Biomedical subjects

R Rothnagel

Publications and source records attributed to R Rothnagel.

7 recordsLinked to original sources

PubMed1996

Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus.

In double-stranded-RNA (dsRNA) viruses found in animals, bacteria and yeast, the genome is transcribed within the structurally intact core of the virion with extraordinary efficiency. The structural organization of the genome and the enzymes involved in the transcription inside any of these viruses, critical for understanding this process, is not known. Here we report what we believe is the first three-dimensional characterization of the viral genome and the transcription complex in a prototypical dsRNA virus. Rotavirus is a large (diameter 1,000 A) icosahedral virus composed of three capsid protein layers and 11 dsRNA segments. It is the most important cause of gastroenteritis in children, accounting for over a million deaths annually. We show that viral dsRNA forms a dodecahedral structure in which the RNA double helices, interacting closely with the inner capsid layer, are packed around the enzyme complex located at the icosahedral 5-fold axes. The ordered RNA accounts for about 4,500 out of a total 18,525 base pairs in the genome, the largest amount of icosahedrally ordered RNA observed in any virus structure to date. We propose that the observed organization of the dsRNA is conducive for an orchestrated movement of the RNA relative to the enzyme complex during transcription.

Cell Line↗

PubMed1996

Rotavirus structure: interactions between the structural proteins.

Structural studies on rotavirus using electron cryomicroscopy and computer image analysis have permitted visualization of each shell in the triple-layered rotavirus structure. Biochemical results have aided our interpretation of the structural organization of these layers and protein interactions seen in the three-dimensional structure, and have provided a better understanding of the structure-function relationships of the rotavirus structural proteins.

Capsid↗

PubMed1995

Expression of tobacco ringspot virus capsid protein and satellite RNA in insect cells and three-dimensional structure of tobacco ringspot virus-like particles.

The capsid protein gene of tobacco ringspot virus (TobRV), which had been modified to contain an amino-terminal methionine codon, was ligated into a baculovirus transfer vector downstream from the polyhedrin promoter. The resulting plasmid was cotransfected with linearized baculovirus DNA into insect cells. Recombinant baculovirus expressed high levels of the TobRV capsid protein that assembled to form virus-like particles that were similar in size and shape to authentic TobRV capsids. These virus-like particles did not encapsidate any RNA, including the capsid protein mRNA. The capsid protein mRNA is a truncated RNA 2, which may lack a putative encapsidation signal. To determine whether an intact packaging substrate could be encapsidated by the TobRV capsid protein, another recombinant baculovirus, concomitantly expressing both capsid protein and TobRV satellite RNA, was constructed. Surprisingly, the vast majority of the satellite RNA molecules expressed from this recombinant baculovirus were ligated in the insect cells to form circular RNA molecules. Like circular forms of satellite RNA generated in planta, these circular satellite molecules remained unencapsidated by the TobRV capsid protein. Computer-generated three-dimensional reconstruction using electron cryomicrographs of the empty virus-like particles allowed the first structural analyses of any nepovirus capsid. This 22-A resolution reconstruction resembled capsids of other members of the picornavirus superfamily. These data support the hypothesis that the nepovirus capsid is structurally analogous to those of the como- and picornaviruses.

Animals↗

PubMed1994

Electron cryomicroscopy and digital image processing of lipoprotein(a).

Electron cryomicroscopy was used to study the structure of human lipoprotein(a) (Lp(a)), a plasma lipoprotein implicated in cardiovascular disease. An individual Lp(a) particle consists of a neutral lipid core within a shell of phospholipid, cholesterol and glycoprotein. In principle, electron cryomicroscopy images of single particles should contain structural detail attributable to the density differences among these components and the surrounding buffer. We observed such structural detail in images of frozen, hydrated Lp(a) particles. Lp(a) particles appeared to be roughly spherical in shape with an average diameter of 210 A. As is generally true for unstained samples in vitreous ice, imaged with a low electron dose, these images have low contrast with low signal-to-noise ratios. To increase the signal-to-noise ratio, we averaged classes of similar particles. We began with a set of 5813 randomly oriented Lp(a) particles and generated classes using a linear multivariate statistical method, followed by hierarchical ascendant classification. Our initial classification, based on only the first eight eigenvectors, separated particles on the basis of gross size and shape. After a rough reference-free alignment step, a second classification used the finer details in the images. This approach yielded class averages with structural detail only faintly visible in the raw, single images.

Freezing↗

PubMed1994

Three-dimensional structure of baculovirus-expressed Norwalk virus capsids.

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.

Baculoviridae↗

PubMed1993

Three-dimensional structure of a membrane-containing virus.

The structure of Sindbis virus was determined by electron cryomicroscopy. The virion contains two icosahedral shells of viral-encoded proteins separated by a membrane bilayer of cellular origin. The three-dimensional structure of the ice-embedded intact Sindbis virus, reconstructed from electron images, unambiguously shows that proteins in both shells are arranged with the same icosahedral lattice of triangulation number T = 4. These studies also provide structural evidence of contact between the glycoprotein and the nucleocapsid protein across the membrane bilayer. The structural organization of Sindbis virus has profound implications for the morphogenesis of the alphaviruses. The observed interactions confirm stoichiometric and specific protein associations that may be crucial for virion stability and predict a mechanism for assembly.

Animals↗

PubMed1993

Three-dimensional visualization of the rotavirus hemagglutinin structure.

Three-dimensional structures of a native simian and reassortant rotavirus have been determined by electron cryomicroscopy and computer image processing. The structural features of the native virus confirm that the hemagglutinin spike is a dimer of VP4, substantiated by in vivo radiolabeling studies. Exchange of native VP4 with a bovine strain equivalent results in a poorly infectious reassortant. No VP4 spikes are detected in the three-dimensional reconstruction of the reassortant. The difference map between the two structures reveals a novel large globular domain of VP4 buried within the virion that interacts extensively with the intermediate shell protein, VP6. Our results suggest that assembly of VP4 precedes that of VP7, the major outer shell protein, and that VP4 may play an important role in the receptor recognition and budding process through the rough endoplasmic reticulum during virus maturation.

Animals↗