Structure of sesbania mosaic virus at 4.7 A resolution

Abstract

TRUCTURE OF SESBANIA MOSAIC VIRUS AT 4.7 Å RESOLUTION

X-ray structures of seven RNA plant viruses—tomato bushy stunt virus (TBSV), southern bean mosaic virus (SBMV), satellite tobacco necrosis virus (STNV), bean pod mottle virus (BPMV), cowpea mosaic virus (CPMV), turnip crinkle virus (TCV), and satellite tobacco mosaic virus (STMV)—are known at reasonably high resolution. These studies have provided detailed information regarding coat protein folding and molecular interactions between protein subunits. All these viruses were initially isolated in temperate regions of the world. In contrast, viruses occurring extensively in tropical regions have not been studied.

Although there is no a priori reason to believe that the latter viruses differ from the former in any substantial manner, detailed studies on tropical viruses will provide information on the geographical distribution and evolution of viruses. Also, it might be possible to infer structural adaptations important for multiplication and stability at higher temperatures. Further, the diversity in molecular interactions in related viruses could provide information on the constraints on virus assembly.

With these objectives, I initiated structural studies on Sesbania mosaic virus (SMV), a plant virus infecting Sesbania grandiflora plants in Andhra Pradesh, India.

The thesis begins with a broad review of the structure and assembly of isometric viruses. Significant results of X-ray diffraction studies on spherical viruses are highlighted, concentrating mainly on plant viruses. In the course of these investigations, it was realized that SMV belongs to the sobemovirus group of plant viruses. SBMV, the type member of this group, has been extensively studied. The complete genomic sequence of the SBMV cowpea strain, the amino acid sequence of its coat protein, and the three-dimensional structure of the virus have been determined. The mechanism of assembly of SBMV has also been investigated. These studies are briefly reviewed.

The information available on SMV at the time when these investigations were initiated is also described. A summary of different methods available at present for X-ray diffraction data collection on single crystals of biological macromolecules and a short description of the Nicolet/Siemens area detector system used for collecting X-ray diffraction data on SMV crystals are also presented in Chapter 1.

The second chapter describes the purification, characterization, and crystallization of SMV. SMV was propagated on Sesbania grandiflora plants and purified from infected leaves by differential centrifugation. The purified virus sedimented as a single band in 10–40% sucrose gradient centrifugation. The virus particles appeared spherical under an electron microscope with an approximate diameter of 30 nm, occasionally penetrated by the negative stain.

The UV absorption spectrum of the purified virus was typical of nucleoprotein complexes with ~32% nucleic acid content. The molecular weight of the coat protein subunit, as determined by SDS polyacrylamide gel electrophoresis, was 31,000. The sedimentation coefficient of the virus in 0.05 M sodium acetate buffer, pH 5.6, or in 0.05 M potassium phosphate buffer, pH 8.0, was 117 S. The sedimentation coefficient dropped to 108 S in 0.05 M potassium phosphate buffer, pH 8.0, containing 10 mM EDTA.

In these properties, SMV resembles several other classes of plant viruses where capsid stability is known to depend on bound divalent cations. Crystals diffracting to better than 3.0 Å resolution were obtained by precipitating the virus (25–50 mg/ml in 0.1 M sodium acetate buffer, pH 5.6, containing 10 mM dithiothreitol and 10 mM magnesium chloride) with ammonium sulfate. The crystals were characterized on an Elliot rotating anode X-ray generator using screenless precession photography. The crystals belong to the rhombohedral space group R3 with a=291 A? a=291 A ? and ?=62? ?=62 ? . The unit cell contains one molecule of the virus particle.

Chapter 3 deals with the strategies used for recording and processing X-ray diffraction data on SMV crystals using a Nicolet/Siemens area detector system. Crystal-to-detector distances of 22–28 cm and oscillation angles of 0.2–0.25° were used for recording the data. The exposure time per frame was 20–30 min. Three independent data sets were collected on native SMV crystals. Large unit cell dimensions and the pseudo-cubic nature of SMV crystals, along with a probable error in the XENGEN package (version 1.3), posed problems in indexing and subsequent processing of X-ray diffraction data. Area detector frames were processed assuming a triclinic system, and reflection intensities related by rhombohedral symmetry were subsequently averaged. This strategy could be generally useful for crystals with pseudosymmetry.

The area detector data gave a merging R-factor of 6–8% (on intensities) for Friedel-equivalent reflections within a data set. The R-factor obtained by averaging the intensities of reflections related by R3 symmetry was 9–15%. Different data sets scaled together gave an R-factor of 15–20% and a correlation coefficient of 0.96–0.98.

The symmetry of the virus particle, as established by self-rotation function studies, and the probable similarity of SMV to other known viruses, as revealed by cross-rotation function studies, are described in Chapter 4. Self-rotation function peaks were consistent with the anticipated icosahedral symmetry of the virus particle. The orientation of the particle in the unit cell was initially determined by self-rotation function peaks and subsequently improved by computations of locked-rotation functions. Cross-rotation function studies strongly indicated a structural similarity between SMV and SBMV. The quality of cross-rotation function peaks was comparable to that of SMV self-rotation function peaks. These results suggested that a model of SBMV suitably placed in the SMV cell could be a valid starting model for the determination of SMV by molecular replacement technique.

Chapter 5 gives an account of the strategies used for the structure determination of SMV. During the initial stages of the project, attempts were made to solve the phase problem using the techniques of both multiple isomorphous replacement (MIR) and molecular replacement (MR). Low-resolution (8 Å) data were collected on crystals of SMV soaked in mercuric chloride and lead acetate. However, attempts for structure determination by MIR were abandoned when it was realized that it is possible to solve the phase problem more easily by MR. A molecule of SBMV suitably placed in the SMV cell was used as a starting model.

Detailed examination of packing revealed that an SBMV-like particle fits the SMV cell with only a few short contacts, although the SMV cell volume is about 25% less than that of SBMV. Phases were computed for both an SBMV polyalanine chain and a full chain model placed in the SMV cell. Phases from the polyalanine model were accepted up to 7.5 Å resolution and refined by density averaging, following established protocols. Phase refinement and extension were carried out in several steps. At the end of refinement, the crystallographic R-factor and correlation coefficient were 15.8% and 0.90, respectively. Phase refinement and extensions were performed using different strategies; however, irrespective of the strategies used, the final phases were found to converge to unique values.

Analysis of the final electron density map (4.7 Å) revealed a polypeptide fold remarkably similar to that of SBMV. The polypeptide chain was easily traceable with the help of the known structure of SBMV. All the helical and most of the ?-strand segments were resolved in the map. There were very few breaks in the electron density map. Occasional overflow of density was observed in ?-strands. The single disulfide bridge of the SBMV coat protein appears to be retained in SMV. Four icosahedrally independent cation binding sites have been tentatively identified. Three of these sites, related by a quasi three-fold axis, are also found in SBMV. The fourth site is situated on the quasi three-fold axis and is not found in SBMV. Aspartic acid residues, which replace Ile 218 of SBMV from the quasi three-fold related subunits, are suitable ligands to the cation at this site (Chapter 6).

Experiments performed to confirm the proposed cation binding sites and the results of corresponding difference Fourier studies are presented in Chapter 7. SMV crystals were grown in the presence of EDTA. These crystals were more fragile and radiation-sensitive when compared to native crystals. X-ray diffraction data at 5.5 Å resolution were collected on these crystals using the Nicolet/Siemens area detector system. A difference Fourier map was computed using the observed differences between the structure factor amplitudes of native and demetallized SMV data and refined phases from the SMV native structure. The difference Fourier map clearly indicated that only the cation in the BC subunit interface was substantially removed by the chelating agent. This is a rather surprising and novel result. The implications of these results on the assembly of viruses, in light of similar experiments performed on other viruses, are also discussed in this chapter.

Future prospects of these investigations are briefly presented in the concluding section.