Deoxyribonuclease activity of normal and poxvirus-infected Hela cells

Vol. 19, No. 1, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DRCXTRIRONUCLRASE ACTIVITT OF NORMAL ANDPOXVIRUS-INPECTED HI&t CRLLW B. R. McAuslan Department of Biology,

Princeton University,

Princeton,

New Jersey

ReceivedJanuary 21, 1965 An increase in the deoxyribonuclease activity

of L-cells

with Vaccinia virus was noted by Hanafusa (1960).

infected

More recently

Magee

1964) has been unable to detect such an increase in activity.

(1963,

Clarification

of these controversial

study of viral

replication

findings may be relevant

and, in particular,

induced DNApolymerase (Magee, 1962). tion of the effect

to the study of poxvirus-

We therefore

of poxvirus infection

increase in DNAaeeactivity

of Cowpox-infected Hela-S3 cells is demonstrable.

the corresponding activity ability

has properties

of uninfected Hela-S

to induce DNAaseactivity

undertook a re-examina-

on DNAaseactivity.

This communication shows that a striking

that the induced enzyme activity

to the

In addition,

we find

that distinguish 3

cells.

it from

However, the

does not appear to be a property of all

poxviruses. The maintenance of cells,

procedures for their

the preparation of cell extracts protein concentration

infection

with virus,

and the method for determination of

have been described (McAuslan and Joklik,

1962).

The virus used was the Brighton strain of cowpox (CP) or the Utrecht strain

of rabbitpox

(RP) purified

by methods described by Joklik

(1962).

The substrate for the enzyme assay was DNA-P32 prepared by Marmurrs method (1961) from X-coli 1 mc P3204 per litre.

* Aided by

U.

R grown to a limit

in tris-glucose

mediumcontaining

Acid soluble counts in the product were practically

S. Public Health Service Grant AI-&CO. 15

BIOCHEMICAL

Vol. 19, No. 1, 1965

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

eliminated by treatment with Norit (Lehman, 1960).

For someexperiments

DNA-P32 (7Opg) in 1.0 ml 0.02 M NaCl containing O.O25Mtris heated (10 mina. 1OO'C) then chilled

to 0'.

ties of DNAaaeof normal and infected

cells,

pH7.5 was

For comparison of the properextracts

were prepared 7

hours post-infection. The kinetics extracts

of the increase in DNAaaeactivity

of infected-cell

under the assay conditions are shown in Fig. 1.

Similar results

have been obtained with cells infected with the Connaught Laboratories strain

of vaccinia virus.

of RP infected cells

No significant

change in the DNAaseactivity

could be detected although thymidine kinase was

induced.

'

I

I

2 HOURS

I

I

4

6

POST

I

8

I

IO

-INFECTION

Fig. 1. Increase in DNAaseactivity of CP-infected Hela cells. (0) Normal cell extracts; (0) infected-cell extracts. ASSay conditions: The reaction mixture (350 ul) contained native DNA-P32 (25 gg; 20,000 CPM); MgC12(2~ moles); glycine-NaOH pR 8.5 (33poles); cell extract 25 pl, 250 pg protein). The mixture was incubated for 40 minutes at 37” then chilled. Carrier DNA (0.1 ml containing 0.25 mg DNA'and 0.5 mg bovine serum albumin) and 0.5 N 0.45 ml) was added. After 5 minute8 at O', the mixture was centriE~b[3 mina. 10,OOCxg) and the supernetant transferred to planchettes for determination of radioactivity. The assay was linear within increeaing protein over the range 50 - 5OOpg and with time, for at least 60 minutes. Activity is expressed as cpm rendered acid soluble under these assay conditions.

16

Vol. 19, No. 1, 1965

The influence rate

demonstrates

of normal

BIOCHEMICAL

of the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

physical

a difference

and CP-infected

state

of the

in substrate

cell

extracts

INCUBATION

substrate

requirements

(Fig.

TIME

IN

on the reaction of the activities

2).

MINUTES

of the physical state of the substrate on DNAase activity Fig. 2. Effect normal and CP-infected cell extracts. (0) infected cell extracts plus native DNA; (WC) infected-cell extracts plus thermally denatured DNA; (A) normal cell extracts plus thermally denatured DNA; (0 ) normal cell extracts plus native DNA. of

To see how many distinguishable measured, in Fig. activity thermal

thermal 2. is

The results being

stability

DNA, is not

stability

curves (Fig.

measured curve

enzyme activities were

plotted

3) indicate

that

by using

at 4’7” for

due to the presence

thermally infected

of excess 17

for

are actually four

cases

probably

only

the one enzyme

denatured

DNA.

cell

the

being

DNAase assayed

enzyme in the assay.

outlined

The unusual with

native

Vol. 19, No. 1, 1965

BIOCHEMICAL

TIME

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IN MINUTES

AT ELEVATED

TEMPERATURE

stability of DNAase activity of Normal and Infected-cell Fig. 3. Thermal extracts. Extracts at the same protein concentration (pH 7.5) were held for various periods at either 47” or 57’ then chilled before assaying as described under Fig. 1. (0) normal extracts plus native DNA; (A) Normal extracts plus denatured DNA; ( 0 ) infected extracts plus native DNA; ( j+) infected extracts plus denatured DNA. The influence

of pli on enzyme activity

is

shown in Table

I.

TABLE I DNA -STATE

ENZYME EETBACT

Native

5

7.0

Normal

1050

375

300

350

Native

Infected

1100

750

2ooo

3325

Denatured

Normal

310

50

870

10.0

10.6

200

180

160

5695

2990

360

145

2250

1600

625

50

were essentially as described for Fig. 1. Buffers used were Acetate at pH5: 0.l.M tris HCl in the range 7 to 9.2 or O.lM glycine in the range 8.5 to 10.6.

0.l.M NaOH

350

7.8

a.5

9.2

9.6

2y3

Assays

The “alkaline” compared

for

Extracts “alkaline” (pH5)

their

DNAase of normal sensitivity

of uninfected

and infected-cell

to various Hela

DNAase activity.

cells

inhibitors

assayed

The corresponding

with “acid”

extracts (Table native

were

II). DNA, show low

DNAase activity

is

Omis-

sion

approximately three times that of the alkaline DNAase. +Iof Mg ions markedly increases this “acid” DNAase activity.

post

infection

with

change

in

“acid”

DNAase was detected. 18

At pH 9.2,

No

Vol. 19, No. 1, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II

$ Maximal

Activity Infected

Normal (pH 8.5,

Standard

assay

Standard

assay + lng Actinomycin

Standard Standard

assay + 10 pg Actinomycin D +t -4-b assay minus Mg , plus Mn

Standard

assay

native

native

DNA as substrate, cells

activity

closely

thesis,

(McAuslan,

did

not affect

vity

is not

DNAase.

follows

those

the

results,

due to viral-induced

as complete

adsorption

Hela

cells.

is

similar

in Pig.

absolute,

uninfected

cells

is 5 fold

The pre-

and post-

tiated

by their

response

vity

A likely

explanation

induced

by infection,

for higher

the

native for

increase

in

thymidine

kinase

syn-

with

or of RP-infected cell

is

extracts.

greater

than

from

the virus

The increase

could

be accounted

inoculum.

(on which

19

virus

stocks

induced

DNAaae has a preferential,

DNA whereas denatured

of uninfected

the DNAase activity DNA than

of the different codes

for

the

for

Further,

activity

heat

cowpox

do

to that

the appearance that

of cells

infection DNAase activities can also +I- ions and to actinomycia D. to Mn for

RNAase

in DNAase acti-

of an RNA inhibitor

hen egg membranes properties

100

of this

the increase

cells

400 times

2 show that

requirement

DNA.

that

of DNAase activity

in its

40

of cell-extracts

of CP-infected

DNAase of fertile

The results perhaps

at least

150

in the DNAase activity

poxvirus-induced

of normal

DNAase is

10

The kinetics

destruction

extracts

50

40

increases

indicating

in alkaline

grown)

for

100

75

S-RNA

Pre-treatment

1963 a,b).

the DNAase activity

were

100

demonstrable.

not inhibit

“alkaline”

D

up to 20 fold are

In vitro,

the

100

500 pg E. coli

plus

of CP-infected

DNA)

for

native

be differen-

DNAese acti-

synthesis

of this

of

Vol. 19, No. 1, 1965

BIOCHEMICAL

enzyme. The DNAaseactivity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of uninfected-

or infected-cell

extracts

under conditions used to assay DNApolymerase (see Green and Pina, 1962; Magee, 1962) is unlikely

to significantly

influence the latter

reaction.

However, the use of a non-DNAaseinducing strain (RP) might be preferable for further

study of viral

induced polymerase.

The failure

of RP to induce

DNAasedoes not appear to be due to the presence of an inhibitor the inoculum or induced by RP infection. bility

We are investigating

present in the possi-

that the CP induced DNAaseinfluences the yield of infectious

pox which, for the Hela cell system, is low compared to the yield

cow-

of infectious

=.

The expert technical edged.'

assistance of Miss Pamela Herde is gratefully

acknowl-

REFERENCES 1 , 602 (1962). Green, M. and Pina, M., Virol 0 Hanafusa T., Bikens Ji -!k 9i -73(1962) 19 1). Joklik W. K. , Virolog . . &. a, 1i79 (1960). Lehman, I . R., -. 604 (1962). Magee, W. , vi.ro~og~~, Magee, W.

Magee,

216 (1963).

W.

Marmur, J McAuslan,

a:nd=liT;; 36 (1962).

McAuslan, B. R.,, . virolony, g, McAuslan, B. R., Virology ,A,

W. K:,SBiochem. Biophys. Research Cornnuns. 162 (l$ja). 383 (1963b).

20