Structural studies on physalis mottle tymovirus

Abstract

X-ray diffraction studies on single crystals of viruses enable the visualization of the structures of intact virus particles at near-atomic resolution. These studies provide detailed information regarding the coat protein folding, molecular interactions between protein subunits, plausible sites of receptor recognition, the role of metal ions in the capsid structure, and the assembly and disassembly processes. The present thesis deals with structural studies on a small spherical plant virus, Physalis mottle virus (PhMV).

PhMV is a highly infectious single-stranded RNA virus with a coat protein architecture based on a T=3 icosahedral lattice and belongs to the tymovirus group of isometric plant viruses. The virus particles have a diameter of about 300 Å. Natural preparations of tymoviruses are found to contain two classes of particles that sediment at different velocities through sucrose density gradients and are called the top (54S) and bottom (115S) components. The top component particles are either devoid of RNA or contain only a small subgenomic RNA, while the bottom component particles contain the full genomic RNA. Polyamines such as spermine, spermidine, putrescine, and cadaverine appear to be responsible for the neutralization of the negative charges on the RNA phosphates. Replacement of these polyamines by monovalent cations leads to the release of nucleic acid and formation of empty protein shells at alkaline pH.

In an earlier phase of work, PhMV purified from infected plant tissue was crystallized in the space group R3 (a=294.56 Å, ?=59.86°). X-ray diffraction data to 3.8 Å resolution were recorded on films by screenless oscillation photography. The films were processed, and the data were merged and scaled (Overall completion 40%; Rmerge =14.6%). The resulting data were used to establish the icosahedral symmetry of PhMV particles and to determine the precise orientation of particles in the crystal cell (Munshi et al., 1987; Hiremath et al., 1990). The gene coding for PhMV coat protein (PhCP) and several of its deletion mutants were cloned and expressed in BL21 (DE3) cells harboring pET-CP. It was shown that the recombinant intact coat protein and several mutants lacking up to 30 amino acids from the N-terminal end could assemble to form empty shells resembling the natural top component (Sastri et al., 1997).

In this study, a polyalanine model of the homologous Turnip yellow mosaic virus structure was shown to be suitable for PhMV structure determination by cross-rotation function studies and structure factor calculations. Phase refinement by density averaging and solvent flattening was carried out to 3.8 Å in 310 cycles of calculation using a modified computational procedure. In the final rounds of averaging (3.8 Å), the electron density was sampled at 480 and 240 grid points along the cell edges of the R3 crystal in the unaveraged and averaged maps, respectively. The overall R-factor and correlation coefficient at the end of molecular replacement calculations were 30.5% and 0.56, respectively. (R defined as 100 * ?(|F? - F?|)/DF? and correlation coefficient as ?((F? - <F?>) (F? - <F?>)/(2(F? - <F?>)²))*(?(F? - <F?>)²) ¹/²). The overall phase change from the starting values for reflections between the resolution limits of 10-3.8 Å was 58°. Molecular models of the three icosahedrally independent subunits were built into the final averaged map using the interactive graphics program O. The final map accounts for all residues, except 1-10 of the A-subunit and the N-terminal residues of B- and C-subunits. The resulting molecular models were refined using X-PLOR. The R-factor in the final round of X-PLOR refinement for 101,747 reflections included in refinement was 32%. The coat protein structure consists of a flexible N-terminal arm (residues 1-27) and an eight-stranded antiparallel ?-barrel (residues 28-188). The virus particles contain three subunits (A-, B-, and C-) in the icosahedral asymmetric unit. The ordered segment of the N-terminal residues of the A-subunits, which constitute 12 pentamers at the icosahedral 5-fold axes, interact with a hexamer formed by B- and C-subunits. In contrast, the N-terminal residues of B- and C-subunits have a very different conformation. The hexamers are held more strongly than pentamers, and hexamer-hexamer contacts are more extensive than pentamer-hexamer contacts. The structure suggests a plausible mechanism for the release of nucleic acid and the formation of empty capsids triggered by a change in the conformation of the N-terminal arm. 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.

Since the encapsidated genetic material lacks the icosahedral symmetry of the protein shell, it is generally found disordered in X-ray structures of virus crystals. Structural studies on crystals of empty capsids could provide information on the key changes in the structure and organization of coat protein subunits that accompany RNA encapsidation. In this study, crystals of the recombinant capsids were obtained by precipitating the capsids at a concentration of 80-100 mg/ml in 0.5 M sodium acetate buffer, pH 5.6, 10 mM dithiothreitol, and 2 mM CaCl? using 2-4% PEG 8000. The empty capsids crystallized in the space group PI with cell dimensions of a=289.64 Å, b=287.72 Å, c=295.23 Å, ?=63.6°, ?=61.4°, and ?=62.9°, respectively. A deletion mutant lacking 26 residues was crystallized under similar conditions. The cell parameters of the deletion mutant, as well as a new PI crystal form of the native particle, are close to those of the recombinant capsids of intact protein. Data to 3.2 Å resolution were recorded on crystals of recombinant capsids using a MAR imaging plate detector system mounted on a Rigaku RU-200 X-ray generator equipped with a 200p. focal cup. These data were merged and scaled using the program XDS (Overall completion 36%; Rmerge=14.9%). The self-rotation function computed using this data clearly revealed the icosahedral symmetry of the empty capsids. The known structure of native PhMV was used as a phasing model for the structure determination of empty shells to 3.2 Å resolution by real-space electron-density averaging, exploiting the 60-fold non-crystallographic redundancy. The overall R-factor and correlation coefficient after 28 cycles of averaging were 22.4% and 0.82, respectively. The electron-density was easily interpretable due to the availability of the native PhMV structure. Polypeptide models of the three icosahedrally independent subunits built into the averaged density using the program O were refined using CNS, imposing strict non-crystallographic symmetry. The R-factor in the final round of refinement for 263,841 reflections was 27.9%. Compared to the native virus, residues 10-28 in the N-terminal arm of the A-subunit, residues 1-9 in the B-subunit, and residues 1-5 in the C-subunit were disordered in the empty capsids. An analysis of the subunit disposition revealed that the virus has expanded radially outward by 1.8 Å. The A-subunits move in a direction that makes 10° to the icosahedral 5-fold axes of symmetry. The B- and C-subunits move along vectors making 12° and 15° to the quasi 6-fold axes of symmetry. The overall tertiary fold in PhCP is conserved, and the quaternary organization of the pentameric and hexameric capsomeres is not altered significantly. However, the contacts are reduced in the empty shells. Thus, encapsidation of RNA appears to cause the ordering of the N-terminal arm in the three subunits of PhMV. Also, several positively charged side chains facing the RNA become ordered. These structural changes in PhMV appear to be larger than the corresponding changes observed in viruses for which both the empty and full particle structures have been determined. These structures provide a molecular basis for understanding the results of extensive stability studies that have been carried out on tymoviruses.

Traditional classification of plant viruses is based on the range of plants susceptible to viral infection. Due to the availability of databases describing plant viral infections, (Plant virus online: http://biology.anu.edu.au/Groups/MES/vide/ ) it is now possible to examine the relationship between the virus class and the profile of susceptible plants. Such an analysis will also provide information on the natural variation in the host ranges of serologically related viruses. Towards this end, the profiles representing the families of plants infected by different plant viruses were constructed. These profiles were clustered using the PELEUP program of the GCG package, and dendrograms were derived using the UPGMA algorithm. These studies show that very different viruses can have similar infection profiles, and hence, these profiles are not robust indicators of virus identity. The wide variation in the host range of serologically related viruses suggests that plant viruses can easily adapt to different hosts.

A part of the results discussed in this thesis has already been reported in the following publications:

S. Sri Krishna, C. N. Hiremath, S. K. Munshi, M. Sastri, H. S. Savithri, and M. R. N. Murthy (1999), Structure of Physalis mottle virus at 3.8 Å resolution: Implications for the viral assembly, J. Mol. Biol., 289, 919-934.

M. Sastri, S. Reddy, S. Sri Krishna, M. R. N. Murthy, & H. S. Savithri (1999), Identification of a discrete intermediate in the assembly/disassembly of Physalis mottle tymovirus through mutational analysis, J. Mol. Biol., 289, 905-918.

M. R. N. Murthy, S. Sri Krishna, M. Sastri, and H. S. Savithri (1999), Structure, stability, and assembly of Physalis mottle tymovirus, Perspectives in Structural Biology, A volume in honour of G. N. Ramachandran (Editors M. Vijayan, N. Yathindra, A. S. Kolaskar) Chapter 34: 467-484, Indian Academy of Sciences, Universities Press.

S. Sri Krishna, M. Sastri, H. S. Savithri, and M. R. N. Murthy, Structural studies on the empty capsids of Physalis Mottle Virus. (Communicated).