Partially folded states of the capsid protein of cowpea severe mosaic virus in the disassembly pathway

Many animal viruses undergo post-assembly proteolytic cleavage that is required for infectivity. The role of maturation cleavage on Flock House virus was evaluated by comparing wild type (wt) and cleavage-defective mutant (D75N) Flock House virus virus-like particles. A concerted dissociation and unfolding of the mature wt particle was observed under treatment by urea, whereas the cleavage-defective mutant dissociated to folded subunits as determined by steady-state and dynamic fluorescence spectroscopy, circular dichroism, and nuclear magnetic resonance. The folded D75N ␣ subunit could reassemble into capsids, whereas the yield of reassembly from unfolded cleaved wt subunits was very low. Overall, our results demonstrate that the maturation/ cleavage process targets the particle for an "off pathway" disassembly, because dissociation is coupled to unfolding. The increased motions in the cleaved capsid, revealed by fluorescence and NMR, and the concerted nature of dissociation/unfolding may be crucial to make the mature particle infectious.Viruses are macromolecular assemblies designed to exert their biological role in a single sequential cycle: 1) assembly inside the cells; 2) release to the environment; 3) attachment to new host cells; 4) disassembly and delivery of genome; and 5) replication of the genome and transcription of new viral proteins. Among these five stages, disassembly of the capsid and unpacking of the nucleic acid is the least understood (1-4). To be effective, disassembly has to occur fast and at the correct time after endocytosis. The switch for this process is usually attributed to the acidic pH inside the endocytic vesicles, but in vitro many viruses are not uncoated by low pH, or the uncoating occurs slowly, not consistent with the requirement for rapid replication. A fundamental property of most animal viruses is the post-assembly maturation, which is required for infectivity. In picornaviruses (5-8) and nodaviruses (9, 10), the maturation cleavage is autocatalytic and is dependent upon appropriate particle assembly. A picornavirus capsid is initially assembled from 60 copies of the subunits VP0, VP3, and VP1 and an RNA molecule. The final processing of picornaviruses occurs after assembly in which the precursor protein VP0 is cleaved to release VP4 and VP2 (11). The cleavage site is not accessible to the surface, and it is proposed that the packaged RNA acts as a nucleophile (12). Like the picornaviruses, the nodaviruses are initially constructed as provirions, which mature to an infectious virion by post-assembly cleavage of ␣ protein into ␤ (43 kDa) and ␥ (4 kDa) subunits.Flock House virus (FHV) 1 is a nonenveloped T ϭ 3 icosahedral insect virus of the family Nodaviridae (Fig. 1). The virus particle consists of 180 copies of the coat protein, which encapsidates the bipartite RNA genome (9,11,13,14). The FHV coat protein expressed in a baculovirus system spontaneously assembles into a virus-like particle (VLP) containing cellular RNA, and the ␣ capsid protein undergoes cleavage into ␤ an...

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“…5A). As previously found with other dissociated capsid proteins (26,27), the monomeric subunits were adsorbed to the column.…”

Section: Cleavage-defective Coat Protein Reassembles Into Capsids-supporting

confidence: 62%

Virus Maturation Targets the Protein Capsid to Concerted Disassembly and Unfolding

Oliveira¹,

Gomes²,

Almeida³

et al. 2000

Journal of Biological Chemistry

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“…We also found that the coat proteins remain bound to the nucleic acid after pressure disassembly of the capsid in comoviruses (26,55), picornaviruses (56), and nodaviruses (27), suggesting again the strong interactions between protein and nucleic acid in virus assembly. These interactions can be disrupted by decreasing temperature to negative values (Ϫ15°C) under pressure, revealing their entropic nature (26).…”

Section: Discussionmentioning

confidence: 61%

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

Poian

Johnson

Silva

2002

Journal of Biological Chemistry

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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...

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“…Recent studies have demonstrated intermediate states in the assembly and disassembly pathway of many viruses, and protein-nucleic acid interactions appear to remain intact under pressure (32,(37)(38)(39). Our results with Sindbis and influenza viruses show that although pressure can preserve most of the physical properties of the envelope, it causes exposure of hydrophobic domains that populate the fusion-active state that may account for the pronounced inactivation.…”

Section: Fusion Of Sindbis Virus With Erythrocyte Ghosts After Virus mentioning

confidence: 56%

“…More recently, bis-ANS has been used to follow changes in the conformation of an enveloped virus as it assumes the fusion-active state (30,31). Bis-ANS has been also used to follow changes in the capsid protein in the disassembly pathway (23,32,33). Fig.…”

Section: Pressure-induced Exposure Of Hydrophobic Domains In En-mentioning

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%

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Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

Gaspar¹,

Silva²,

Gomes³

et al. 2002

Journal of Biological Chemistry

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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...

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Partially folded states of the capsid protein of cowpea severe mosaic virus in the disassembly pathway

Cited by 51 publications

References 43 publications

Virus Maturation Targets the Protein Capsid to Concerted Disassembly and Unfolding

Virus Maturation Targets the Protein Capsid to Concerted Disassembly and Unfolding

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

Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

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