Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction

Opalka, Natacha; Tihova, Mariana; Brugidou, Christophe; Kumar, Abhinav; Beachy, Roger N.; Fauquet, Claude; Yeager, Mark

doi:10.1006/jmbi.2000.4043

Cited by 32 publications

(33 citation statements)

References 40 publications

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“…At the twofold axes, slabs are oriented in an antiparallel fashion and are separated by ϳ50 Å. Similar cage-like structures have been observed in several other RNA viruses and virus-like particles (12,20,23,29,40,44,45).…”

Section: Rcnmv Divalent-cation Contentsupporting

confidence: 68%

Removal of Divalent Cations Induces Structural Transitions in Red Clover Necrotic Mosaic Virus , Revealing a Potential Mechanism for RNA Release

Sherman

Guenther

Tama

et al. 2006

J Virol

114

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The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 Å by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 ؎ 30 Ca 2؉ ions bound to the capsid and 420 ؎ 25 Mg 2؉ions thought to be in the interior of the capsid. Depletion of both Ca 2؉ and Mg 2؉ ions from RCNMV leads to significant structural changes, including (i) formation of 11-to 13-Å-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca 2؉ and Mg 2؉ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.The Tombusviridae family consists of small, icosahedral plant viruses that are transmitted through the soil and infect their hosts via the root system (34). Given that these viruses must survive harsh environments, it is not surprising that they possess unusually stable and robust capsids. This high degree of virion stability raises the question of how the intracellular environment triggers disassembly or minimally exposes the genome for translation. Structural analyses of several plant viruses have revealed that maintenance of a stable capsid conformation is dependent on the presence of divalent cations bound to the capsid (25,33,36). It has been a longstanding hypothesis that swelling and other conformational changes induced by ion extraction from virions are critical for a productive viral life cycle (13). For Tomato bushy stunt virus (TBSV), the type species of the genus Tombusvirus within the Tombusviridae family, it was hypothesized that Ca 2ϩ ions would be released from viral capsids within infected cells, leading to virion swelling sufficient to expose the viral genome.Crystallographic studies of TBSV (28) revealed a capsid formed by 180 chemically identical capsid protein (CP) subunits in three quasiequivalent conformations (A, B, and C). Each CP subunit is composed of three distinct structural domains, which include the RNA-interacting (R), shell (S), and protruding (P) domains. The conformational differences that distinguish the A, B, and C subunits are localized within the hinge regions between the respective S and P domains. These hinges point either down (in A-B dimers) or up (in C-C dimers). In addition, the loop that ...

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Section: Rcnmv Divalent-cation Contentsupporting

confidence: 68%

Removal of Divalent Cations Induces Structural Transitions in Red Clover Necrotic Mosaic Virus , Revealing a Potential Mechanism for RNA Release

Sherman

Guenther

Tama

et al. 2006

J Virol

114

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“…A current hypothesis is that calcium binding by virus particles will disrupt pit membranes between mature and differentiating xylem vessels or tracheids, so enabling the virus to traffic through the membrane. This hypothesis was proposed on the basis of immunochemical analyses of the colonization of rice plants by Rice yellow mottle virus (Opalka et al, 1998) and on the well-known role of calcium in the stabilization of Sobemovirus isometric capsids (Hsu et al, 1976;Opalka et al, 2000). It is known that calcium binding stabilizes TMV particles (Gallagher & Lauffer, 1983;Namba et al, 1989), and putative calcium binding sites have been described for CGMMV, watermelon strain (Wang & Stubbs, 1994), which has the same amino acid sequence in the CP as the SH strain (Ugaki et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…Dubois et al, 1994;Fribourg et al, 1987;Khan et al, 1994; Urban et al, 1989;Verchot et al, 2001). Further evidence supporting xylem transport has been reported in a few instances (Dubois et al, 1994;Opalka et al, 2000; Schneider & Worley, 1959a, b). As well as the growing number of reported cases, one aspect of virus systemic movement via the xylem that still needs to be addressed is exactly how viruses translocate from the non-living tracheary elements to living cells (i.e.…”

Section: Introductionmentioning

confidence: 92%

“…from the apoplast to the symplast) for the establishment of systemic infection. To explain this, a model by which the virus chelates calcium in the pit membrane between tracheids, relaxing the membrane's structure, and then moves into living immature tracheids has been proposed for Rice yellow mottle virus (Opalka et al, 2000). Hence, systemic movement of plant viruses through the xylem is a relatively unexplored process.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of the systemic colonization of cucumber plants by Cucumber green mottle mosaic virus

Moreno

Thompson

García‐Arenal

2004

Journal of General Virology

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Systemic movement of Cucumber green mottle mosaic virus (CGMMV) in cucumber plants was shown to be from photoassimilate source to sink, thus indicating phloem transport. Nevertheless, CGMMV was not detected by immunocytochemical procedures in the intermediary cell-sieve element complex in inoculated cotyledons, where photoassimilate loading occurs. In stem internodes, CGMMV was first localized in the companion cells of the external phloem and subsequently in all tissues except the medulla, therefore suggesting leakage of the virus from, and reloading into, the transport phloem during systemic movement. In systemically infected sink leaves, CGMMV was simultaneously detected in the xylem and phloem. Interestingly, CGMMV accumulated to high levels in the differentiating tracheids of young leaves implying that the xylem could be involved in the systemic movement of CGMMV. This possibility was tested using plants in which cell death was induced in a portion of the stem by steam treatment. At 24 6C, steam treatment effectively prevented the systemic movement of CGMMV, even though viral RNA was detected in washes of the xylem above the steamed internode suggesting that xylem circulation occurred. At 29 6C, CGMMV systemically infected steam-treated cucumber plants, indicating that CGMMV can move systemically via the xylem. Xylem transport of CGMMV was, however, less efficient than phloem transport in terms of the time required for systemic infection and the percentage of plants infected. INTRODUCTIONSystemic transport through the vascular system is a crucial step in plant virus infection. Most plant viruses are thought to move systemically through the phloem in parallel with photoassimilate transport (reviewed by Haywood et al., 2002;Lucas & Wolf, 1999;Nelson & Van Bel, 1998;Oparka & Turgeon, 1999;Thompson & Schulz, 1999). Phloem transport includes the loading (entry) of the virus into the phloem at source tissues, its circulation in the transport phloem and its unloading (exit) from the phloem at the sink tissues. Photoassimilate loading occurs presumably in the minor veins of source organs (Nelson & Van Bel, 1998;Santa Cruz, 1999) and in the case of cucurbits is thought to follow a symplastic route involving the specialized intermediary cell-sieve element complex (Turgeon, 1996). Knowledge of phloem circulation of plant viruses is far from complete, yet it is known that it parallels photoassimilate flow (Leisner et al., 1992;Oparka & Turgeon, 1999). The requirement of a functional capsid protein (CP) for systemic movement is common but not universal, depending on specific virus-host interactions (e.g. Dalmay et al., 1992;McGeachy & Barker, 2000;Petty & Jackson, 1990;Ryabov et al., 2001), and for most viruses the precise form in which the virus is transported has not been determined. Phloem unloading in the sink organs also parallels photoassimilate unloading and proceeds basipetally in the developing leaf during the sink-to-source transition (Cheng et al., 2000;Leisner et al., 1992; Roberts et al., 1997; Santa Cruz e...

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“…Cryo-transmission electron microscopy ͑TEM͒ [21][22][23] and x-ray crystallography [24][25][26] reveal, at angstrom-level resolution, highly symmetrical supramolecular assemblages. The structural information corresponds mainly to the rigid protein shell, or capsid, which encapsulates the viral genome.…”

Section: Introductionmentioning

confidence: 99%

Electrostatics of capsid-induced viral RNA organization

Forrey

Muthukumar

2009

The Journal of Chemical Physics

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We have addressed the role of electrostatics in the formation of genome structure in the Pariacoto virus, where substantial experimental data are available. We have used Langevin dynamics simulation of a coarse-grained model, based on the published crystal structure of the rigid portion of the Pariacoto capsid and including flexible N-terminal protein arms, attached to the rigid capsid at the appropriate locations. The inclusion of charged residues in our model was dictated solely by the location of charges inherent in the Pariacoto sequence itself. Although the viral genome and other exogenous RNA sequences used in experimental studies can assume secondary structures, we have intentionally used uniformly charged flexible polyelectrolyte lacking predetermined secondary structures as the substitute for the viral genome, in order to see whether the same final assembled genome structure emerges without invoking secondary RNA structures. The intent of our study was to investigate the internal environment presented by the capsid proteins of Pariacoto virus, specifically whether the topological features and electrostatic potential at the inner capsid surface can induce complexation of generic negatively charged polyelectrolyte into structures similar to those observed experimentally with packaged RNA. We find that the charge decoration on the interior of the capsid templates the assembly of the flexible polyelectrolyte, allowing hybridizationlike folding of similarly charged strands, and eventually organizing dodecahedral assembly of the polymer. Our results from a generic flexible polyelectrolyte for the assembled structure and bimodal monomer distribution are remarkably matched to that of the viral RNA found experimentally. Results of our work can be interpreted primarily as a consequence of electrostatics, as consideration of base-pairing has been omitted. We propose that our work supports the growing body of evidence that electrostatic interactions play a crucial role in RNA viral assembly and structure.

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Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction

Cited by 32 publications

References 40 publications

Removal of Divalent Cations Induces Structural Transitions in Red Clover Necrotic Mosaic Virus , Revealing a Potential Mechanism for RNA Release

Removal of Divalent Cations Induces Structural Transitions in Red Clover Necrotic Mosaic Virus , Revealing a Potential Mechanism for RNA Release

Analysis of the systemic colonization of cucumber plants by Cucumber green mottle mosaic virus

Electrostatics of capsid-induced viral RNA organization

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