Chapter 47
VIRUS GENETICS: PLANT AND ANIMAL VIRUSES

PRE-LECTURE ASSIGNMENT
1. Quickly review notes for the previous lec-

ture,

2. Suggested readings:

a. General genetics textbooks
Sinnott, Dunn, and Dobzhansky: Chap.
27, pp. 377-378.
Srb and Owen: Chap. 19, pp. 400-402.
b. Additional references
Burnet, F. M., and Stanley, W. M.
Editors, 1959. The viruses. Vol. 1,
General virology. 609 pp. Vol. 2,
Plant and bacterial viruses. 408 pp.
Vol. 3, Animal viruses. 428 pp. New
York: Academic Press.
Fraenkel-Conrat, H., and Williams,
R. C. 1955. Reconstitution of active
tobacco mosaic virus from its inactive
protein and nucleic acid components.
Proc. nat. Acad. Sci., U.S., 41: 690-
698. Reprinted in: "Classic papers in
genetics", J. A. Peters, Ed. 1959.
Englewood Cliffs, N.J.: Prentice-Hall,
Inc.
Luria, S. E. 1953. General virology.
427 pp. New York: John Wiley & Sons,

Lecturer—J. LEDERBERG

a. When propagated on intact host animals,
quantitation of particles is expensive
and time-consuming,

b. Samples to be titrated may be plated
onto agar layers seeded with susceptible
tissue culture cells. Clearing plaques
are produced as by bacteriophage. This
kind of technique is very useful.

. Unfortunately, many viruses (like influ-

enza virus) do not produce sufficient cyto-
pathic effect to produce detectable plaques
on such agar plates. For these viruses,
the techniques of limit-dilution must still
be used.

. Influenza virus

a. This has been adapted to grow on cells
lining the fluid cavities of the chick
embryo.

b. A sample of virus to be tested is suffi-
ciently diluted, and then aliquots inno-
culated into a series of eggs.

c. After 48 hours or so, the eggs are
harvested to determine the fraction
which contained a virus particle.

d. If near-limit dilutions are used (so the
probability is low that an aliquot con-

Inc. tains a virus particle), one can esti-
mate the virus content of the entire
LECTURE NOTES sample.
A. Plant and animal viruses are e. At near-limit dilutions, the virus par-

1. of great economic and medical significance. ticles harvested from an egg are pro-~

2. difficult to study for technical reasons. bably from one clone.
B. Determination of an infective unit Cc. Life cycle of animal viruses
1. This is a chief difficulty. 1. Influenza virus cycle is used as an ex-

. Plant virus

a. To titrate a virus attacking leaves, a
sample is rubbed on the leaf surface.

b. Only a small fraction of the virus par-
ticles find and penetrate susceptible
cells and give a demonstrable lesion.

. Poliomyelitis virus

ample, whose general features may apply
also to other animal and, to some extent,
plant viruses.

2, The mammalian host cell (Fig. 47-1) has

a. a flexible shape,
b. an ambiguous margin, and
c. an outermost mucoid coat which acts as
a substrate for an enzyme located on the
virus surface. This coat constitutes,

therefore, a virus receptor.

. The influenza virus has

a. an inner core of RNA genetic material,
and

b. an outer protein coat containing the mu-
cin-reacting enzyme.

. The virus cannot attach if the mucoid coat

is stripped by specific enzyme or periodate
treatment.

. After attachment, the particle enters the

cell, perhaps by being engulfed via the
cell's normal pseudopodial activity.

Once inside the cell, the particle enters an
eclipse phase (see Chap. 46) and multiplies
vegetatively, at which time the cell's RNA
content increases.

. After some hours intact particles are grad-

ually liberated.
a. Evidence indicates that the influenza

viral coat is added during emergence
from the host.

b. This coat contains some material made
(by the host -- therefore host cell-speci-
fic) before infection and some made after
infection (by host and virus together).

D. Consequences of mixed infections

1.

Burnet and others used MEL and WSE

strains of influenza virus which differ in

their markers.

a. Serologically, WSE is W and MEL is A.

b. WSE is inactivated by ovomucin (c) while
MEL is not (C).

c. WSE is pathogenic when placed on the
egg's chorio-allantoic membrane (e)
while MEL is not (E).

. Egg membranes are multiply-infected with

mixtures of the two strains.

. Phenotyp* mixing (Burnet)

From such mixed infections, daughter
particles are neutralized almost per-
fectly efficiently by antiserum for either
strain. .

b. The coats show this phenotypic mixing
even though the genomes within them are
either WSE or MEL.

c. This effect, then, is not due to genetic
recombination.

. Heterozygosis (Burnet)

a. The virus from mixed infections is har-
vested and clones obtained via limit-di-
lution (see B5).

b. Some clones contain more than one
genetic type.

 

Figure 47-1

c. This heterozygosis may be explained
either by adhesion of two, whole, genet-
ically-different particles which act as a
unit on limit-dilution, or by one particle
containing two different genomes.

d. Since there has been no exchange of
parts of the genetic material in forming
a stable clone, this also is not a com-
plete phenomenon of genetic recombina-
tion.

5. Genetic recombination
a. Influenza virus

Mixed infections give particles which
yield pure clones that are stable recom-
binants (Ac or W C).

b. Vaccinia virus (Fenner)

Similar evidence was obtained, for this
more complex virus, in experiments in-
volving markers for hemagglutinins,
heat resistance, virulence, and pock
character.

Poliomyelitis viruses

1. No evidence has been obtained so far for
genetic recombination in these viruses.

2. If found, recombination would greatly ac~
celerate the deletion of the specific adapta~
tion to the human host which these viruses
possess.

3. Present work is limited to mutational stud-
ies.

Chemical separation of viral components

1. Aqueous phenol solution destroys the pro-
tein but leaves the nucleic acid intact
(Schramm, using animal viruses).

2. Tobacco mosaic virus protein is removed,

265
G.

I.

266

almost unchanged, by moderate alkalinity.

Infection by nucleic acid

1. RNA, minus demonstrable protein, has
been shown to be capable of infecting tobac-
co and mammalian cells.

2, After vegetative multiplication, the mature
particles formed have the protein coats that
would be specified by this RNA when intro-
duced by virus.

3. Thus virus protein plays no part in replica-
tion either of the genetic material or of it-
self.

4, As compared to nucleic acid in virus, puri-
fied nucleic acids have infectivities up to
several percent.

5. Isolated RNA is more susceptible to ribonu-
clease, temperature, and pH, and less sus-
ceptible to detergents, than is intact virus.

6. Recent evidence suggests isolated DNA
from bacteriophage is infective.

Reconstitution experiments with tobacco mo-

saic virus (TMV)

1. TMV is a long linear particle; the outside is
protein, built up of stacks of monomeric
blocks; the inside is spiralled RNA.

2. Fraenkel-Conrat has reconstituted essen-
tially the original virus by mixing, under
certain conditions, its separate protein and
RNA.

3. Using two strains of TMV, the standard
(TMV) and Holmes ribgrass (HR), he was
able to construct virus with TMV RNA and
HR protein.

a. The behavior of these particles was
somewhat like that of animal viruses
showing phenotypic mixing.

b. However, particles have such low infec-
tivity in ‘plants that mixed infections,
which could produce phenotypic mixing,
do not occur,

c. Such a reconstituted particle is inacti-
vated not by anti-TMV serum but by
anti-HR serum. The progeny of such a
particle are typical TMV.

d. Corresponding results were obtained
from reconstituted particles having HR
RNA and TMV coats.

Biologically-active nucleic acids have not yet

been synthesized in the laboratory.

1. The success of systems using DNA as a
primer for synthesizing more DNA (Korn-
berg; see Chap. 41) is a giant step in this
direction.

2, The genetic behavior of RNA motivates at-
tempts to replicate it in vitro.

 

J.

"It should be stressed that DNA and RNA are
not unique materials. They have the same re~
lationship to the information contained in them
as carbon black does to the words in a diction-
ary."

POST-LECTURE ASSIGNMENT

1. Read the notes immediately after the lec-

ture or as soon thereafter as possible,
making additions to them as desired.

2. Review the reading assignment.
3. Be able to discuss or define orally or in

writing the items underlined in the lecture
notes.

4, Complete any additional assignment,
QUESTIONS FOR DISCUSSION

47. 1. What are the comparative advantages and cluding statement, quoted in J in the lecture

disadvantages of influenza and poliomyelitis notes,
viruses as material for genetic investigation ?

47. 2. Why is the WSE strain of tobacco mosaic
virus called an egg-adapted strain?

47, 3. Is the technique of limit-dilution used in
assaying tobacco mosaic virus? Explain.

47. 4. In what respects do the host cells of bac-
teriophage, on the one hand, and of plant and
animal viruses, on the other hand, differ
from each other with respect to virus?

47. 5. What evidence was presented that geneti-
cally recombinant clones of influenza virus
are stable and pure?

47, 6. How can the different consequences of
mixed infections with influenza virus be dis-
tinguished from each other?

47. 7. Specifically what would you do experimen-
tally, in order to benefit mankind, if the
phenomenon of genetic recombination was
discovered in the poliomyelitis viruses?

47. 8. What evidence does Fraenkel-Conrat's
experiments furnish that RNA is the sole or
primary determinant of the coat protein of
the tobacco mosaic virus?

47. 9. Cite evidences for the exact replication of
RNA. -

47.10. What genetic attributes does RNA share
with DNA?

47.11. In what respects have genetic investiga-
tions using plant and animal viruses been
more fruitful, to date, than those using bac-
teriophages ?

47.12. How would you proceed to determine
whether a nucleic acid synthesized in vitro
was biologically active?

47.13. In what respects has our understanding of -
the genetics of higher organisms been aided

by the genetic study of microorganisms ?

47.14. Discuss the meaning of Lederberg's con-

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