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

Labbé, M.; Baudoux, Pierre; Charpilienne, Annie; Poncet, Didier; Cohen, Jean

doi:10.1099/0022-1317-75-12-3423

Cited by 64 publications

(54 citation statements)

References 26 publications

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“…2A). Rotavirus is a trilayered virus, which contains the VP2 protein in its innermost layer (Charpilienne et al, 2001;Labbe et al, 1994). When baculovirus assembly is performed in the The properties of Dnm1p-GFP assemblies in wt, mdv1⌬, fis1⌬, and caf4⌬ cells, expressing Dnm1p-GFP and mtDsRed were semi-automatically determined.…”

Section: Resultsmentioning

confidence: 99%

Fis1p and Caf4p, but not Mdv1p, determine the polar localization of Dnm1p clusters on the mitochondrial surface

Schauß

Bewersdorf

Jakobs

2006

Journal of Cell Science

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The mitochondrial division machinery consists of the large dynamin-related protein Dnm1p (Drp1/Dlp1 in humans), and Fis1p, Mdv1p and Caf4p. Proper assembly of Dnm1p complexes on the mitochondrial surface is crucial for balanced fission and fusion events. Using quantitative confocal microscopy, we show that Caf4p is important for the recruitment of Dnm1p to the mitochondria. The mitochondrial Dnm1p assemblies can be divided into at least two morphologically distinguishable fractions. A small subset of these assemblies appear to be present as Dnm1p-spirals (or rings) that encircle tubule constrictions, with seldom more than seven turns. A larger fraction of the Dnm1p assemblies is primarily present at one side of the mitochondrial tubules. We show that a majority of these mitochondria-associated Dnm1p clusters point towards the cell cortex. This polarized orientation is abolished in fis1Δ and caf4Δ yeast cells, but is maintained in mdv1Δ cells and after disruption of the actin cytoskeleton. This study suggests that Caf4p plays a key role in determining the polarized localization of those Dnm1p clusters that are not immediately involved in the mitochondrial fission process.

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Section: Resultsmentioning

confidence: 99%

Fis1p and Caf4p, but not Mdv1p, determine the polar localization of Dnm1p clusters on the mitochondrial surface

Schauß

Bewersdorf

Jakobs

2006

Journal of Cell Science

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“…This protein was or was not pretreated with 1 g of tTG at 37°C for 1 h. BSA (0.2 g), tTG (1 g), and the core protein (0.2 g, with or without tTG treatment) were spotted onto a piece of nitrocellulose membrane that had been prewetted in H 2 O. After pre-blocking at 25°C for 1 h in standard binding buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.04% BSA, 0.04% polyvinylpyrrolidone, and 0.04% Ficoll), the membrane was incubated with the RNA probe in 1 ml of standard binding buffer containing 50 mM NaCl and 100 units of RNasin at 25°C for 45 min (40). The membrane was then washed three times with standard binding buffer for 5 min each at 25°C and analyzed with a PhosphorImager.…”

Section: Methodsmentioning

confidence: 99%

Post-translational Modification of the Hepatitis C Virus Core Protein by Tissue Transglutaminase

Strohecker

2001

Journal of Biological Chemistry

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The hepatitis C virus (HCV) core protein is a structural protein that packages the viral genomic RNA. In this study, we demonstrate that a stable core protein dimer could be produced in liver cells. The production of this protein could be enhanced by calphostin C and serum deprivation. This protein was determined to be the core protein dimer because of its reactivity with the anti-core antibody, its similar electrophoretic mobility compared with that of the core protein dimer generated by cross-linking with glutaraldehyde, and its increase in size by a hemagglutinin tag fused to the core protein sequence. This core protein dimer was highly stable and resistant to SDS and ␤-mercaptoethanol. The enzyme that mediated the formation of this stable core protein dimer was determined to be the tissue transglutaminase (tTG) because, first, tTG could be activated by calphostin C and serum deprivation; second, the formation of this dimer was suppressed by monodansylcadaverine, a tTG inhibitor; and third, the core protein could be crosslinked by tTG in vitro. Thus, the HCV core protein represents the first known viral structural protein substrate of tTG. The post-translational modification by tTG reduced the RNA binding activity of the core protein, raising the possibility that tTG may regulate the biological functions of the HCV core protein.

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“…Both of these proteins have affinity for RNA, but only in the case of VP1 has a sequence-dependent activity been detected (Labbe et al 1994;Tortorici et al 2003). Although we have successfully used electrophoretic mobility shift assays (EMSA) to identify RdRP-recognition signals at the 3¢-end of rotavirus (+)RNAs, similar attempts to identify an interaction of the polymerase with the 5¢-recognition signal have been unsuccessful so far (Tortorici et al 2003; data not shown).…”

Section: Wwwrnajournalorg 141mentioning

confidence: 99%

A base-specific recognition signal in the 5′ consensus sequence of rotavirus plus-strand RNAs promotes replication of the double-stranded RNA genome segments

Tortorici

Shapiro²,

Patton³

2005

RNA

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Replication of the segmented double-stranded (ds)RNA genome of rotavirus requires the viral RNA-dependent RNA polymerase (RdRP) to use 11 different (+)RNAs as templates for (À) strand synthesis. Complementary sequences proximal to the 5¢ and 3¢ termini are predicted to direct cyclization of the (+)RNAs by forming panhandle structures from which short highly conserved terminal sequences protrude as single-stranded tails. Cell-free replication assays indicate that such structural organization of the 5¢-and 3¢-ends is required for efficient dsRNA synthesis. Multiple specifically recognized elements exist at the 3¢-end that promote dsRNA synthesis including RdRP-recruitment signals and a (À) strand initiation sequence. In contrast to the 3¢-end, the role of the 5¢-end has been less well defined. In this study, we determined that the 5¢-end contains a base-specific recognition signal that plays an important role in the assembly of the RdRP and cofactors into a stable initiation complex for (À) strand synthesis. The 5¢ recognition signal is associated with the G2 residue of the 5¢-consensus sequence, a residue that shows absolute conservation among all rotavirus groups (A, B, and C) examined to date. From our results, we suggest that rotavirus (+)RNA cyclization, although likely initiated by 5¢-3¢ nucleotide complementarity, may be stabilized by RdRP-dependent bridging. Given that synthesis of the (À) strand on the (+)RNA template will disrupt 5¢-3¢ nucleotide interactions, RdRP-dependent bridging may be the sole mechanism by which the dsRNA product can be held in the necessary cyclized conformation required for efficient multiple rounds of transcription.

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

Cited by 64 publications

References 26 publications

Fis1p and Caf4p, but not Mdv1p, determine the polar localization of Dnm1p clusters on the mitochondrial surface

Fis1p and Caf4p, but not Mdv1p, determine the polar localization of Dnm1p clusters on the mitochondrial surface

Post-translational Modification of the Hepatitis C Virus Core Protein by Tissue Transglutaminase

A base-specific recognition signal in the 5′ consensus sequence of rotavirus plus-strand RNAs promotes replication of the double-stranded RNA genome segments

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