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

+ve strand RNA

-ve strand RNA

ssRNA

dsRNA

DNA

ssDNA

dsDNA

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

CMV: 17; PVY: 16

TMV: 14; PVX: 76

6. Glutamine

CMV: 20; PVY: 23

TMV: 16; PVX: 33

11. Leucine

CMV: 26; PVY: 10

TMV: 12; PVX: 19

16. Serine

CMV: 32; PVY: 10

TMV: 16; PVX: 31

2. Arginine

CMV: 24; PVY: 11

TMV: 11; PVX: 18

7. Glutamic acid 12. Lysine

CMV: 18; PVY: 13

TMV: 2; PVX: 22

17. Tryptophane

CMV: 1 ; PVY: 2

TMV: 3; PVX: 9

3. Asparatic acid

CMV: 30; PVY: 22

TMV: 18; PVX: 42

8. Glycine

CMV: 16; PVY: 13

TMV: 6 ; PVX: 23

13. Methionine

CMV: 8; PVY: 8

TMV: 0 ; PVX: 15

18. Tyrosine

CMV: 11; PVY: 6

TMV: 4; PVX: 4

4. Asparagines 9. histidine

CMV: 4; PVY: 4

TMV: - ; PVX: 4

14. Phenylalanine

CMV: 7 ; PVY: 5

TMV: 8; PVX: 22

19. Threonine

CMV: 17; PVY: 13

TMV: 16; PVX: 58

5. Cystein

CMV: 0; PVY: 1

TMV: 1; PVX: 5

10. Isoleucine

CMV: 16; PVY: 12

TMV: 9 ; PVX: 21

15. Proline

CMV: 18; PVY: 11

TMV: 8 ; PVX: 34

20. Valine

CMV: 22; PVY: 13

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)

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

The Transmission Electron Microscope (TEM)

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

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

Fine structures determination

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

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

These help the CP and n/a

to be held together

precisely

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

Methods for studying stabilizing bonds

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

Workflow for solving the

structure of a molecule by X-

ray crystallography.

A protein crystal seen under amicroscope.

Crystals used in X-ray crystallography may

be smaller than a millimeter across.

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 of

spots (called reflections) can be used to

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

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

TMV rod is 18 nanometers (nm) X 300 nm

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.

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.

Properties of coat proteins

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 (3.2 kb), RNA2 (2.9

kb), RNA3 (2.1 kb), RNA4

(0.9 kb)

RNA1 RNA2 RNA3 RNA4

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