Search PubMed⌕ Search

Biomedical subjects

B V Prasad

Publications and source records attributed to B V Prasad.

At least 37 records · Page 2Linked to original sources
PubMed1997

Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes.

An essential oncogenic determinant of subgroup D human adenovirus type 9 (Ad9), which uniquely elicits estrogen-dependent mammary tumors in rats, is encoded by early region 4 open reading frame 1 (E4 ORF1). Whereas Ad9 E4 ORF1 efficiently induces transformed foci on the established rat embryo fibroblast cell line CREF, the related subgroup A Ad12 and subgroup C Ad5 E4 ORF1s do not (R. T. Javier, J. Virol. 68:3917-3924, 1994). In this study, we found that the lack of transforming activity associated with non-subgroup D adenovirus E4 ORF1s in CREF cells correlated with significantly reduced protein levels compared to Ad9 E4 ORF1 in these cells. In the human cell line TE85, however, the non-subgroup D adenovirus E4 ORF1s produced protein levels higher than those seen in CREF cells as well as transforming activities similar to that of Ad9 E4 ORF1, suggesting that all adenovirus E4 ORF1 polypeptides possess comparable cellular growth-transforming activities. In addition, searches for known proteins related to these novel viral transforming proteins revealed that the E4 ORF1 proteins had weak sequence similarity, over the entire length of the E4 ORF1 polypeptides, with a variety of organismal and viral dUTP pyrophosphatase (dUTPase) enzymes. Even though adenovirus E4 ORF1 proteins lacked conserved protein motifs of dUTPase enzymes or detectable enzymatic activity, E4 ORF1 and dUTPase proteins were predicted to possess strikingly similar secondary structure arrangements. It was also established that an avian adenovirus protein, encoded within a genomic location analogous to that of the human adenovirus E4 ORF1s, was a genuine dUTPase enzyme. Although no functional similarity was found for the E4 ORF1 and dUTPase proteins, we propose that human adenovirus E4 ORF1 genes have evolved from an ancestral adenovirus dUTPase and, from this structural framework, developed novel transforming properties.

Adenovirus E4 Proteins↗
PubMed1996

The structure of aquareovirus shows how the different geometries of the two layers of the capsid are reconciled to provide symmetrical interactions and stabilization.

BACKGROUND: Aquareoviruses are important pathogens of aquatic animals and have severe consequences in aquaculture. These viruses belong to the family Reoviridae. A structural feature common to members of the Reoviridae is a multilayered capsid, formed by several concentric icosahedral shells with different protein compositions. How these proteins, which often are present in unequal stoichiometries, interact between icosahedral layers to stabilize the capsid is not well understood. RESULTS: We have determined the three-dimensional structure of aquareovirus to 23 A resolution using electron cryomicroscopy and computer image analysis. The protein capsid is composed of two structurally distinct icosahedral layers: an outer layer approximately 100 A thick, with incomplete T=13 left-handed symmetry, surrounds an inner layer 600 A in diameter that has T=1 symmetry and is perforated by channels near the fivefold axes. There are 120 subunits, arranged in dimers, in the inner layer, each of which interacts with two of the 600 subunits in the outer layer. A separate set of closely interacting proteins forms the fivefold axes of the virus structure, forming continuous density throughout both layers of the capsid. Comparison of full and empty (lacking RNA) virus structures reveals an RNA shell that lies directly beneath the inner layer. CONCLUSIONS: Our aquareovirus structure displays marked similarity to the mammalian reovirus intermediate subviral particles, suggesting a close evolutionary relationship. However, the noticeable distinction is that aquareovirus lacks the hemagglutinin spike observed in reovirus. The T=1 inner layer organization observed in the aquareovirus appears to be common to other members of the Reoviridae. Such organization may be of fundamental significance in the endogenous transcription of the genome in these viruses.

Animals↗
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

Three-dimensional structure of scaffolding-containing phage p22 procapsids by electron cryo-microscopy.

The procapsids of bacterial viruses are the products of the polymerization of coat and scaffolding subunits, as well as the precursors in DNA packaging. Electron cryo-microscopy has been used to study the three-dimensional structures of bacteriophage P22 procapsids containing wild-type and mutant scaffolding proteins. The scaffolding mutant structure has been resolved to 19 A resolution and agrees with the 22 A resolution wild-type procapsid reconstruction. Both procapsid reconstructions contain an outer icosahedral coat protein shell and an inner scaffolding protein core. The outer core protein forms a T = 7 icosahedral lattice with distinctive channels present at the centers of the pentons and hexons. In addition, the hexons display a prominent skew. Computational isolation of the skewed hexon shows the presence of a local 2-fold axis that reduces the number of unique conformations in the asymmetric unit to four at this resolution. We have classified the four unique subunits into three distinct classes, based upon the shape of the upper domain and the presence of a channel leading to the inner coat protein surface. In addition, at the inner surface of the coat protein, finger-like regions that extend towards the scaffolding protein core are present in two of the subunits. The finger-like regions suggest the presence of an ordered interaction between the inner coat protein and the scaffolding protein. However, an icosahedral scaffolding protein shell is not formed, and the innermost scaffolding protein core does not pack with icosahedral symmetry.

Bacteriophage P22↗
PubMed1996

Structure of Norwalk virus.

Norwalk virus is the major cause of epidemic viral gastroenteritis of humans. Attempts to grow this virus in laboratory cell lines have been unsuccessful. However, the Norwalk virus capsid protein, when expressed in insect cells infected with a recombinant baculovirus, spontaneously assembles into virus-like particles. We have determined the 3-dimensional structure of baculovirus-expressed Norwalk virus using electron cryomicroscopy and computer image reconstruction to a resolution of approximately 22 A. These particles, having a diameter of 380 A exhibit T = 3 icosahedral symmetry. The 3-dimensional structure is composed of 90 dimers of the 58000 molecular weight (58 K) capsid protein, each of which forms an arch-like capsomere. The structure of the protein subunit is modular with three distinct domains. The distal globular domain that appears bilobed is connected to the lower shell domain by a central stem-like domain. We also have been able to grow crystals of the baculovirus-expressed Norwalk virus particles suitable for high resolution X-ray crystallography.

Capsid↗
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

Protein subunit structures in the herpes simplex virus A-capsid determined from 400 kV spot-scan electron cryomicroscopy.

The three-dimensional structure of the A-capsid of herpes simplex virus type-1 has been determined to a resolution of approximately 26 A by using 400 kV spot-scan electron cryomicroscopy and computer image reconstruction techniques. The density map of the capsid has revealed several new structural details in the protein subunits of pentons, hexons, and triplexes. Our structural analysis has provided further evidence for the assignment of the four major capsid proteins to these various subunits. VP5, a 150 kDa major capsid protein that makes up both the penton and the bulk of the hexon subunits, has three domains: an upper diamond-shaped domain, a middle stem-like domain, and a lower anchoring domain. Structural differences are noticeable between the VP5 subunits in various quasi-equivalent environments. A horn-shaped mass density present at the distal end of each hexon subunit but missing from the penton subunit has been assigned to VP26, a minor 12 kDa protein. The six types of triplexes have similar, but not identical, features that include two legs and an upper domain that has a tail, which are interpreted to be formed from two copies of VP23 (36 kDa) and one copy of VP19c (57 kDa), respectively. Each triplex has two arms that interact with the adjacent VP5 subunits, and the modes of interaction vary among the quasi-equivalent triplexes. The 25 A-thick floor of the capsid is formed by the close association of the lower domains of subunits from the hexons, pentons, and triplexes. The interior of the capsid is accessible through the trans-capsomeric channels and the holes at the base of each triplex. These openings may play a role in the transport of genomic DNA and scaffolding proteins during capsid morphogenesis.

Animals↗
PubMed1994

Three-dimensional structure of calicivirus.

The Caliciviridae comprise a new family of single-stranded RNA viruses. While human caliciviruses cause gastroenteritis, the animal caliciviruses cause a wide range of diseases. We have determined the three-dimensional structure of a primate calicivirus using electron cryomicroscopy and computer image-processing techniques. Calicivirus is one of the rare animal viruses whose capsid is made of a single structural protein. The three-dimensional structure of the virus is distinct from that of any other animal virus. However, there are several architectural similarities with plant viruses such as tomato bushy stunt virus and turnip crinkle virus. The calicivirions are 405 A in diameter and exhibit T = 3 icosahedral symmetry. The main features of the three-dimensional structure are the 32 large surface hollows, 50 A deep and 90 A wide, at the icosahedral 5-fold and 3-fold axes, and the 90 distinctive arch-like capsomeres surrounding these hollows at the local and strict 2-fold axes. Each capsomere is a dimer of the capsid protein. Despite noticeable differences, the three quasi-equivalent subunits show common structural features: the upper bilobed domain, the central stem domain, and the lower shell domain. The 2-fold related capsid proteins interact through the bilobed domains to form the top of the arch. The structural differences between the connectors of the stem and the shell domain among the three subunits suggest the presence of a hinge region that may facilitate the capsid protein to adapt to the three quasi-equivalent environments of the T = 3 icosahedral structure. The shell domains of the pentavalent and hexavalent capsid proteins associate to form a continuous shell between the radii of 115 and 150 A. A beta-barrel structure has been suggested for the shell domain. The mass density in the inner shell between the radius of 85 and 110 A may contain a portion of the capsid protein interacting with the RNA. The features between the 45 and 85 A radius are suggestive of ordered RNA.

Animals↗
PubMed1994

Characterization of rotavirus VP2 particles.

Rotavirus particles consist of three concentric proteinaceous capsid layers. The innermost capsid (core) is made of VP2. The genomic RNA and the two minor proteins VP1 and VP3 are encapsidated within this layer. Empty rVP2 particles are produced when insect cells are infected with a recombinant baculovirus which contains the bovine Rf rotavirus gene 2 (Labbé et al., 1991, J. Virol. 65, 2946-2952). Analysis of expressed rVP2 particles by SDS-PAGE showed these particles were composed of three major VP2-related proteins, called bands A, B, and C, with apparent molecular weights of 94K, 85K, and 77K, respectively. N-Terminal amino acid sequence analysis of each band showed that band A and band B were blocked, and band C lacked 92 amino acids from the N terminus. Bands B and C were predicted to also lack an approximately 10K peptide fragment from the C terminus. Electron microscopy (EM) showed negatively stained rVP2 particles to be spherical with icosahedral symmetry, 520 +/- 20 A in diameter. Highly concentrated rVP2 particles were converted to unusual forms, including elongated bristly structures, helix-like structures, and sheet-like helix structures. These unusual forms apparently resulted from a structural conversion of individual rVP2 particles. This conversion was reversible both in solution or on a collodion-carbon-coated grid support. The reconstituted rVP2 particles possessed normal morphology and reacted with purified VP6 to form rVP2/6 empty double-layered (previously called single-shelled) virus-like particles with an association constant Ka approximately 10(11) M-1. Native viral core particles lacking RNA were obtained by dialysis of full cores prepared from purified SA11-4F rotavirus double-layered particles against a hypotonic buffer in the presence of EDTA. EM showed both the full and empty native viral cores to be spherical with icosahedral symmetry. Highly concentrated SA11-4F full and empty cores also were converted into elongated and bead-like structures. However, in contrast to rVP2 particles, the conversion of SA11-4F cores was not reversible. These results provide some helpful clues to understanding VP2 functions, the assembly of VP2 particles, the assembly of VP2/6 double-layered particles, and the transport of metabolites inside and outside of the core particle.

Amino Acid Sequence↗
PubMed1994

Structural studies of viruses by electron cryomicroscopy.

Electron cryomicroscopy is a unique biophysical technique for studying molecular structures of viruses which are difficult to analyze by x-ray diffraction. The structural information derived from the low resolution reconstructions of viruses has so far been useful to understand various functional properties of the viruses such as antibody neutralization, receptor binding and assembly. Electron cryomicroscopy has enabled the visualization of the four core alpha helices of the coat protein in tobacco mosaic virus. This represents the highest resolution detail of a virus studied by electron cryomicroscopy. The prospects of attaining similar resolution beyond 10 A for spherical viruses as well are encouraging, with newly available instrumentation, data collection and processing procedures.

Cryopreservation↗
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↗
PubMed1993

Three-dimensional transformation of capsids associated with genome packaging in a bacterial virus.

Three-dimensional structures of the empty procapsid and the mature capsid of the Salmonella bacteriophage P22 have been determined to a resolution of 28 A using electron cryomicroscopy and computer image processing. The coat subunits in both the structures are arranged as pentamers and hexamers on a T = 7 icosahedral lattice. The two structures display significant differences in shape, size and intersubunit interactions. The empty procapsid is spherical in contrast to the distinctly larger and polyhedral mature capsid. The empty procapsid structure exhibits holes at all the quasi sixfold positions that are absent in the mature capsid. These holes may be the exit ports for scaffolding subunits. Detailed comparisons of the two structures indicate that extensive structural changes take place during maturation in all seven quasi-equivalent subunits. These changes cause flattening of the icosahedral facets, capsid expansion and closing of the holes. This process results in a stable and impenetrable capsid that protects the bacterial genome.

Bacteriophage P22↗
PubMed1993

Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].

The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.

Amino Acid Sequence↗
PubMed1993

Teaching electron diffraction and imaging of macromolecules.

Electron microscopic analysis can be used to determine the three-dimensional structures of macromolecules at resolutions ranging between 3 and 30 A. It differs from nuclear magnetic resonance spectroscopy or x-ray crystallography in that it allows an object's Coulomb potential functions to be determined directly from images and can be used to study relatively complex macromolecular assemblies in a crystalline or noncrystalline state. Electron imaging already has provided valuable structural information about various biological systems, including membrane proteins, protein-nucleic acid complexes, contractile and motile protein assemblies, viruses, and transport complexes for ions or macromolecules. This article, organized as a series of lectures, presents the biophysical principles of three-dimensional analysis of objects possessing different symmetries.

Biophysics↗