Characterization of Rotavirus VP2 Particles

Zeng, Chi; Labbé, M.; Cohen, Jean; Prasad, B. V. Venkataram; Chen, Dayue; Ramig, Robert F.; Estes, Mary K.

doi:10.1006/viro.1994.1265

Cited by 59 publications

(60 citation statements)

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“…When protease inhibitors are not added before lysis of insect cells, core-like particles are made of a VP2 cleavage product (Labb6 et al,199 l) that is also identified in empty viral particles (Brfissow et al, 1990). Terminal sequencing of the cleavage product showed that it lacks amino acids 1 to 92 from these corelike particles (Zeng et al, 1994). These results, and data presented here, indicate that the site of pseudocore assembly and the RNA binding sites are located in different domains of the proteins.…”

Section: Discussionsupporting

confidence: 59%

Identification of the nucleic acid binding domain of the rotavirus VP2 protein

Labbé

Baudoux

Charpilienne

et al. 1994

Journal of General Virology

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The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for doublestranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified singleshelled particles.

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Section: Discussionsupporting

confidence: 59%

Identification of the nucleic acid binding domain of the rotavirus VP2 protein

Labbé

Baudoux

Charpilienne

et al. 1994

Journal of General Virology

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“…and mixed multimers (bristly helix-like structures and sheet-like structures, etc.) in addition to Tϭ1 core-like particles (7,25). Therefore, whether a decamer is sufficient to activate a VP1 monomer remains to be experimentally confirmed.…”

Section: Discussionmentioning

confidence: 99%

Rotavirus VP2 Core Shell Regions Critical for Viral Polymerase Activation

McDonald

Patton

2011

J Virol

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The innermost VP2 core shell of the triple-layered, icosahedral rotavirus particle surrounds the viral genome and RNA processing enzymes, including the RNA-dependent RNA polymerase (VP1). In addition to anchoring VP1 within the core, VP2 is also an essential cofactor that triggers the polymerase to initiate double-stranded RNA (dsRNA) synthesis using packaged plus-strand RNA templates. The VP2 requirement effectively couples packaging with genome replication and ensures that VP1 makes dsRNA only within an assembling previrion particle. However, the mechanism by which the rotavirus core shell protein activates the viral polymerase remains very poorly understood. In the current study, we sought to elucidate VP2 regions critical for VP1-mediated in vitro dsRNA synthesis. By comparing the functions of proteins from several different rotaviruses, we found that polymerase activation by the core shell protein is specific. Through truncation and chimera mutagenesis, we demonstrate that the VP2 amino terminus, which forms a decameric, internal hub underneath each 5-fold axis, plays an important but nonspecific role in VP1 activation. Our results indicate that the VP2 residues correlating with polymerase activation specificity are located on the inner face of the core shell, distinct from the amino terminus. Based on these findings, we predict that several regions of VP2 engage the polymerase during the concerted processes of rotavirus core assembly and genome replication.

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“…Plasmid and Recombinant Baculovirus Constructs-The plasmid pBS2C24⌬ (22), constructed for the expression of VP2 with the first 92 amino acids deleted, was modified to remove the extra methionine. The linker TCTAGAGGATCC was added to provide an XbaI site and a flexible linker, consisting of the amino acids SRGS between VP2 and the fused protein.…”

Section: Methodsmentioning

confidence: 99%

“…VP2 binds to viral RNA, and its nucleic acid binding domain is located between amino acids (aa) 1 1 and 132 (3). The bond between Gln-92 and Lys-93 in VP2 is a protease-accessible site (4). It has been shown that coexpression of capsid viral proteins in the baculovirus system results in the assembly of virus-like particles (VLP) (2).…”

mentioning

confidence: 99%

Individual Rotavirus-like Particles Containing 120 Molecules of Fluorescent Protein Are Visible in Living Cells

Charpilienne¹,

Nejmeddine²,

Berois³

et al. 2001

Journal of Biological Chemistry

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151

156

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Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.

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Characterization of Rotavirus VP2 Particles

Cited by 59 publications

References 0 publications

Identification of the nucleic acid binding domain of the rotavirus VP2 protein

Identification of the nucleic acid binding domain of the rotavirus VP2 protein

Rotavirus VP2 Core Shell Regions Critical for Viral Polymerase Activation

Individual Rotavirus-like Particles Containing 120 Molecules of Fluorescent Protein Are Visible in Living Cells

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