonstrate that high pressure inactivates two membrane-enveloped viruses, influenza and Sindbis, by trapping the particles in a fusion-intermediate state. The pressure-induced conformational changes in Sindbis and influenza viruses were followed using intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, and fusion, plaque, and hemagglutination assays. Influenza virus subjected to pressure exposes hydrophobic domains as determined by tryptophan fluorescence and by the binding of bis-8-anilino-1-naphthalenesulfonate, a well established marker of the fusogenic state in influenza virus. Pressure also produced an increase in the fusion activity at neutral pH as monitored by fluorescence resonance energy transfer using lipid vesicles labeled with fluorescence probes. Sindbis virus also underwent conformational changes induced by pressure similar to those in influenza virus, and the increase in fusion activity was followed by pyrene excimer fluorescence of the metabolically labeled virus particles. Overall we show that pressure elicits subtle changes in the whole structure of the enveloped viruses triggering a conformational change that is similar to the change triggered by low pH. Our data strengthen the hypothesis that the native conformation of fusion proteins is metastable, and a cycle of pressure leads to a final state, the fusion-active state, of smaller volume.Enveloped viruses utilize regulated membrane fusion to introduce their genomes in the cytoplasm of the host cell. The fusion is mediated by surface envelope proteins of the virus in response to a trigger (1, 2). Once triggered, the fusion process leads to a conformational change that promotes the interaction of a specific sequence (fusion peptide) with the target membrane and initiates membrane fusion. Membrane fusion is crucial in other biological functions such as myotube formation, fertilization, and trafficking of endocytic and exocytic vesicles within eukaryotic cells (1, 3). Many enveloped animal viruses have been studied as models for understanding the mechanism of membrane fusion. While the fusion proteins of many viruses reveal significant similarity in their putative fusogenic conformation, such as those of influenza virus (hemagglutinin HA2), 1 human immunodeficiency virus (gp41 protein), Moloney murine leukemia virus (TM protein), and Ebola virus (GP2 protein) (4), the events of membrane fusion for other virus families (e.g. Flaviviridae and Togaviridae) are beginning to be understood. Alphavirus and the flavivirus fusion proteins appear to have evolved from a common ancestor (5) and possess a similar new class of membrane fusion proteins that do not form coiledcoils (6 -8).Sindbis and influenza are enveloped viruses that first enter a cell by endocytosis and then fuse with the cellular membrane in response to acidic conditions. Sindbis virus is the prototype of the Alphavirus genus, Togaviridae family. The Alphavirus spike is...

“…Metastability has been observed for serine protease inhibitors (40,41), heat shock transcription factors (42), viral envelope glycoproteins (43), and proteolitically processed capsid proteins of nonenveloped viruses (11). Here, we characterize the metastable state of the ribonucleoprotein capsid by perturbing it with hydrostatic pressure or urea.…”

mentioning

confidence: 99%

“…Enveloped viruses enter cells by protein-mediated fusion of the membrane enclosing the virus particle with the cellular membrane, which ultimately results in release of the nucleocapsid into the cell for processing (3). In nonenveloped viruses, biophysical studies have been carried out to understand how the overall stability depends on protein-protein interactions (4 -7), protein-nucleic acid interactions (8,9), and proteolysis maturation (10,11). Here, the alphavirus Mayaro was used as a simple model of enveloped viruses to compare the properties of the whole and nucleocapsid particles deprived of the lipid bilayer and transmembrane glycoproteins.…”

mentioning

confidence: 99%

The Metastable State of Nucleocapsids of Enveloped Viruses as Probed by High Hydrostatic Pressure

Gaspar¹,

Terezan²,

Pinheiro³

et al. 2001

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Enveloped viruses fuse their membranes with cellular membranes to transfer their genomes into cells at the beginning of infection. What is not clear, however, is the role of the envelope (lipid bilayer and glycoproteins) in the stability of the viral particle. To address this question, we compared the stability between enveloped and nucleocapsid particles of the alphavirus Mayaro using hydrostatic pressure and urea. The effects were monitored by intrinsic fluorescence, light scattering, and binding of fluorescent dyes, including bis(8-anilinonaphthalene-1-sulfonate) and ethidium bromide. Pressure caused a drastic dissociation of the nucleocapsids as determined by tryptophan fluorescence, light scattering, and gel filtration chromatography. Pressure-induced dissociation of the nucleocapsids was poorly reversible. In contrast, when the envelope was present, pressure effects were much less marked and were highly reversible. Binding of ethidium bromide occurred when nucleocapsids were dissociated under pressure, indicating exposure of the nucleic acid, whereas enveloped particles underwent no changes. Overall, our results demonstrate that removal of the envelope with the glycoproteins leads the particle to a metastable state and, during infection, may serve as the trigger for disassembly and delivery of the genome. The envelope acts as a "Trojan horse," gaining entry into the host cell to allow release of a metastable nucleocapsid prone to disassembly.