Composition & Architecture of plant viruses. P.N. Sharma Department of Plant Pathology, CSK HPKV, Palampur (H.P.)

Transcription

1 Composition & Architecture of plant viruses P.N. Sharma Department of Plant Pathology, CSK HPKV, Palampur (H.P.)

2 Plant Viruses Classification, Morphology, Genome, and Structure

3 Importance Detailed knowledge of virus structure is important to understand different aspects of virology e.g. how virus survive, infect, spread, replicate and how they are related with one other. Knowledge of virus architecture has increased greatly with the invention of EM, optical defraction, X-ray crystallography procedures; Molecular techniques Chemical information about viruses

4 Morphology of Viruses About ½ of all known plant viruses are elongate (flexuous threads or rigid rods). About ½ of all known plant viruses are spherical (isometric or polyhedral). A few viruses are cylindrical bacillus-like rods.

5 Chemical composition of plant viruses Protein( Capsid) Capsomere Nucleic acids RNA +ve strand RNA -ve strand RNA ssrna dsrna DNA ssdna dsdna

6 Viral Composition Proteins 60-95% of the virion Repeating subunits, identical for each virus type but varies from virus to virus and even from strain to strain TMV subunits amino acids with a mass of 17,600 Daltons (17.6 kda, kd or K) TYMV 20,600 Dalton protein Nucleic acid is 5-40% of the virion Spherical viruses: 20-40% Helical viruses: 5-6%

7 Viral Composition Nucleic acid (5-40%) represents the genetic material, indispensable for replication Nucleic acid alone is sufficient for virus replication Fraenkel-Conrat, Schramm Protein (60-95%) protects virus genome from degradation facilitates movement through the host and transmission from one host to another

8 A/a composition of capsid proteins of some viruses 1. Alanine CMV: 17; PVY: 16 TMV: 14; PVX: Arginine CMV: 24; PVY: 11 TMV: 11; PVX: Asparatic acid CMV: 30; PVY: 22 TMV: 18; PVX: Glutamine CMV: 20; PVY: 23 TMV: 16; PVX: Leucine CMV: 26; PVY: 10 TMV: 12; PVX: Glutamic acid 12. Lysine CMV: 18; PVY: 13 TMV: 2; PVX: Glycine CMV: 16; PVY: 13 TMV: 6 ; PVX: Asparagines 9. histidine CMV: 4; PVY: 4 TMV: - ; PVX: 4 5. Cystein CMV: 0; PVY: 1 TMV: 1; PVX: Isoleucine CMV: 16; PVY: 12 TMV: 9 ; PVX: Methionine CMV: 8; PVY: 8 TMV: 0 ; PVX: Phenylalanine CMV: 7 ; PVY: 5 TMV: 8; PVX: Proline CMV: 18; PVY: 11 TMV: 8 ; PVX: Serine CMV: 32; PVY: 10 TMV: 16; PVX: Tryptophane CMV: 1 ; PVY: 2 TMV: 3; PVX: Tyrosine CMV: 11; PVY: 6 TMV: 4; PVX: Threonine CMV: 17; PVY: 13 TMV: 16; PVX: Valine CMV: 22; PVY: 13 TMV: 14; PVX: 27 Total CMV: 287 PVY: 203 TMV: 158 PVX: 463

9 %age of protein & n/a in some viruses %age of protein & n/a in some viruses Virus n/a (%) Protein (%) TMV 5 95 PVX 6 94 PVY 5 95 CpMV CMV TRSV 40 60

10 Viral Ultrastructure Terminology for virus components Capsid is the protein shell that encloses the nucleic acid Capsomers are the morphological units seen on the surface of particles and represent clusters of structure units Capsid and enclosed nucleic acid is called the nucleocapsid The virion is the complete infectious virus particle Caspar, D. L. D. and Klug, A. (1963) "Structure and Assembly of Regular Virus Particles." In Viruses, Nucleic Acids, and Cancer, 17th Annual Symposium on Fundamental Cancer Research, University of Texas, Williams and Wilkins, Baltimore, pp

11 Watson and Crick In 1956 proposed: Amount of the virus nucleic acid was insufficient to code for more than a few proteins of limited size Therefore the protein shell must be of identical subunits Subunits had to be arranged to provide each with an identical environment, i.e., symmetrical packing

12 Virus Architecture Detailed knowledge of virus structure is important to understand different aspects of virology Knowledge of virus architecture has increased greatly with the innovation like EM, optical defraction, X-Ray crystallography procedures, mol. techniques and chemical nature of the virus.

13 Various feature of viruses can be estimated by studying: Chemical & biochemical studies Size of particles Hydrodynamics Laser scattering has been used to determine the radii of spherical viruses E.M. X-ray crystallography it gives accurate estimates of radius of icosahedral viruses but condition is that the virus should be able to form stable crystals.

14 Electron microscopy In 1924 L. de BROGLIE discovered the wave-character of electron rays thus giving the prerequisite for the construction of the electron microscope. Invented by M. KNOLL and E. RUSKA (Technische Universität Berlin, 1932). One of the first biological objects observed was the tobacco mosaic virus (TMV). The first picture of a cell was published in 1945 by K. R. PORTER, A. CLAUDE and E. F. FULLAM (Rockefeller Institute, New York). The Transmission Electron Microscope (TEM) The Scanning electron microscope (SEM)

15 The Transmission Electron Microscope (TEM)

16 The Transmission Electron Microscope (TEM) A 1973 Siemens electron microscope, EM developed by E. Ruska 1933

17 Fine structures determination E.M. Metal shadow preparations: using heavy metals, it enhances the contrast of particles Freeze drying: useful about surface details particularly with lipid protein bilayer mambranes (Large viruses) Negative staining: the use of electron dense stains is more important than heavy metals shadowing for morphological details. Such stains may be +ve or ve

18 Positive stains React chemically with and are bound to virus surface e.g. various Osmium, lead and uranyl compounds and phosphotungustic acid (PTA) are used under appropriate conditions. However, the chemical reaction may alter or disintegrate the virus so ve stains are more important Negative stains: Fine structures determination They do not react with the virus but penetrate available spaces on the surfaces or with in virus particle e.g. Uranylacetate or Potassium phosphotungstate (KPT) are used near ph 5.0

19 Fine structures determination Thin sections Cryo EM X-ray crystallography analysis Neutron small angle scattering: neutron scattering by virus solution is a method by which low resolution information can be obtained about structure of virus. E.g. important for radii of isometric particles Mass spectrography Serological method's Gel diffusion ELISA ISEM

20 Methods for studying stabilizing bonds The primary structure of viral CP & n/a depends upon covalent bonds. Three kinds of interactions are involved in viruses : Protein : protein Protein : RNA RNA : RNA These help the CP and n/a to be held together precisely In addition, small molecules e.g. divalent metal ions (CA2+ in particular) have marked effects on the stability of some viruses. These interactions determine how much the virus is stable How it might be assembled during virus synthesis How viral n/a is released following infection of cell

21 Methods for studying stabilizing bonds The stabilizing interactions are hydrophobic bonds, H= bonds, salt linkage etc. these interactions cab be studied by: X-ray crystallography Stability to chemicals and physical agents: e.g. Phenol, urea, temperature and detergents etc. Chemical modification of CP: a/a changes Removal of ions: in viruses whose structure are stabilized by Ca2+ ions can be affected by their removal e.g. in isometric particles, CA2+ ions removal by EDTA causes swelling of the particles. So this phenomenon can give information about the kind of bond important fro virus stability.

22 Methods for studying stabilizing bonds Circular dichroism: Spectra can be used to obtain estimates of the extent of a- helix and B- structure in a viral protein subunit. n/a tests

23 Architecture of rod shaped viruses Crick & Watson (1956) put forwarded a hypothesis regarding structures of small viruses (TYMV & TMV) that: Viral RNA enclosed in CP Naked RNA is infectious Basic requirement is protein shell to protect n/a etc. In rod shaped viruses, the protein subunits are arranged in a helical manner regardless of protein subunit number into a helical array.

24 X-ray crystallography X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and diffracts into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a threedimensional picture of the density of electrons within the crystal. From this electron density we can determined: the mean positions of the atoms in the crystal, as well as their chemical bonds, their disorder and various other information.

25 X-ray sources The brightest and most useful X-ray sources are synchrotrons A protein crystal seen under amicroscope. Crystals used in X-ray crystallography may be smaller than a millimeter across. Workflow for solving the structure of a molecule by X- ray crystallography.

26 Diffractometer A Diffractometer is a measuring instrument for analyzing the structure of a material from the scattering pattern produced when a beam of radiation or particles (as X rays or neutrons) interacts with it. Principle Because it is relatively easy to use electrons or neutrons having wavelengths smalle r than a nanometer, electrons and neutrons may be used to study crystal structure in a manner very similar to X-ray diffraction. Electrons do not penetrate as deeply into matter as X-rays, hence electron diffraction reveals structure near the surface; neutrons do penetrate easily and have an advantage that they possess an intrinsic magnetic moment that causes them to interact differently with atoms having different alignments of their magnetic moments. An X-ray diffraction pattern of a crystallized enzyme. The pattern o spots (called reflections) can be used to determine the structure of the enzyme.

27 TMV TMV particles are: Rigid helical rods 300 nm long X 18 nm dia 95% protein & ~5% n/a (RNA) ssrna Extremely stable structure Retain infectivity at room temp. for ~50 years Naked RNA is highly unstable like others.

28 Detailed worked by using X-ray defraction gave details of arrangement of protein subunits and RNA in rod. The particles comprises ~2130 subunits that are closely packed in a helical array. The pitch of helix is 2.3 (fig.) and the RNA chain is compactly coiled in a helix following that of the protein subunits There are 49 nt. & 161/3 protein subunits per turn The PO4 of the RNA are at about 4nm from the rod axis. The helix of TMV is right handed (Finch, 1972)

29 TMV architecture Negatively stained particles revealed that : One end of the rod can be seen as concave The other end is convex 3 end of the RNA is at the convex end & 5 at concave end (Wilson wt al. 1976; Butler et al., 1977) A central canal with a radius of ~2nm becomes filled with stain in vely stained preparations Short Rods: of variable length & <300nm, causes problem of end to end aggregation etc.

30 SYMPTOMS OF TMV

31 Rod shaped particles Helix (rod) e.g., TMV TMV rod is 18 nanometers (nm) X 300 nm

32 PARTICLE STRUCTURE TMV rod is 18 nanometers (nm) X 300 nm Tobacco mosaic virus is typical, well-studied example Each particle contains only a single molecule of RNA (6395 nt) and 2130 copies of the coat protein subunit (158 aa; 17.3 kda) 3 nt/subunit subunits/turn 49 subunits/3 turns TMV protein subunits + nucleic acid will self-assemble in vitro in an energy-independent fashion Self-assembly also occurs in the absence of RNA

33 Tobacco mosaic virus

34 Properties of coat proteins CP consists of 158 amino acid with a mol. Wt of ~17-18 KDa. Fibre defraction have determined the structure to 2.0oA resolution (Namba et al., 1989) The protein has high proportion of secondary structures with 50%of the residues form four a- helices and 10% of residues in B-turns. The four closely parallel and antiparallel a- helices (residues 20-32, 38-48, & ) make up the core of the subunits. And the distal end of the four helices are connected transversely by a narrow and twisted strip of b-sheet.

35 Properties of coat proteins The central part of the subunit distal to the b- sheet is a cluster aromatic residues (Phe12, Trp17, Phe62, Tyr70, Tyr139, Phe144) forming a hydrophobic patch. The N- & C- termini of the protein are to the outside of the particle The polypeptide chain is in a flexible or disordered state below a radius in t particle of about 4nm so that no structure is revealed in this region.

36 Properties of coat proteins One of the reassembly product of TMV protein subunit is a double disk containing two rings of 17 protein subunits and in this region the details of the inter subunit contacts can be determined (by X-ray crystallography) (Klug et al.; Bloomer et al., 1978). The subunits of the upper ring in the disk are flat and in the lower ring are tilted down ward toward the centre of the disk with three regions of contact between the subunits.

37 Plant viruses are diverse, but not as diverse as animal viruses probably because of size constraints imposed by requirement to move cell-to-cell through plasmodesmata of host plants

38 Viral Morphological Groups Cubic (icosahedral) Helical Horne, R. W. & Wildy, P. (1961). Symmetry in virus architecture. Virology 15,

39 Icosahedral arrangement is typical in virus structure An icosahedron has 20 triangular (equilateral) faces (facets), 12 vertices, and a 5:3:2 axes of rotational symmetry

40 Isometric viruses Icosahedron (sphere) e.g., BMV

41 Tobacco necrosis virus, 26 nm in diameter

42 BROME MOSAIC VIRUS Type member of the Bromovirus genus, family Bromoviridae Virions are nonenveloped icosohedrals (T=3), 26 nm in diameter, contain 22% nucleic acid and 78% protein RNA1 RNA2 RNA3 RNA4 BMV genome is composed of three positive sense RNAs separately encapsidated RNA1 (3.2 kb), RNA2 (2.9 kb), RNA3 (2.1 kb), RNA4 (0.9 kb)

43 Francki, Milne & Hatta Atlas of Plant Viruses, vol. I. Three-dimensional image of Turnip yellow mosaic virus (TYMV) reconstructed from EM

44 Tobacco mosaic virus First virus crystallized (1946 Stanley was awarded the Nobel prize) First demonstration of infectious RNA (1950s) First virus to be shown to consist of RNA and protein First virus characterized by X-ray crystallography to show a helical structure First virus genome to be completely sequenced

45 Tobacco mosaic virus (TMV), 300 nm Potato virus Y (PVY), 740 nm

46 Cocoa swollen shoot virus, Badnavirus Maize streak virus, Geminiviridae