Prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoEM structures

Background: Dengue virus along with the other members of the flaviviridae family has reemerged as deadly human pathogens. Understanding the mechanistic details of these infections can be highly rewarding in developing effective antivirals. During maturation of the virus inside the host cell, the coat proteins E and M undergo conformational changes, altering the morphology of the viral coat. However, due to low resolution nature of the available 3-D structures of viral assemblies, the atomic details of these changes are still elusive. Results: In the present analysis, starting from C alpha positions of low resolution cryo electron microscopic structures the residue level details of protein-protein interaction interfaces of dengue virus coat proteins have been predicted. By comparing the preexisting structures of virus in different phases of life cycle, the changes taking place in these predicted protein-protein interaction interfaces were followed as a function of maturation process of the virus. Besides changing the current notion about the presence of only homodimers in the mature viral coat, the present analysis indicated presence of a proline-rich motif at the protein-protein interaction interface of the coat protein. Investigating the conservation status of these seemingly functionally crucial residues across other members of flaviviridae family enabled dissecting common mechanisms used for infections by these viruses. Conclusions: Thus, using computational approach the present analysis has provided better insights into the preexisting low resolution structures of virus assemblies, the findings of which can be made use of in designing effective antivirals against these deadly human pathogens.

Nucleotide sequence of the 3terminal region of belladonna mottle virus (renamed Physalis mottle virus) RNA and analysis of the evolutionary relationships among tymoviral coat proteins

The 3' terminal 1255 nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3' terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addiition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Carbon-13 NMR studies of lipid mobilization pattern in germinating groundnut (Arachis hypogaea L.) seeds

The products of lipid mobilization in groundnut (Arachis hypogaea L.) seeds as a function of time immediately after imbibition are monitored by 13C NMR. Different parts of the embryonic axis, namely,the radicle, hypocotyl, and plumule, exhibit characteristic time dependent 13C NMR spectra observed at 24-h intervals after imbibition. The various stages in the transformation of storage lipids present in different parts of the embryonic axis are clearly demonstrated. The transformaton of storage lipids is completed first in the radicle followed by the hypocotyl and finally the plumule. A mechanism of the transformation of the storage lipids is discussed.

Interaction of Sesbania mosaic virus movement protein with the coat protein - implications for viral spread

Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N- terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement.

Structure of Sesbania Mosaic Virus at 4·7 Å Resolution and Partial Sequence of the Coat Protein

Sesbania mosaic virus (SMV) is a plant virus infecting Sesbania grandiflora plants in Andhra Pradesh, India. Amino acid sequence of the tryptic peptides of SMV coat protein were determined using a gas phase sequenator. These sequences showed identical amino acids at 69% of the positions when aligned with the corresponding residues of southern bean mosaic virus (SBMV).Crystals diffracting to better than 3 Å resolution were obtained by precipitating the virus with ammonium sulphate. The crystals belonged to rhombohedral space group R3 with α = 291·4 Å and α = 61·9°. Three-dimensional X-ray diffraction data on these crystals were collected to a resolution of 4·7 Å, using a Siemens-Nicolet area detector system. Self-rotation function studies revealed the icosahedral symmetry of the virus particles, as well as their precise orientation in the unit cell. Cross-rotation function and modelling studies with SBMV showed that it is a valid starting model for SMV structure determination. Low resolution phases computed using a polyalanine model of SBMV were subjected to refinement and extension by real-space electron density averaging and solvent flattening. The final electron density map revealed a polypeptide fold similar to SBMV. The single disulphide bridge of SBMV coat protein is retained in SMV. Four icosahedrally independent cation binding sites have been tentatively identified. Three of these sites, related by a quasi threefold axis, are also found in SBMV. The fourth site is situated on the quasi threefold axis. Aspartic acid residues, which replace Ile218 of SBMV from the quasi threefold-related subunits are suitable ligands to the cation at this site

Microtensile testing of a free-standing Pt-aluminide bond coat

A finite element method (FEM)-based study has been carried out for the design of flat microtensile samples to evaluate tensile properties of Pt-aluminide (PtAl) bond coats. The critical dimensions of the sample have been determined using a two-dimensional elastic stress analysis. In the present testing scheme, the ratio of the dimensions of the holding length to the fillet radius of the sample was found important to achieve failure within the gage length. The effect of gage length and grip head length also has been examined. The simulation predictions have been experimentally verified by conducting microtensile test of an actual PtAl bond coat at room temperature. The sample design and testing scheme suggested in this study have also been found suitable for evaluation of tensile properties at high temperature. (C) 2010 Elsevier Ltd. All rights reserved.

Primary structure of sesbania mosaic virus coat protein: its implications to the assembly and architecture of the virus

Sesbania mosaic virus (SMV) is a plant virus that infects Sesbania grandiflora plants in Andhra Pradesh, India. The amino acid sequence of the coat protein of SMV was determined using purified peptides generated by cleavage with trypsin, chymotrypsin, V8 protease and clostripain. The 230 residues so far determined were compared to the corresponding residues of southern bean mosaic virus (SBMV), the type member of sobemoviruses. The overall identity between the sequences is 61.7%. The amino terminal 64 residues, which constitute an independent domain (R-domain) known to interact with RNA, are conserved to a lower extent (52.5%). Comparison of the positively charged residues in this domain suggests that the RNA-protein interactions are considerably weaker in SMV. The residues that constitute the major domain of the coat protein, the surface domain (S-domain, residues 65-260), are better conserved (66.5%). The positively charged residues of this domain that face the nucleic acid are well conserved. The longest conserved stretch of residues (131-142) corresponds to the loop involved in intersubunit interactions between subunits related by the quasi 3-fold symmetry. A unique cation binding site located on the quasi 3-fold axis contributes to the stability of SMV. These differences are reflected in the increased stability of the SMV coat protein and its ability to be reconstituted with RNA at pH 7.5. A major epitope was identified using monoclonal antibodies to SMV in the segment 201-223 which contains an exposed helix in the capsid structure. This region is highly conserved between SMV and SBMV (70%) suggesting that it could represent the site of an important function such as vector recognition.

Synthesis of Trilaurin by Developing Pisa Seeds (Actinodaphne hookeri)

The developing seeds of Actinodaphne hookeri were investigated to delineate their ability to synthesize large amounts of trilaurin. Until 88 days after flowering the embryos contained 71% neutral lipids (NL) and 29% phospholipids (PL) and both these components contained C-16:0, C-18:0, C-18:2, and C-18:3 as the major fatty acids (FA). At 102 days after flowering the seeds began to accumulate triacylglycerols (TAG) and to synthesize lauric acid (C-12:0). By 165 days after flowering, when the seeds were mature, they contained about 99% NL and 1% FL. At this stage the TAG contained exclusively C-12:0, while the PL consisted of long-chain fatty acids (LCFA) only. Leaf lipids in contrast did not contain any C-12:0. Experiments on [1-C-14]acetate incorporation into developing seed slices showed that at 88 days after flowering only 4% of the label was in TAG, 1% in diacylglycerols (DAG), and 87% in FL. One hundred two days after flowering seeds incorporated only 2% of the label into TAG, 30% into DAG, and 64% into FL. In contrast at 114 days after flowering 71% of the label was incorporated into TAG, 25% into DAG, and only 2% into FL. Analysis of labeled FA revealed that up to 102 days after flowering it was incorporated only into LCFA, whereas at 114 days after flowering it was incorporated exclusively into C-12:0. Furthermore, 67% of the label in PL at 114 days after flowering was found to be dilaurylglycerophosphate. Analysis of the label in DAG at this stage showed that it was essentially in dilaurin species. These observations indicate the induction of enzymes of Kennedy pathway for the specific synthesis of trilaurin at about 114 days after flowering, Homogenates of seeds (114 days after flowering) incubated with labeled FA in the presence of glycerol-3-phosphate and coenzymes A and ATP incorporated 84% of C-12:0 and 61% of C-14:0, but not C-16:0, C-18:2, and C-18:3, into TAG. In contrast the LCFA were incorporated preferentially into FL. It is concluded that, between 102 and 114 days after flowering, a switch occurs in A. hookeri for the synthesis of C-12:0 and trilaurin which is tissue specific. Since the seed synthesizes exclusively C-12:0 at 114 days after flowering onwards and incorporates specifically into TAG, this system appears to be ideal for identifying the enzymes responsible for medium-chain fatty acid as well as trilaurin synthesis and for exploiting them for genetic engineering. (C) 1994 Academic Press, Inc.

Crystallization and preliminary X-ray diffraction analysis of a galactose-specific lectin from the seeds of Butea monosperma

The galactose-specific lectin from the seeds of Butea monosperma has been crystallized by the hanging-drop vapour-diffusion technique. The crystals belonged to space group P1, with unit-cell parameters a = 78.45, b = 78.91, c = 101.85 A, alpha = 74.30, beta = 76.65, gamma = 86.88 degrees. X-ray diffraction data were collected to a resolution of 2.44 A under cryoconditions (100 K) using a MAR image-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the coordinates of several structures of legume lectins as search models indicate that the galactose-specific lectin from B. monosperma forms an octamer.

Sibling rivalry between seeds within a fruit: Some population genetic models

Competition between seeds within a fruit for parental resources is described using one-locus-two-allele models. While a �normal� allele leads to an equitable distribution of resources between seeds (a situation which also corresponds to the parental optimum), the �selfish� allele is assumed to cause the seed carrying it to usurp a higher proportion of the resources. The outcome of competition between �selfish� alleles is also assumed to lead to an asymmetric distribution of resources, the �winner� being chosen randomly. Conditions for the spread of an initially rare selfish allele and the optimal resource allocation corresponding to the evolutionarily stable strategy, derived for species with n-seeded fruits, are in accordance with expectations based on Hamilton�s inclusive fitness criteria. Competition between seeds is seen to be most intense when there are only two seeds, and decreases with increasing number of seeds, suggesting that two-seeded fruits would be rarer than one-seeded or many-seeded ones. Available data from a large number of plant species are consistent with this prediction of the model.

Spatial distribution of oil in groundnut and sunflower seeds by nuclear magnetic resonance imaging

The nuclear magnetic resonance imaging technique has been used to obtain images of different transverse and vertical sections in groundnut and sunflower seeds. Separate images have been obtained for oil and water components in the seeds. The spatial distribution of oil and water inside the seed has been obtained from the detailed analysis of the images. In the immature groundnut seeds obtained commercially, complementary oil and water distributions have been observed. Attempts have been made to explain these results.

Crystallization and preliminary X-ray diffraction analysis of a galactose-specific lectin from the seeds of Butea monosperma

The galactose-specific lectin from the seeds of Butea monosperma has been crystallized by the hanging-drop vapour-diffusion technique. The crystals belonged to space group P1, with unit-cell parameters a = 78.45, b = 78.91, c = 101.85 A, alpha = 74.30, beta = 76.65, gamma = 86.88 degrees. X-ray diffraction data were collected to a resolution of 2.44 A under cryoconditions (100 K) using a MAR image-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the coordinates of several structures of legume lectins as search models indicate that the galactose-specific lectin from B. monosperma forms an octamer.