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Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release.

ABSTRACT: The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A 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 Ca2+ ions bound to the capsid and 420 +/- 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-A-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ 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.

SUBMITTER: Sherman MB

PROVIDER: S-EPMC1641784 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release.

Sherman Michael B MB Guenther Richard H RH Tama Florence F Sit Tim L TL Brooks Charles L CL Mikhailov Albert M AM Orlova Elena V EV Baker Timothy S TS Lommel Steven A SA

Journal of virology 20060818 21

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A 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 ...[more]

PMID: 16920821

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Project description:Members of the Tombusviridae family have highly similar structures, and yet there are important differences among them in host, transmission, and capsid stabilities. Viruses in the Tombusviridae family have single-stranded RNA (ssRNA) genomes with T=3 icosahedral protein shells with a maximum diameter of ∼340 Å. Each capsid protein is comprised of three domains: R (RNA binding), S (shell), and P (protruding). Between the R domain and S domain is the "arm" region that studies have shown to play a critical role in assembly. To better understand how the details of structural differences and similarities influence the Tombusviridae viral life cycles, the structures of cucumber leaf spot virus (CLSV; genus Aureusvirus) and red clover necrotic mosaic virus (RCNMV; genus Dianthovirus) were determined to resolutions of 3.2 Å and 2.9 Å, respectively, with cryo-electron microscopy and image reconstruction methods. While the shell domains had homologous structures, the stabilizing interactions at the icosahedral 3-fold axes and the R domains differed greatly. The heterogeneity in the R domains among the members of the Tombusviridae family is likely correlated with differences in the sizes and characteristics of the corresponding genomes. We propose that the changes in the R domain/RNA interactions evolved different arm domain interactions at the β-annuli. For example, RCNMV has the largest genome and it appears to have created the necessary space in the capsid by evolving the shortest R domain. The resulting loss in RNA/R domain interactions may have been compensated for by increased intersubunit β-strand interactions at the icosahedral 3-fold axes. Therefore, the R and arm domains may have coevolved to package different genomes within the conserved and rigid shell.IMPORTANCE Members of the Tombusviridae family have nearly identical shells, and yet they package genomes that range from 4.6 kb (monopartite) to 5.3 kb (bipartite) in size. To understand how this genome flexibility occurs within a rigidly conserved shell, we determined the high-resolution cryo-electron microscopy (cryo-EM) structures of cucumber leaf spot virus and red clover necrotic mosaic virus. In response to genomic size differences, it appears that the ssRNA binding (R) domain of the capsid diverged evolutionarily in order to recognize the different genomes. The next region, the "arm," seems to have also coevolved with the R domain to allow particle assembly via interactions at the icosahedral 3-fold axes. In addition, there are differences at the icosahedral 3-fold axes with regard to metal binding that are likely important for transmission and the viral life cycle.

Project description:Calcium-independent phospholipase A(2)? (iPLA(2)?) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)? gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)? in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)? resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)? mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)? selective), but not its enantiomer, (S)-BEL (iPLA(2)? selective) or pyrrolidine (cytosolic PLA(2)? selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)? activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)?. Intriguingly, Ca(2+)-induced iPLA(2)? activation was completely inhibited by long-chain acyl-CoA (IC(50) ?20 ?m) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)? is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.

Dataset Information

Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release.

Publications

Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release.

Similar Datasets

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