Translocation portals for the substrates and products of a viral transcription complex: the bluetongue virus core

Diprose, Jonathan M.

doi:10.1093/emboj/20.24.7229

Cited by 73 publications

(57 citation statements)

References 26 publications

(56 reference statements)

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“…Assembly of the VP7 layer onto the VP3 layer plugs these pores, which only opens up when the core becomes transcriptionally active in the presence of nucleoside triphosphates (rNTPs) (21). Therefore, we hypothesized that the addition of VP7 to the putative subcores might protect the incorporated 10 ssRNAs.…”

Section: Resultsmentioning

confidence: 99%

In vitro reconstitution of Bluetongue virus infectious cores

Lourenco

Roy

2011

Proc. Natl. Acad. Sci. U.S.A.

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Bluetongue virus (BTV) is a vector-borne, nonenveloped icosahedral particle that is organized in two capsids, an outer capsid of two proteins, VP2 and VP5, and an inner capsid (or core) composed of two major proteins, VP7 and VP3, in two layers. The VP3 layer (subcore) encloses viral transcription complex (VP1 polymerase, VP4 capping enzyme, VP6 helicase) and a 10-segmented double-stranded (dsRNA) genome. Although much is known about the BTV capsids, the order of the core assembly and the mechanism of genome packaging remain unclear. Here, we established a cell-free system to reconstitute subcore and core structures with the proteins and ssRNAs, demonstrating that reconstituted cores are infectious in insect cells. Furthermore, we showed that the BTV ssRNAs are essential to drive the assembly reaction and that there is a distinct order of internal protein recruitment during the assembly process. The in vitro engineering of infectious BTV cores is unique for any member of the Reoviridae and will facilitate future studies of RNA-protein interactions during BTV core assembly.assembly pathway | Orbivirus | engineered particle

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Section: Resultsmentioning

confidence: 99%

In vitro reconstitution of Bluetongue virus infectious cores

Lourenco

Roy

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Addition of an extra negative charge at the VP6-VP2 contact region could therefore inhibit the necessary conformational change of VP2. A recent study of the viral transcription complex of bluetongue virus (6) clearly shows that VP7 binds nucleoside triphosphates providing a substrate sink. This binding is associated with slight conformational changes.…”

Section: Discussionmentioning

confidence: 99%

Identification of Rotavirus VP6 Residues Located at the Interface with VP2 That Are Essential for Capsid Assembly and Transcriptase Activity

Charpilienne

Lepault

Rey

et al. 2002

J Virol

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Rotavirus has a complex triple-layered icosahedral capsid. The external layer consists of VP7 and VP4, the intermediate layer consists of VP6 trimers, and the internal layer consists of VP2. Double-layered particles (DLP) derived from the virus by solubilization of VP4 and VP7 are transcriptionally competent and extrude capped mRNA from their vertices. Analysis of the pseudoatomic model of the VP6 layer, obtained by placing the atomic structure of VP6 into electron microscopy reconstructions of the DLP, has identified the regions of the protein involved in interactions with the internal layer. To study the role of VP6 both in the assembly of DLP and in transcription, 13 site-specific substitution mutations of VP6, targeting the contacts between the two inner layers, were constructed and expressed in the baculovirus system. The effects of these mutations on VP6 expression, trimerization, and formation of macromolecular assemblies were investigated. Using either in vitro reconstituted DLP derived from purified viral cores and recombinant VP6 or in vivo self-assembled virus-like particles resulting from the coexpression of VP2 and VP6 in the baculovirus-Sf9 system (VLP2/6), we have identified the amino acids essential for recovery of transcription or assembly. All VP6 mutants formed stable trimers which, like wild-type VP6, assembled into tubular structures. The ability of VP6 to interact with VP2 was examined by several assays, including electron microscopy, coimmunoprecipitation, purification of VLP2/6, and monitoring of the transcriptase activity of reconstituted DLP. Of the 13 VP6 mutants examined, 3 were unable to assemble with VP2 and 3 others partially assembled. These mutants either did not rescue the transcriptase activity of core particles or did so only marginally. Four mutants as well as the wild-type VP6 assembled and transcribed very well. Three mutants assembled well on cores but, surprisingly, did not rescue the transcriptase activity of reconstituted DLP. Our results indicate that hydrophobic interactions between VP6 and VP2 residues are responsible for the stability of the DLP. They also show that subtle electrostatic interactions between VP6 and the underlying transcriptase machinery can be essential for mRNA synthesis.Rotaviruses are members of the Reoviridae family of segmented, double-stranded RNA viruses. Viruses in this family are nonenveloped, and their complex capsids contain several concentric protein layers displaying icosahedral symmetry. Structural studies by X-ray crystallography have revealed the complex interactions between the innermost layers of some of the Reoviridae, like bluetongue virus (BTV) (9), as well as the structure of the single-layered core particle of reoviruses (20). In the case of rotaviruses, the crystal structure of the doublelayered particle (DLP) has not yet been reported but a pseudoatomic model has been derived for the middle layer by use of an approach combining X-ray crystallography and electron microscopy (18). As is the case for BTV, the two innermost lay...

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“…These mRNA molecules are capped by the guanylyl-transferase and transmethylase activities of VP4 [92] and leave the particles via channels situated at the five fold axes of the core particle [68]. The viral mRNA serve as templates for translation in viral proteins, starting within two hours post infection [20]. Viral positive RNA are directed to VIB where the correct encapsidation of the different segments (nature and numbers) within the VP3 shell may involve interactions with the helicase VP6 [89], the ssRNA binding NS2 protein [53], and the VP1 and VP4 proteins.…”

Section: Btv Life Cyclementioning

confidence: 99%

Bluetongue virus: virology, pathogenesis and immunity

et al. 2008

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-Bluetongue (BT) virus, an orbivirus of the Reoviridae family encompassing 24 known serotypes, is transmitted to ruminants via certain species of biting midges (Culicoides spp.) and causes thrombo-hemorrhagic fevers mainly in sheep. During the 20th century, BTV was endemic in sub-tropical regions but in the last ten years, new strains of BTV (serotypes 1, 2, 4, 8, 9, 16) have appeared in Europe leading to a devastating disease in naive sheep and bovine herds (serotype 8). BTV enters into insect cells via the viral inner core VP7 protein and in mammalian cells via the external capsid VP2 haemagglutinin, which is the major determinant of BTV serotype and neutralization. BTV replicates in mononuclear phagocytes and endothelial cells where it induces expression of inflammatory cytokines as well as apoptosis. BTV can remain as nonreplicating entities concealed in erythrocytes for up to five months. Homologous protection against one BTV serotype involves neutralizing antibodies and T cell responses directed to the external VP2 and VP5 proteins, whereas heterologous protection is supported by T cells directed to the NS1 non structural protein and inner core proteins.

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Translocation portals for the substrates and products of a viral transcription complex: the bluetongue virus core

Cited by 73 publications

References 26 publications

In vitro reconstitution of Bluetongue virus infectious cores

In vitro reconstitution of Bluetongue virus infectious cores

Identification of Rotavirus VP6 Residues Located at the Interface with VP2 That Are Essential for Capsid Assembly and Transcriptase Activity

Bluetongue virus: virology, pathogenesis and immunity

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