The Structure of the Rotavirus Inner Capsid Studied by Electron Microscopy of Chemically Disrupted Particles (original) (raw)
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Journal of General Virology, 1985
The major structural polypeptide of rotaviruses is p45K (VP6), which forms the morphological subunit of the inner capsid. Such subunits show a trimeric structure when examined with the electron microscope. Treatment of single-capsid rotavirus particles with 1.5 M-CaCI~ removes p45K, resulting in the generation of smooth cores. Sucrose density gradient centrifugation analysis of the removed p45K revealed that it has a sedimentation coefficient close to 7.3S, compatible with an oligomeric (possibly trimeric) structure. Polyacrylamide gel electrophoresis under reducing or non-reducing conditions indicated that p45K has intramolecular but not intermolecular disulphide bonds, suggesting that interactions between p45K monomers may be due to some other type of association, such as hydrophobic or charge interactions. Velocity sedimentation of infected cell extracts revealed that native p45K also behaves as an oligomeric protein. Such results were confirmed using p45K partially purified by DEAE-cellulose chromatography. The evidence obtained indicated that all p45K present in the virion is in the oligomerie form, not associated by disulphide bonding, and that most native p45K present in the infected cells is also in the oligomeric form, probably as a consequence of early protein-protein interaction in rotavirus morphogenesis.
Biological research, 1994
The structural relationship between VP6 (inner capsid polypeptide) and the viral core was studied using chemical cross-linking with dithiobis(succinimidyl propionate). Crosslinked single shelled and reconstituted rotavirus particles, suggest the existence of a complex organization of VP6 molecules in the inner capsid and a direct interaction with the core polypeptide VP3. The inhibition of the recovery of RNA polymerase activity associated with the reconstitution of the single shelled particle in the presence of antiVP6 monoclonal antibodies indicates that a VP6 domain between amino acids 56 and 58 seems to be important in viral transcription. A VP6 gene temperature-sensitive mutant (ts G) carrying a mutation affecting assembly of single shelled particles was used in reconstitution experiments. The mutant was able to recover RNA polymerase activity at restrictive temperature. Wild type cores or VP6 were able to reconstitute the particle with both the mutant cores and VP6. These resu...
Composition and Topography of Structural Polypeptides of Bovine Rotavirus
Journal of General Virology, 1981
The composition and topography of the structural polypeptides of bovine rotavirus was studied by polyacrylamide gel electrophoresis of radioactively labelled virus grown in LLC-MK2 cells and by lactoperoxidase-catalysed iodination of single-and double-capsid particles. Bovine rotavirus was found to possess at least six structural polypeptides, three of them associated with the inner capsid (p 102K, p9lK and p45K) and the others with the outer capsid (p84K, p37K and p34K). The most abundant polypeptide of the inner capsid was p45K, which accounted for approx. 80% of the protein mass, followed by p91K (approx. 20% of the protein mass) and pl02K (approx. 1% of the protein mass). Polypeptide 45K is not readily available for iodination, indicating that it is partially covered by p91K, which is the most exposed polypeptide of the inner capsid. The number of polypeptide molecules per single capsid particle (calculated on the basis of an RNA content of 16%) was estimated to be approx. 6 molecules of pl02K, 140 of p91K and 989 of p45K. The stoichiometry and degree of exposure of outer capsid polypeptides was more difficult to establish, even when it appears that p84K and p34K are the most exposed components.
Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction
The Journal of Cell Biology, 1990
The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a "bristled" outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a &...
Virology, 1989
The segmented double-stranded (ds)RNA genome of the rotaviruses is replicated asymmetrically with viral mRNA serving as the template for minus-strand RNA synthesis . To identify intermediate structures in rotavirus replication, subviral particles (SVPs) purified from the cytoplasm of simian rotavirus SAI 1 -infected cells were assayed for RNA polymerase activity in a cell-free system that supports viral RNA replication . Intact SVPs containing newly made RNA were resolved by electrophoresis under nondenaturing conditions on 0 .6% agarose gels (50 mMTris-glycine, pH 8 .8) . This gel system was found to separate without disrupting SA11 single-and double-shelled virions and virion-derived core particles . SVPs from the cell-free system that contained newly made dsRNA migrated in the agarose gels at positions between virien-derived cores and intermediate of single-and double-shelled virions . SVPs containing newly made dsRNA were eluted from the gel and analyzed for protein content by electrophoresis on polyacrylamide gels . The results showed that three distinct types of replication intermediates (Rls) were present in SA11-infected cells . The smallest intermediate (precore RI, 45 nm, 220 S) contained the structural proteins Viol, VP3, and VP9 and the nonstructural proteins NS53, NS35, and NS34 . A second intermediate (core RI, 60 nm, 310 S) contained the core proteins VP1, VP2, and VP3 and the proteins VP9, NS35 and NS34 . The largest RI (single-shelled RI, 75 nm, 420 S) contained the inner shell proteins VP1, VP2, VP3, and VP6 and the proteins VP9, NS35 and NS34 . Analysis of the formation and turnover of Rls in infected cells pulse-labeled with 36 S-amino acids supports a hypothesis that rotavirus single-shelled particles are assembled in vivo by the sequential addition of VP2 and VP6 to precore Rls consisting of VP1, VP3, VP9, NS35, and NS34 . n19e9AieademicPress . Inc .
Rotavirus Proteins: Structure and Assembly
Rotavirus is a major pathogen of infantile gastroenteritis. It is a large and complex virus with a multilayered capsid organization that integrates the deter minants of host specificity, cell entry, and the enzymatic functions necessary for endogenous transcription of the genome that consists of 11 dsRNA segments. These segments encode six structural and six nonstructural proteins. In the last few years, there has been substantial progress in our understanding of both the structural and functional aspects of a variety of molecular processes involved in the replication of this virus. Studies leading to this progress using of a variety of structural and biochemical techniques including the recent application of RNA interference technology have uncovered several unique and intriguing features related to viral morphogenesis. This review focuses on our current understanding of the structural basis of the molecular processes that govern the replication of rotavirus.
Journal of Virology, 1996
It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral in...
Journal of …, 2008
Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T ؍ 13 to T ؍ 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.