Structural studies on the empty capsids of Physalis mottle virus.

The three-dimensional crystal structure of the empty capsid of Physalis mottle tymovirus has been determined to 3.2 A resolution. The empty capsids crystallized in the space group P1, leading to 60-fold non-crystallographic redundancy. The known structure of Physalis mottle virus was used as a phasing model to initiate the structure determination by real-space electron-density averaging. The main differences between the structures of the native and the empty capsids were in residues 10 to 28 of the A-subunit, residues 1 to 9 of the B-subunit and residues 1 to 5 of the C-subunit, which are ordered only in the native virus particles. An analysis of the subunit disposition reveals that the virus has expanded radially outward by approximately 1.8 A in the empty particles. The A-subunits move in a direction that makes 10 degrees to the icosahedral 5-fold axes of symmetry. The B and C-subunits move along vectors making 12 degrees and 15 degrees to the quasi 6-fold axes. The quaternary organization of the pentameric and hexameric capsomeres are not altered significantly. However, the pentamer-hexamer contacts are reduced. Therefore, encapsidation of RNA appears to cause a reduction in the particle radius concomittant with the ordering of the N-terminal arm in the three subunits. These structural changes in Physalis mottle virus appear to be larger than the corresponding changes observed in viruses for which both the empty and full particle structures have been determined.

Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T=3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T=3 capsid assembly. In contrast, the deletion of even a single residue from the C terminus (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.

Three-dimensional structure of physalis mottle virus: implications for the viral assembly.

The structure of the T=3 single stranded RNA tymovirus, physalis mottle virus (PhMV), has been determined to 3.8 A resolution. PhMV crystals belong to the rhombohedral space group R 3, with one icosahedral particle in the unit cell leading to 20-fold non-crystallographic redundancy. Polyalanine coordinates of the related turnip yellow mosaic virus (TYMV) with which PhMV coat protein shares 32 % amino acid sequence identity were used for obtaining the initial phases. Extensive phase refinement by real space molecular replacement density averaging resulted in an electron density map that revealed density for most of the side-chains and for the 17 residues ordered in PhMV, but not seen in TYMV, at the N terminus of the A subunits. The core secondary and tertiary structures of the subunits have a topology consistent with the capsid proteins of other T=3 plant viruses. The N-terminal arms of the A subunits, which constitute 12 pentamers at the icosahedral 5-fold axes, have a conformation very different from the conformations observed in B and C subunits that constitute hexameric capsomers with near 6-fold symmetry at the icosahedral 3-fold axes. An analysis of the interfacial contacts between protein subunits indicates that the hexamers are held more strongly than pentamers and hexamer-hexamer contacts are more extensive than pentamer-hexamer contacts. These observations suggest a plausible mechanism for the formation of empty capsids, which might be initiated by a change in the conformation of the N-terminal arm of the A subunits. The structure also provides insights into immunological and mutagenesis results. Comparison of PhMV with the sobemovirus, sesbania mosaic virus reveals striking similarities in the overall tertiary fold of the coat protein although the capsid morphologies of these two viruses are very different.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles.

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(+/-2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T=3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T=3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

In vitro and in vivo studies on slow release propranolol hydrochloride suppositories. Pharmacokinetic and pharmacodynamic evaluation.

Matrix-based slow release (SR) compressed propranolol HCl (25 mg) suppositories were formulated using PEG 4000 and either stearic acid or bees wax at different concentrations (5, 7.5 and 10%). In vitro studies revealed good slow release characteristics from the suppositories. Their in vivo performances--pharmacokinetics and pharmacodynamics--were evaluated in rabbits. Different pharmacokinetic parameters were determined from the plasma concentration-time profiles using a model-independent computer programme, RAMKIN. The relative bioavailability of propranolol (CAS 525-66-6) from three SR suppositories containing stearic acid, 7.5 and 10% and bees wax, 5%, was 86.4, 87.8, and 83.6% respectively. Pharmacodynamic response (beta-blockade) was assessed by determining the degree of reduction of isoprenaline-induced tachycardia at different time intervals. A minimum concentration of 40-60 ng/ml drug in plasma was maintained during 1-10 h, and there has been a minimum of about 40-50% of beta-blockade during 1-9 h post administration. A good correlation between pharmacokinetic and pharmacodynamic profiles was observed.

Solvent extraction of chromium: A review.

The separation of chromium by solvent extraction is reviewed.

Precipitation of beryllium in presence of succinate by the urea hydrolysis technique.

Beryllium can be precipitated as a dense and easily filterable precipitate in presence of succinate by the urea hydrolysis technique. Calcium, magnesium and manganese do not interfere to the extent that they do in the ammonia precipitation. One advantage over the basic sulphate method is that the oxide is obtained at a considerably lower temperature. However, when ammonia salts are present, the basic sulphate method is to be preferred.

Study of beryllium oxinate by the homogeneous precipition method.

Beryllium oxinate has been proeipitated from homogeneous solutions and the nature of the complex has been studied by thermo gravimetric, infrared and X-ray methods. A structural formula has been proposed on the basis of the results.

Precipitation of beryllium from homogeneous solution in presence of sulphate by hydrolysis of urea.

Beryllium can be precipitated from homogeneous solution as a dense and easily filterable precipitate by the hydrolysis of urea, in the presence of sulphate. The method has been applied to the gravimetric determination of beryllium as oxide. The nature of the precipitate thus obtained has been examined by thermogravimetric, infrared, photomicrographic and X-ray methods.