Effect of interferon on synthesis of ssRNA in reovirus type 3-infected L cell cultures

Vol. 47, No. 5, 1972

BIOCHEMICAL

EFFECT

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INTERFERON ON SYNTHESIS

OF

OF ssRNA IN REOYIRUS TYPE 3-INFECTED

L CELL CDLTDRES

C.J. GAIJNTT Department of Microbiology Arizona Medical Center, University of Arizona Tucson, Arizona 85724

Received May 8, 1972 Summary: Interferon treatment of L cell cultures prior to infection with reovirus type 3 resulted in a dose-dependent reduction in synthesis of infectious virus and single-stranded ribonucleic acids (ssRNA). New species ssRNA were synthesized in interferon-treated infected cell cultures, including ssRNA species larger and smaller than those normally found in infected cells. Infection

of L cells

stranded

ribonucleic

molecules

serve

ribosomes

extracted

from

infected

acid

from

molecules

synthesized

--in vitro

transcribes

each of the

in the

using

molecules

interferon.

exclusively

purified

genomic

(6).

length

of ssRNA should To this

end,

the present

that

released

with

denatured

double-stranded

Reovirus

which

dsRNA.

studies

polyextracted geonomic were

an RNA polymerase

ten unique

suggests

ssRNA

ssRNA molecules

contain

into

the

from

ssRNA molecules

that

ssRNA species ssRNA also

ssRNA plays

of the effect

in understanding study

suggest

(1,2).

of genomic

be useful

of single-

cells

(7,8)

and therefore

facts

s&WA have been

(3,4,5).

evidence

in synthesis

Several

dsRNA segments

Recent

of reovirus

3 results

reovirions

in the synthesis

the replication synthesis

RNA.

reovirus-infected

of comparable

length

type

(s&WA).

messenger

hybridized

RNA (dsRNA)

precursor

reovirus

molecules

as reovirus

cells

corresponding

with

of

tha of

serves

a prominent

ro

of interferon

o

the mechanism

of action

was initiated.

MATERIALS --AND METHODS L cells paration

of stock

previously (lo),

were

grown

in

virus

and plaque

described

and concentrated

(3).

suspension

assay

Interferon

by use of

culture

of reovirus

was prepared

(NH4)2S04 1228

Copyright 01972,by

AcademicPress,Inc.

as previously

(11).

type

described 3, Dearing

in L cells The interferon

a

(9). strain,

Prewas

by use of MM virus fluids

were

o

BIOCHEMICAL

Vol. 47, No. 5,1972

centrifuged

at 40,000

irridiation

with

a GE germicidal

vesicular

stomatitis

virus:

formation

of plaques

by 50%.

interferon

assayed

as one unit

with

interferon

L cells

for

x g for

modified

for

the kinetics

interferon-treated

pg/ml

is not

without

dodecyl

5 to 20% sucrose filters

in Minimal

for

2.25 hr sulfate

13).

(SDS)

gradients

containing

and trichloroacetic

infected

heated

for

instant

lysine

(12)

The method

VGM).

dsRNA is

following

exposure

followed

defined to 10

D (2 pg/ml) to 1 pCi/ml

cytoplasm

with

phenol

and centrifuged

Fractions were

Act

by exposure from

and

SSRNA was ob-

with

2 min at 70°,

counts

the

(Joklik's

serum,

cells

0.5% SDS (3).

acid-insoluble

of

(2).

ssRI?A was extracted

(9),

infection

Tritium-labeled

30 min at 36'.

45 min at 7 hr pi

(9,

incubated

ssRNA and dsRNA in control

material

of dsRNA by treating for

calf

previously

to acid-insoluble

reduced

were

is

Medium

medium,

of reovirus

was described

time

Essential

growth

for

Zero

fetal

D) (virus

cultures

(20 ug/ml) for

(2).

using which

cultures

Procedures

described

by

mouse reference

Cell

to infection.

D (Act

was assayed of interferon

conditions.

1% heat-inactivated

ribonulcease

and cycloheximide

and sodium

these

cultures

hydrolized

labeling

of uridine-5-'H

under

dilution

6 hr followed

of the international

of synthesis

L cell

of pancreatic

tained

units

to pH 2 for

The interferon

was that

have been

containing

of actinomycin

as RNA that

Three

exposed

(11).

one unit

cell

and 0.5 up/ml measuring

lamp

culture

of infected

formula)

1 hr at 4',

16 to 18 hr prior

in suspension

of suspension

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

into

collected

determined

onto

(3).

RESULTS Effect in L cell prior

cultures.

to infection.

yields range

of various

were

measured

of inhibition

shown in Table inhibitions of interferon Effect

concentrations Cell

cultures

The infected

1.

Increasing

of yields varied

of interferon

exposed were

technique.

concentrations

different

but

the

on yields

level

of reovirus

to interferon harvested

for

Two experiments

3

16 to 18 hr

which on virus

of interferon of inhibition

type

at 24 hr pi and virus

of interferon

the concentration

of virus in

were cultures

by the plaque of various

of interferon

show the yields

are

resulted

in greater

at a given

concentration

experiments.

on the kinetics

of synthesis

1229

of viral

ssRNA and dsRNA.

Vol. 47, No. 5, 1972

TABLE 1.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EFFECT OF INTERFERON ON YIELDS OF REOVIRUS TYPE 3 IN L CELL CULTURRS

Interferon Concentration (units ml)

Virus Expt.

(XC)* Expt.

500

0.3

100

0.3

13.4

20

1.1

19.0

4

3.9

36.5

100

0

*

Yields 1

%C = Virus yields per cultures in Expt

2

100

cell were 750 and 1300 PFU per cell 1 and 2, respectively.

in control

~ssRNAP/l /’

I

/

I

p--e--anRNA(IF,

i

/’

’

d’

:

//

I P’

5

7

9

TIME

II

(HR

13

I5

pi)

Fig. 1. Time course of synthesis of reovirus ssRNA and dsRNA in interferontreated and control cell cultures. Cell cultures were exposed to 10 or zero units of interferon for 18 hr prLor to fnfectfon. The methods for labeling, extracting and identrfying SSRNA and dsRNA are described in Materials and Methods. Each point in a curve represents the cummulative total of counts in that class of RNA up to that time (hr pi). All values were corrected for incorporation of 3H-counts into RNA of that class in uninfected cells. Virus yield from cells in the interferon-treated culture was 23% of the control cell culture yield of 650 PFU/cell. 1230

BIOCHEMICAL

Vol. 47, No. 5, 1972

The rates cell

of synthesis

cultures

rates

of synthesis

treated size

were

cell both

of both

ssRNA and dsRNA in control

similar

until

of both

classes

cultures.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cells

10 hr pi,

in

as shown

of viral

in Fig

RNA were

1.

reduced

the interferon-treated

ssRNA and dsRNA by 13 hr pi.

and interferon-treated After

reduced

the

in interferon-

culture

Interferon

10 hr pi,

ceased

synthesis

to syntheof dsRNA and

ssRNA by 57 and 32%, respectively. Effect

of various

synthesized

in infected

and 3H-labeled dependent

concentrations L cell

of synthesis

Increasing

the concentration

inhibition

of synthesis

range

in inhibition

Cell

cultures.

SSRNA was prepared

inhibition

of interferon

of synthesis

cultures

as described

were

in Materials

of ssRNA by interferon

of interferon of ssRNA.

on the amount

These

resulted

treated

with

and Methods. was observed

were

comparable

selected

interferon A dose-

(Table

in an increasingly

experiments

of SSRNA for

of ssRNA

2).

greater to show the

concentrations

of interfero

EFFECT OF INTERFERON ON SYNTHESIS OF ssPJIA IN L CELL CULTURES

TABLE 2.

INFECTED WITH REOVIRUS TYPE 3* Acid-insoluble 3H-counts (CPM) in ssRNA+

%C -

40

609

33

4

1072

57

0

1872

100

50

4980

74

5

6225

93

0

6712

100

Interferon Concentration (units/ml) Expt.

Expt.

A

B

* L cell cultures for 16 to 17 hr presence of Act cell from cell and 567 PFU/cell on.

were prior D and cultures from

incubated with interferon at the indicated concentration to infection. ssRNA was labeled with 3H-uridine in the cycloheximide. Virus yields were 39, 952, and 1286 PFU/ treated with 40, 4 or 0 units of interferon and 315, 347, cell cultures treated with 50, 5 or 0 units/ml of interfer-

+ The counts are in phenol-SDS-extracted ssRNA. of each sample were hydrolzed to arichloroacetic ribonuclease.

1231

Greater than acid-soluble

95% of the 3H-counts material by

Vol. 47, No. 5, 1972

Since

the same batch

periments, doses

the

time

of interferon

disparity

of interferon

at the

L cell

that

plays

sedimentation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and same stock

in levels

suggest

of infection

Velocity treated

BIOCHEMICAL

of inhibition differences

a role

analysis

cultures.

in

CPM

200

CPM

200

in the physiological determination

of virus

sswA

of reovirus

10 FRACTION

was used

in these

of SSRNA synthesis

of reovirus

Centrifugation

virus

20

synthesized type

two ex-

by comparable state

of the ccl

yields. in interferon-

3 ssRNA molecules

in

30

NUMBER

Fig. 2. Centrifugation of ssRNA extracted from interferon-treated reovirusinfected L cell cultures in sucrose gradients. Suspension cultures of L cells were exposed to vartous concentrations of interferon (units/ml are denoted by the number in the upper rfght corner of each Fig) for 18 hr. Techniques for labeling, extracting and centrifuging SSRNA onto 5 to 20% sucrose gradients are described in Materials and Methods. Virus yields in cultures exposed to 1, 3, 10, 30 and 100 units/ml of interferon were 71, 23, 12, 9 and 5% of the control yield of 250 PFU/cell. The bottom of the gradient is to the left in all Fig. The arrows represent positions to which 28 and 18s KB cell ribosomal RNA sediment

1232

Vol. 47, No. 5, 1972

BIOCHEMICAL

linear

gradients

5 to 20% sucrose

classes

of ssRNA molecules

and 14.0s

(3,6,

9).

treated

at 36'

tracted

from uninfected

The results

Approximately

to different

equal

tracted

acid-insoluble

several

similar

drawn.

not detected

amounts

(1)

In L cell

were

were

applied

summarized

cultures

exposed

out

gradient

labeling

analysis

of RNA excell

synthesized are

by 3H-counts

to most

cultures.

in L cell

shown

in Fig

2.

in phenol-SDS-ex-

to each gradient. 3.

these

(10 ug/ml)

(50 units/ml)

in Table

in

coefficients

of interferon

of ssRNA, as judged

carried

used for

sedimentation

of ssRNA species

concentrations

are

that

by sucrose

analyses

18.5

ribonuclease

or interferon-treated

materials,

experiments

with

of 24.5,

95% of ssRNA analyzed

conditions

size-

interferon-

by pancreatic

ssRNA species

gradient

than

different

were

reovirus-infected

Greater

Under

control

of sucrose

from

to digestion

4 to 6s were

exposed

TABLE 3.

analyses

cultures.

experiments,

than

were

sedimentation

L cell

greater

cultures

velocity

30 min incubation.

ssRNA in these

of three coefficients

was susceptible

after

the resolution

sedimentation

ssRNA extracted

and control

experiments

allows

of average

Similar

on 3H-uridine-labeled

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The results

Several

general

concentrations

of

conclusions

of interferon,

EFFECT OF INTERFERON ON NUMBER OF SIZE CLASSES OF ssRNA SYNTHESIZED IN REOVIRUS-INFECTED

Number of ssRNA size-classes

L CELLS*

Concentrations of interferon (units/ml)

Virus Yield (XC) in Expt. No. -3 -, , L 2 4

7

I

3

0

100

100

100

100

r

1

100

4

l-5

71

87

94

74

76

5

3-10

23

-

15

-

-

6

a-40

44

-

-

54

7

40

8

100

12,

9

3

-

5----

* Number of size-classes of centrifuged into linear 5 in the legend for Fig 2. profiles shown in Fig No.

ssRNA was determined from the RNA profiles of ssRNA to 20% sucrose gradients under the conditions described The data shown for Expt. No. 1 was taken from the RNA 2.

1233

BIOCHEMICAL

Vol. 47, No. 5, 1972

the

three

normal

size-classes

of ssRNA in each class increased. treated

cell

increased

cultures

with

classes

of reovirus

were

(2) Aberrant

similarly

size

of infectious by increasing

aberrant

size-classes

synthesized class

per

virus

reduced

produced

doses

of ssFWA synthesized

amounts

cell

(3)

cultures

of low molecular

the

i.e.

control

infected

cell

cell

size-classes of aberrant with

as yields

in numbers

size-classes

larger

than

cultures

(4)

As the

synthesized

RNA.

(5)

ssRNA extracted

to interferon

doses

that

resulted

had been exposed

greater

inhibition

of virus

yields

gave variable

sizedose

increased

TCA-insoluble

which

of

of ssRNA

weight

cultures

were

the largest

cultures.

siz

the

of virus

was found

were

was

correlation

Some of the aberrant

in infected

was increased,

aberrant

an inverse

an increase

cell

the amounts

in interferon-

The number

culture,

of interferon,

of ssRNA.

of these

showed

per

although

synthesized

of interferon.

culture

in interferon-treated

of interferon

and amount

concentrations

detected,

as the dose of interferon

of ssRNA were

and the number

increasing

ssRNA were

reduced

classes

of ssRNA synthesized

titer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

cell

in a 95% or

ssRNA patterns

in sucrose

gradient:

DISCUSSION Treatment

of L cells

reduced

synthesis

species

of ssRNA.

to synthesize cells

infected

with

VSV (16)

that

of normal

with

against

that implies

of early

viral

treatment

that

viruses

the antiviral

of L cells

prior

than

the largest

are viral.

The

existance

up to 5u (18)

of dsRNA released

from

dsRNA templates. and 7.7u gently

(19) disrupted

1234

activity

progress

of interferon presented

resulted

have been virions.

strands

herein

in synthesis

found

to check of large

Large

in

The studies

ssRNA species

ssRNA molecules be large

(17).

3

reported

RNA species

to infection

in

type of new

were

The results

are

must

interferon

transcriptases.

larger

reovirus

synthesis

messenger

and vaccinia

experiments

there

dsEWA in lengths studies

the virion-bound

were

homologous

showed

with

and permitted

with

VSV (16)

(17)

to infection

Hybridization

cells.

that

treated amounts

SV40 (15),

that

prior

ssRNA species

cultures reduced

interferon

the large

interferon

viral

and vaccinia

of SSRNA molecules infected

Cell

markedly

was directed showed

with

in untreate

the assumption

ssRNA molecules of reovirus

seen in electron

microscopic

Therefore,

dsRNA segments

BIOCHEMICAL

Vol. 47, No. 5,1972

are most probably could

serve

linked

that

large

has been reported more

than

Since

molecules

could

polyribosomes

(2).

the hypothesis

precursor

with

alters

also

which

(2)

equal

phenol

ssRNA molecules is correct,

replication

of termination

serve

the results of reovirus

not

by interfering

of ssRNA

accomodate

of viral in report with

12

ssRNA in

extracted

the large from

herein

suggest

of transcription

as precursors of this

containing

or 18.0s

presented

in synthesis portions

L cells

size-classes

ssRNA molecules

and SDS, the results

incomplete

three

to 24.5

are normally

in an increase

and

Data

polyribosomes

of ssRNA should

of the 14.0s

process

resulted

viral

ssRNA molecules

ssRNA molecules

virions

in reovirus-infected

of the

(14.0s)

into

ssRNA molecules.

that

quantities

of 14.0s

may represent

that

found

size-class

the normal

dsRNA segments inhibits

of large

the presence

As large

treatment

ssRNA molecules

interferon

for

cells

interferon

genomic

linkage

to incorporation

may be present

et al.

contained

account

reovirus-infected

If

Ward,

the smallest

at maximum,

prior

transcription

ssRNA molecules

(2).

ribosomes

Interferon

for

30 ribosomes

molecules.

that

in some manner

as templates

suggesting

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of small

of ssRNA. (4 to 6s)

ssRNA molecules. the synthesis indicate synthesis

of

that of

ssRNA molecules. ACKNOWLEDGEMENTS

This work was supported by Grant DRG-1036 from the Damon Runyon Memorial Fund for Cancer Research, Inc. and by U.S. Public Health Service Research Grant AI-09040 from the National Institute of Allergy and Infectious Diseases. I thank Maria Cline and James Kiernat for excellent technical assistance and Marshall Dinowitz and Christopher Mathews for a critical reading of the manuscript. REFERENCES 1. 2.

Prevec, L. and Graham, A.F., Science, 154, 522 (1966). Ward, R., Banerjee, A.K., La Fiandra, A., and Shatkin, A.J., J. Virol., 2, 61. (1972). 3: Prevec, L.,Watanabe, Y., Gauntt, C.J., and Graham, A.F., J. Virol., 2, 289. (1968). 4. Bellamy, A.R., and Joklik, W.K., J. Mol. Biol., 29, 19. (1967). 5. Millward, S., and Nonoyama, M., Cold Spring Harbor Symposia on Quantitative Biology, XXXV, 773. (1970). 6. Banerjee, A.K., and Shatkin, A.J. J. Virol., 2, 1. (1970). 7. Sakuma, S. and Watanabe, Y., J. Virol. S, 190. (1971). 8. Schonberg, M., Silverstein, S.C., Levin, D.H., and Acs, G., Proc. Nat. Acad. Sci. U.S.A. , 6J, 505. (1971). 9. Watanabe, Y., and Graham, A.F. J. Virol. 1, 665. (1967). 10. Giron, D.J., Allen, P.T., Pindak, F.F., and Schmidt, J.P. Appl. Micro., 21, 387. (1971).

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Vol. 47, No. 5, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

11. Gauntt, C.J. and Lockart, R.Z., Jr. J. Bacterial., 91, 176. (1966). 12. Loh, P.C., and Oie, H.K., J. Virol., 5, 890. (1969). 13. Shatkin, A.J., and Rada, B., J. Virol., 1, 24. (1967). 14. Warner, J.R., Knopf, P., and Rich, A., Proc. Nat. Acad. Sci. U.S.A., 9, 122. (1963). 15. Oxman, M.N., and Levin, M.J., Proc. Nat. Acad. Sci. U.S.A., 68, 299. (1971). 16. Marcus, P.I., Englehardt, D.L., Hunt, J.M. and Sekellick, M.J., Science 174, 593. (1971). 17. Bialy, H.S., and Colby, C., J. Virol., 2, 286. (1972). 18. Granboulan, N., and Niveleou, A., J. Microsc., 5, 23. (1967). Z Naturforsch, G, 159. (1967). 19. Dunnebacke, T.H., and Kleinschmidt, A.K.,

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