Composition & Architecture

of plant viruses

P.N. Sharma
Department of Plant Pathology,
CSK HPKV, Palampur (H.P.)
Plant Viruses

Classification, Morphology, Genome,

and Structure
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
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.
Chemical composition of plant viruses

 Protein( Capsid)
 Capsomere
 Nucleic acids
 RNA  DNA
 +ve strand RNA  ssDNA
 -ve strand RNA
 dsDNA
 ssRNA

 dsRNA
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 - 158 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%
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
A/a composition of capsid proteins of some viruses

1. Alanine 6. Glutamine 11. Leucine 16. Serine

CMV: 17; PVY: 16 CMV: 20; PVY: 23 CMV: 26; PVY: 10 CMV: 32; PVY: 10
TMV: 14; PVX: 76 TMV: 16; PVX: 33 TMV: 12; PVX: 19 TMV: 16; PVX: 31
2. Arginine 7. Glutamic acid 12. Lysine 17. Tryptophane
CMV: 24; PVY: 11 CMV: 18; PVY: 13 CMV: 1 ; PVY: 2
TMV: 11; PVX: 18 TMV: 2; PVX: 22 TMV: 3; PVX: 9
3. Asparatic acid 8. Glycine 13. Methionine 18. Tyrosine
CMV: 30; PVY: 22 CMV: 16; PVY: 13 CMV: 8; PVY: 8 CMV: 11; PVY: 6
TMV: 18; PVX: 42 TMV: 6 ; PVX: 23 TMV: 0 ; PVX: 15 TMV: 4; PVX: 4
4. Asparagines 9. histidine 14. Phenylalanine 19. Threonine
CMV: 4; PVY: 4 CMV: 7 ; PVY: 5 CMV: 17; PVY: 13
TMV: - ; PVX: 4 TMV: 8; PVX: 22 TMV: 16; PVX: 58
5. Cystein 10. Isoleucine 15. Proline 20. Valine
CMV: 0; PVY: 1 CMV: 16; PVY: 12 CMV: 18; PVY: 11 CMV: 22; PVY: 13
TMV: 1; PVX: 5 TMV: 9 ; PVX: 21 TMV: 8 ; PVX: 34 TMV: 14; PVX: 27
Total CMV: 287 PVY: 203 TMV: 158 PVX: 463
%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 31-33 67-69
CMV 18 82
TRSV 40 60
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. 27-39.
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
 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.
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.
 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)
 The Transmission Electron Microscope (TEM)
  The Transmission Electron Microscope (TEM)

A 1973 Siemens electron microscope, EM developed by E. Ruska 1933

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
 Fine structures determination

 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:
 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
 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
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
: These help the CP and n/a
 Protein : protein to be held together
 Protein : RNA precisely
 RNA : RNA
 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
 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.
 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
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.
 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 three-
dimensional 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.
X-ray sources
 The brightest and most
useful X-ray sources
are synchrotrons

A protein crystal seen under amicroscope. Workflow for solving the

Crystals used in X-ray crystallography may structure of a molecule by X-
be smaller than a millimeter across. ray crystallography.
 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 An X-ray diffraction pattern of a
causes them to interact differently with atoms having crystallized enzyme. The pattern o
spots (called reflections) can be used to
different alignments of their magnetic moments. determine the structure of the enzyme.
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.
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)
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.
SYMPTOMS OF TMV
Rod shaped particles

Helix (rod)
e.g., TMV

TMV rod is 18 nanometers

(nm) X 300 nm
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
 16.33 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
Tobacco mosaic virus
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, 74-88 & 114-
134) 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.
 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.
 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.
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
Viral Morphological Groups

 Cubic (icosahedral)
 Helical
Horne, R. W. & Wildy, P.
(1961). Symmetry in virus
architecture. Virology 15,
348–373
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
Isometric viruses

Icosahedron
(sphere) e.g., BMV
Tobacco necrosis virus, 26 nm in diameter
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
• BMV genome is composed
of three positive sense RNAs
separately encapsidated
RNA1 RNA2 RNA3 RNA1 (3.2 kb), RNA2 (2.9
RNA4
kb), RNA3 (2.1 kb), RNA4
(0.9 kb)
Francki, Milne & Hatta. 1985 Atlas of Plant Viruses, vol. I.

Three-dimensional image of Turnip yellow mosaic virus (TYMV)

reconstructed from EM
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
Tobacco mosaic virus (TMV), 300 nm

Potato virus Y (PVY), 740 nm

 Maize streak virus,
Geminiviridae

Cocoa swollen shoot virus,

Badnavirus