J. gen. Virol. (I969), 4, 571-576
57I
Printed in Great Britain
A Host-cell D N A Function Involved in the Replication o f
Avian Tumour Viruses and o f Fowl-plague Virus
By J. ZAVADA*
Institute of Virology, Czechoslovak Academy of Science, Bratislava
(Accepted 2 December 1968)
SUMMARY
The multiplication of fowl-plague virus was much less sensitive to
actinomycin D in chick cellsinfected with avian myeloblastosis virus and in
hamster cells, transformed by Rous sarcoma virus than in comparable
normal cultures. In addition, reproduction of fowl-plague virus was less
effectively suppressed by u.v.-irradiation of cells carrying avian myeloblastoffs or Rous sarcoma virus than of control cultures. These findings
could indicate that avian myeloblastosis and Rous sarcoma viruses induce
a function of host-cell D N A , whose products can be utilizedin reproduction
of fowl-plague virus.
INTRODUCTION
The reproduction of most R N A viruses,including Newcastle disease virus,is known
to be resistant to actinomycin D (Barry, 1964). Nevertheless, some of RNA-containing viruses are inhibited by actinomycin D: viz. Rous sarcoma virus (Temin, 1963,
1964a; Bader, 1964, 1965), influenza viruses, including fowl-plague virus (Barry, Ives
& Cruickshank, 1962; Barry, 1964) and routine leukaemia viruses (Bases & King,
1967; Duesberg & Robinson, 1967). Double-stranded R N A of poliovirus is more
sensitive than single-stranded R N A (Koch, Quintrell & Bishop, ~967).
Sensitivity or resistance of RNA-viruses to actinomycin D is also reflected by
u.v.-sensitivityof the host cellsto grow the virus.The capacity of chick cellsto support
multiplication of Rous sarcoma virus was found to be extremely sensitiveto u.v. light
(Rubin & Temin, 1959) as was the capacity to reproduce influenza viruses (Barry,
1964). The capacity of chick-embryo cells to support multiplication of Newcastle
disease virus is highly resistant to u.v. light (Rubin & Tcmin, 1959; Rosenberg &
Rosenbergovfi, 1962).
The simplest explanation of these findings isthat a host-cellcoded function, necessary
for virus reproduction, is induced with Rous sarcoma, influenza and routine Icukaemia
viruscs.If thishost cellfunction isc o m m o n to allthree viruses,preinfcction era cellwith
one type should ensure that the subsequent multiplication of either of remaining two in
that cell is more resistant to the effects of actinomycin, because the essential host cell
function is already present. Also the inactivation of cellulargenes by u.v. lightshould
prevent virus reproduction in a cell where the genes are repressed and their products
absent but not in a cell where the products are already induced by another virus.
The following systems were chosen for study: RIF-frec chick embryo cellsand cells
fi'om the same batch infected with avian myeloblastosis virus. The virus multiplies
and matures in the cells,but it does not transform them effectively.The baby hamster
* Present address: Imperial Cancer Research Fund, Lincoln's Inn Fields,London. W.C. 2
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J. ZAVAOA
kidney cell line BHK/2I, C 13 (BHK) (Macpherson & Stoker, 1962), the same cells transformed by Rous sarcoma virus, strain SCHMIDT-Rm'PIN,clone SR7 (Macpherson, I965),
and a strain derived from Rous sarcoma virus-transformed hamster embryo culture
(RSH) were used (Vesel3~ & Svoboda, 1965). Both of these cell lines are Rous sarcoma
virus-transformed, but the virus does not mature in them. In these systems the presence
of avian myeloblastosis or Rous sarcoma virus, does not interfere with fowl-plague
virus. Fowl-plague virus was chosen as a representative of the influenza group, because
it multiplies well in cell cultures of several different species and plaques may be
produced in these cells.
METHODS
Viruses. Fowl plague virus, strain DOBSON,was obtained from the collection of this
Institute. It was adapted to B H K cells by IO passages; afterwards it was cloned by
plaques in B H K cells and then regrown in allantoic cavity of chick embryo. Stock
infectious allantoic fluid was dispensed in sister ampoules and kept at - 2 5 °. A single
virus batch was used in all experiments; no ampoule was thawed more than once.
This virus stock contained 3 x lOs p.f.u.]ml.; no decrease of infectivity was detected
during 6 months of storage. Haemagglutination titre of this batch was I : lO24. Avian
myeloblastosis virus, strain BAI-A, was kindly provided by Dr J. ~dman, Prague. Virus
was propagated by intravenous inoculation of I-day-old chickens. After the development of myeloblastosis the animals were bled into citrate solution. Infectious plasma
served as virus stock; before infection of cell cultures the plasma was dialyzed against
phosphate-buffered saline.
Cells. Chick embryo cells were prepared by trypsinization of Io-day-old embryos.
RIF-free eggs were a generous gift of Dr I. H l o ~ n e k , Prague. One batch of cells was
split in two parts. One of them was infected as primary culture with avian myeloblastosis virus (o.I ml. plasma per lO7 cells). Both control and avian myeloblastosis
virus-infected chick cells were passaged at the same intervals; for experiments the 3rd
and 4th passage in vitro were employed. B H K and SR7 cells were kindly provided by
Dr I. A. Macpherson, Glasgow. RSH cell line was obtained from Dr D. ~imkoviE,
Bratislava. All these cells were grown in ETC medium (Macpherson & Stoker, 1962),
consisting of 8 vol. of Eagle's medium (Difco), I vol. of heated calf serum and I vol.
of Tryptose-phosphate broth (Difco). Experiments with actinomycin D and with u.v.
light were performed on confluent cell cultures, 3 days old, in 5 cm. Petri dishes.
Chick embryo ceils, both normal and preinfected with avian myeloblastosis virus,
were seeded in 5 ml. of medium containing lO6 cells/dish. All three lines of hamster
cells were seeded in 5 ml., containing 7 x lO5 cells/dish. Confluent cultures contained
(in terms of lO6 cells/5 cm. dish): chick cells, either normal or infected, 3"0 to 3"4,
B H K 4"8 to 5"3, SR7 5"7 to 6.1, RSH 5.2 to 6.0.
Plaque assay of fowl-plague virus was carried out in confluent chick embryo primary
cell cultures, 2 days old, in 5 cm. Petri dishes. Each dish was seeded with 5 × lO5 cells
in 5 ml. of medium. Virus dilutions were prepared in phosphate-buffered saline, pH 7"2,
containing 1% of heated calf serum. Virus inoculum 0"5 ml. per dish was adsorbed to
the cultures for I hr at room temperature, with occasional shaking. After addition
of agar overlay, the cultures were incubated for three days at 37 °, stained with neutral
red 1 : IO,OOO in agar and the plaques subsequently scored.
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Cell D N A function common to R S V , A M V and F P V
573
U.v.-experiments employed a Philips T U V - 3 o w germicidal tube; the dose was
monitored using a u.v. dose-meter (for 2537 .~), manufactured in this Institute by
Dr E. Mrena. To avoid the possibility of photoreactivation, experiments were performed
under dim yellow light.
Actinomycin D was a gift of Merck, Sharp and Dohme, Rahway, N.J.
RESULTS
Actinomycin D
Confluent cultures of respective cells, grown in 5 cm. Petri dishes, were used. After
removing the growth medium, fowl-plague virus at a multiplicity of infection of
approximately o.I p.f.u/cell was adsorbed to the cultures for 3o min. at room temperature. Unattached virus was removed, and the cultures were rinsed twice with
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Fig. I
phosphate-buffered saline containing I °//o calf serum. ETC medium containing
concentrations of actinomycin D ranging from o to I.O #g./ml. was added (5 ml./
culture) and the cultures were incubated at 37 ° for 40 hr. At this time, the virus content
of the medium was determined by plaque assay. Fowl plague virus production was
less inhibited by actinomycin D in cells preinfected with avian myeloblastosis or with
Rous sarcoma virus than in the respective controls (Fig. I). Under these experimental
conditions, fowl-plague virus yield (in terms of p.f.u./cell) in cultures without actinomycin D was similar in all types of cells used: chick cells RIF-free, 38; chick cells
infected with avian myeloblastosis virus, 32; BHK, 2i; SR7, 27; RSH, 8.
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574
J. Z~,VADA
Inactivation of capacity by u.v. light
Experiments with u.v. light were carried out in a similar way. Confluent cultures of
cells were grown in Petri dishes; after removing the medium the cultures were rinsed
twice with phosphate-buffered saline to remove residual growth medium, which might
shield the cells from u.v. rays, and the cultures were exposed to various u.v. doses.
After irradiation, the cultures were infected with fowl-plague virus at m.o.i, approximately o.I p.f.u./cell. After 30 min. of adsorption, the inoculum was removed and
the cultures rinsed twice, and 5 ml. of medium was added to each culture. After 40 hr
incubation at 37 ° the medium was harvested and the yield of fowl-plague virus was
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Fig. 2
determined. The capacity to support the multiplication of fowl plague virus was more
u.v.-resistant in cells preinfected with avian myeloblastosis or Rous sarcoma virus
than in comparable normal cultures (Fig. 2). Also in this experiment the yield o f
fowl plague virus was comparable in all cultures, ranging from I4 to 3o p./cell.f.u.
Similar experiments were done with actinomycin D and with u.v. light using m.o.i.
c. 5 and o.oI p.f.u./cell. In experiments using 5 p.f.u./cell virus yield was determined
after 20 hr incubation. All these experiments yielded results very similar to those
presented above. At this multiplicity, however, virus yield in cultures untreated with
either actinomycin D or u.v. light was in most instances less than I p.f.u./cell and
it differed considerably between different types of cells; it was lowest in BHK.
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Cell D N A function common to R S V , A M V and F P V
575
DISCUSSION
The results presented here are consistent with expectation following from the model
suggested in the Introduction. Nevertheless, there is no conclusive evidence that the
model is correct. It will be necessary to show which gene products coded by the host
cell are involved in the replication of both avian myeloblastosis or Rous sarcoma
and fowl-plague viruses. There is known an example of host-cell coded and virusinduced enzyme, the uncoating enzyme of poxviruses (Joklik, 1964).
Avian leucosis viruses and myxoviruses have certain similarities, but these may be
superficial. They include similarities in the multiplication cycle, abortive infections,
defectiveness and to a certain extent a similarity in the structure of the virions. It has
recently been reported that fowl plague virus grown in mixed infection with avian
myeloblastosis virus showed double neutralization with antisera against both fowlplague and avian myeloblastosis viruses (Z~fvada & Rosenbergov~i, I968), suggesting
that phenotypic mixing had taken place. Nevertheless, there are some definite differences; whereas the reproduction of Rous sarcoma virus is sensitive to 5-iododeoxyuridine and cytosine-arabinoside, fowl-plague virus is resistant. This probably indicates that
Rous sarcoma virus reproduction requires both functioning and replicating host-cell
DNA, whereas fowl-plague virus requires only DNA functioning (Bader, i965).
Some alternative explanations have been proposed for the facts found in the case
of Rous sarcoma virus. To explain the unusually high u.v.-resistance of the infectivity
of Rous sarcoma virus and the high u.v.-sensitivity of cell capacity to support its
growth it was suggested that there is a genetic homology between the virus and the host
cell (Rubin & Temin, I959). Whether this explanation is true or not, in vitro hybridization of RNA extracted from avian myeloblastosis virus with DNA from uninfected
chick cells apparently shows a relationship (Harel et al. 1966; Wilson & Bauer, I967).
The exact meaning of this finding for the viral replication cycle remains to be defined.
It has also been suggested that the RNA of Rous sarcoma virus after entering the
cell serves as a template for a DNA copy which behaves as a provirus and in turn
makes RNA strands (Temin, 1963, I964a, b, 1967). This hypothesis has so far not been
supported by convincing experimental data.
A third explanation is that it is not an essential enzyme that is coded by cellular
DNA, but some type of RNA specified by host-cell DNA (Montagnier, I968). This
possibility should be considered seriously in devising further experiments.
Whatever the mechanism of the findings reported here, it is clear that they represent
a new type of interaction between two viruses, analogous to complementation.
The author is grateful to Dr I. A. Macpherson, Glasgow, and Dr J. Huppert, Paris,
for their critical reading of the manuscript, and to Mrs Eva Vrbov~i for expert technical
assistance.
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