Biology 303: Microbiology

Plant Viruses and Viroids

BUTLER, P. JONATHAN G., & AARON KLUG. The assembly of a virus. Scientific American
239(5): 62–69, November 1978 (Offprint No. 1412).
DIENER, T. O. Viroids. Scientific American 244(1): 66–73, 1981 (Offprint No. 1488).
FRAENKEL-CONRAT, HEINZ. Rebuilding a virus. Scientific American 194(6): 42–47, June
1956 (Offprint No. 9).
LEVY, JAY A., HEINZ FRAENKEL-CONRAT, & ROBERT A. OWENS. Virology. 3rd ed.
Englewood Cliffs, NJ: Prentice Hall, 1994, pp. 47–55.

I. Tobacco mosaic virus

A. History

1. Charles Chamberland (1884) developed the porcelain bacterial

filter.

2. Adolph Mayer of the Netherlands was the first to transmit the

disease experimentally and to name the disease (1886)

3. Dimitri Ivanowski (1892, 1893) showed that porcelain-filtered leaf

extracts from mosaic plants induced disease, but disregarded the
experimental observation, and even eleven years later insisted that
"the bacterial nature of the infection is scarcely to be doubted."

4. Martinus Willem Beijerinck (1898) obtained the same results, but

came to a different conclusion: he postulated a new type of infec-
tious agent, a contagium vivum fluidum, which is replicated only in
living cells. This was arguably the birth of virology.

5. Francis O. Holmes (1929) developed the assay for tobacco mosaic

virus: pricking the leaf surface with insect pins dipped in sap from
infected plants. The resultant lesions are proportional to the virus
content of the extracts.

6. C. G. Vinson & A. M. Petre (1929) partially purified tobacco mosaic

virus.

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7. Wendell M. Stanley (1935, 1936) "crystallized" the material, and
concluded that it was a protein (he detected no phosphorus). The
needle-like structures are now known as paracrystals, because
they have only one-dimensional order. This was taken as the ulti-
mate proof that the virus was not a living cell, but only a macromo-
lecular complex. Stanley received the Nobel Prize for his work in
1946.

8. Fred C. Bawden & Norman W. Pirie (1937, 1938) suggested that

the presence of 0.5% phosphorus and 2.5% carbohydrate in the
crystalline material was not artifactual, but rather indicated the
presence of “nucleic acid of the ribose type.”

9. TMV was the first virus to be viewed in the electron microscope, by

Kausche, Pfankuch, & Ruska in 1939.

10. The first X-ray crystallographic analyses in 1941 suggested that the
rod-shaped virus consists of many orderly, helically arranged
protein molecules, and by the 1950s Don Caspar, Ken Holmes, and
Rosalind Franklin had determined that RNA resides in the interior of
the TMV particle.

11. In the 1950s Heinz Fraenkel-Conrat showed that TMV could be

dissociated into its constituent protein and nucleic acid molecules,
then reassociated to reform infective virus particles. In 1957
Fraenkel-Conrat and Bea Singer published their famous mixed
reconstitution experiment, taken as the final proof that nucleic acids
encode protein structure, and not the opposite.1

B. Heinz Fraenkel-Conrat discovered he could disassemble and reas-

semble the virus by adding and removing detergents.

1 FRAENKEL-CONRAT, H., & B. SINGER. Virus reconstitution II. Combination of protein and nucleic acid
from different strains. Biochimica et Biophysica Acta 24: 540–548, 1957.

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Figure 8-1. Tobacco mosaic virus particles partially degraded with

detergent. The thin threads emerging from some of the rods are nucleic
acid. X150,000 (FRAENKEL-CONRAT, 1956).

Figure 8-2. Tobacco mosaic virus broken down with detergents,

then reconstituted by removal of the detergent at pH 7 and ionic
strength 0.5. Note appearance of disks. x130,000 (FRAENKEL-CONRAT,
1956).

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Figure 8-3. Tobacco mosaic virus particles fully reconstituted from

viral protein and nucleic acid at neutral pH. x150,000 (FRAENKEL-
CONRAT, 1956).

Figure 8-4. Ionic strength and pH dependence of aggregation of

viral protein and nucleic acid. At low pH, the protein will aggregate
into a helix without the viral RNA, but at pH 7, disks will not aggregate
into helices without viral RNA. At high pH and low ionic strength, only
small aggregates of protein appear (BUTLER & KLUG, 1978).

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Figure 8-5. Aggregation of protein disks into a helix at low pH, in

the absence of nucleic acid (BUTLER & KLUG, 1978).

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1. The electron microscopic data led to increasingly refined models of
these protein disks.

Figure 8-6. Looped chains of tobacco mosaic virus protein may be

arranged in two layers, making a molecule 23 Ångstroms thick
(FRAENKEL-CONRAT (1956).

Figure 8-7. Lock washer configuration of protein subunits allowed

to aggregate at neutral pH. The disk was estimated to be 150
Ångstroms across, with a central hole 50 Ångstroms in diameter.
(FRAENKEL-CONRAT, 1956).

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2. X-ray crystallography confirmed the structure of the disk.

Figure 8-8. Model of disk structure deduced from 2.8-Ångstrom X-

ray crystallographic analysis (BUTLER & KLUG, 1978).

3. Because both degraded (see Fig. 8-1) and growing (see Fig. 8-9)
viral particles seem to have two strands of RNA protruding from the
virion, it was hypothesized that the assembly begins around a hair-
pin loop of the viral RNA.

Figure 8-9. Growing virus particles have two RNA "tails" at one
end. The particle seems to elongate primarily at the end of the particle
opposite the tails (Butler & Klug, 1978).

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Figure 8-10. Putative hairpin loop in the tobacco mosaic virus

genome (BUTLER & KLUG, 1978).

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4. These observations have led to a model of assembly involving first
nucleation, then elongation.

Figure 8-11. Nucleation of the tobacco mosaic virus begins with the
insertion of the hairpin loop into the central hole of a disk (a). The
RNA intercalates between the two layers of subunits of the disk (b), and
induces the disk to flip into the lock washer configuration (c). More disks
begin to add to the nucleating complex (d) (BUTLER & KLUG, 1978).

Figure 8-12. Elongation of the tobacco mosaic virus particle occurs

at the end distal to that with the protruding RNA (a). The 5' end of
the RNA feeds into the central hole at the bottom as the RNA interacts
with a new disk at the top (b). The new disk flips into the lock washer
configuration (c) and adds to the growing viral particle (d) (BUTLER &
KLUG, 1978).

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5. In this manner the helical virion is assembled, consisting of one
piece of single-stranded RNA 6,395 nucleotides long, lying within a
groove formed by the helical array of coat protein subunits. The
pitch of the helix is 2.3 nm and there are 16.33 coat protein mole-
cules per turn, for a total of 130 turns or 2,123 coat protein
molecules.

Figure 8-13. Helical structure of the tobacco mosaic virus.

The virus consists of a single long strand of RNA (color), repre-
senting perhaps four genes, packed between the turns of a heli-
cal protein made up of 2,123 identical subunits (BUTLER & KLUG,
1978).

Figure 8-13. The intact tobacco

mosaic virus particle (FRAENKEL-
CONRAT, 1956).

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C. Structure of the tobacco mosaic virus genome

1 6395
5' cap tRNA HIS 3'
ORF1 126 kDa (ORF2 183 kDa)

AUG 69 leaky UAG 3417 UAA 4917 6395

I1 Subgenomic RNA ppp 5' tRNA HIS 3'
(2991 nt) ORF3 54 kDa

AUG 3495 UAA 4917 6395

I2 Subgenomic RNA ppp 5' tRNA HIS 3'
(1500 nt) AUG 4903 UAA 5709

ORF4 30 kDa 6395

Coat Protein Subgenomic RNA 5' cap tRNA HIS 3'
(692 nt) AUG 5712 UGA 6191

ORF5 17.6 kDa

Figure 8-14. Structure of the tobacco mosaic viral genome. ORF1

(and ORF2) encode the polymerase. The product of ORF3 is uncertain.
ORF4 encodes a protein necessary for cell-to-cell movement of the virus.
ORF5 encodes the coat protein (LEVY et al., 1994, p. 53).

II. Other plant viruses (Prescott Overhead 27, Fig. 16.9)

A. Nonenveloped (overwhelming majority)

1. ssRNA genomes

a) Alfalfa mosaic virus group

b) Bromovirus (causes brome mosaic)

c) Nepovirus (tobacco ringspot)

d) Comovirus (cowpea mosaic)

e) Dianthovirus (carnation ringspot)

f) Pea enation mosaic virus group

g) Etc., etc., etc.

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2. dsRNA genomes

a) Reoviridae (a crossover group with animal viruses)

3. dsDNA genomes

a) Caulimovirus

4. ssDNA genomes

a) Geminivirus

B. Enveloped

1. ssRNA

a) Rhabdoviridae (again, a possible crossover group with

animal viruses!)

b) Tomato spotted wilt virus group

III. Some general principles

A. All carried by vectors

1. Insects

2. Soil nematodes (e.g. tomato ringspot virus)

3. Fungi (e.g. tomato necrosis virus)

B. Vertical transmission through

1. Seeds

2. Tubers

3. Pollen

C. Spread from cell to cell through plasmodesmata

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IV. Viroids: pure RNA. No protein.

A. Potato spindle-tuber viroid

1. Covalently closed loop of 359 nucleotide residues

2. No translational product

3. 200–10,000 copies per cell

4. Replicated by a host RNA-dependent RNA polymerase

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