Mitochondrial alterations associated with avian reticuloendotheliosis virus (strain T) pathogenicity

BIOCHEMICAL

Vol. 48, No. 3, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MITOClIONDRIAL ALTERATIONS ASSOCIATED WITH AVIAN RETICULOEI~DOTRELIOSIS VIRUS (STRAIN T) PATIIOGENICITY W. X. Balcavage 132 K. L. Uaxte$-Gabbardl M. Ko M. Rea2 F. Padgettl A. S. Levine1 1

Indiana University School of Iiedicine Terre Haute Center for Medical Education at Indiana State University Terre Haute, Indiana 47809 and 2 Department of Chemistry Indiana State University Terre Haute, Indiana 47809

Received

June 27,1972

This report indicates that reticuloendotheliosis virus (REV) induces morphological, physical and enzymatic alterations in chick target organ mitoSuccinoxidase activity of infected mitochondria is decreased at 5 chondria. days post-REV-infection, but ADP:O ratios are unchanged. Calcium accumulation is decreased from 100 nMoles/mg of protein in controls to 50 nMoles/mg of protein in infected mitochondria. Mitochondria from infected tissue exhibit a buoyant density of 1.193 gm/cc, significantly greater than the buoyant density of 1.167 gm/cc for control mitochondria. Elitochondria isolated from REV-infected livers were effective in transmitting reticuloendotheliosis when inoculated intraperitoneally into day old chicks. Electron microscopy of tissue sections indicates that infected cell mitochondria are enlarged with a less dense matrix relative to controls. Purified mitochondria from infected livers contained up to 16 particles having the size and shape of unenveloped(naked) C-type RIIA tumor virus. It liosis

has previously is

exhibits

been shown that

associated oncogenic

(2, 3, 4, 5, 6). mitochondria

with

reticuloendotheliosis

properties

and is

Previous

and the

attempts

oncogenic unsuccessful.

crease

of mitochondria

this rapidly

characteristic dividing

has been non-neoplastic

Copyright 0 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

a well

date

605

to

the

embryonic)

cell

(1).

(9,

tissue.

virus particle tumor

cell

(7, 81, but

of Warburg

have observed

a de-

10, 11,

but

phenomenon

similar

This

between

investigators

neoplastic

reticuloendothe-

RNA C-type

a relationship

from the work

per

(e.g.,

(REV)

characterized

Numerous

attributed

neoplastic virus

to derive

event

have been relatively in the number

viral-induced

12),

seen in

Vol. 48, No. 3, 1972

Other or the

BIOCHEMICAL

investigators

rate

Chang,

proteins

from

et al.

within

shown that

mitochondria in inducing

by AR-130

cells.

In this

cells,

while

which

suggests

REV replication.

prepared

from

and are capable intra-peritoneal

jection

as

dard.

chicks

density

and refractive

from the

tissue

are

challenge

of REV target respect

contain

and organs

is that

viral-like

of 0.25

are

mito-

inclusions

hatched

chicks

(19)

gradients

were

prepared

of gradient

by the

(23).

Electron

was performed

by negative

a bovine

for

were

microscopy

staining

at 25'C

Protein

was stsn-

by Baxter-Gabbard

microscopy of density

techniques

of

serum albumin

determined

electron

in-

performed (20).

days

by

homogenate

were

as described

fractions

samples

settled

described

with

described

by intra-peritoneal

studies

as previously (21)

aged one to seven

and the medium

Respiratory

tissue.

and Kies

of Padgett

(17)

ml of a 10% gravity

by Murphy

indices

of chicks

was induced

Tissue

mitochondria

AR-130, future

to newly

livers

and Lomb 3L refractometer. procedure

has

enzymological

in this

of Schneider

polarograph

described

Sucrose

Oda (13)

AND MElYIODS

Infected

and spleen

sn Interscience

estimated

(22)

(18).

dsy old liver

prepared

the method

and Hagihara

P&specific with

were using

into

mitochondria

viral-like

injection.

Mitochondria

Chance

physical,

reticuloendotheliosis

MATERIALS

post-hatching

against

organs

membrane

In addition,

and protection

target

of neoplastic

observed

from hepatoma,

significant

REV-infected

of transmitting

tissue.

have

membranes

that

Highly

mitochondria

(16)

preliminary

DNA (13)

in mitochondrial

et al.

of neoplastic

we present

into

alterations

Kara,

hyperimmunization

in mitochondrial

(14)

described

or mitochondrial

evidence

for

have

communication

morphological

after

(15)

mitochondria

effective

an increase

of 3H-thymidine

neoplastic

inclusions

chondria

described

of incorporation

tissue.

a locus

have

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

using were

a Bausch prepared

gradient

purified

(4).

RESULTS Initial involved

the

observations

on mitochondria

polarographic

determination

derived

from REV infected

of mitochondrial

606

succinoxidase

tissue activity.

BIOCHEMICAL

Vol. 48, No. 3, 1972

40

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o . REV INFECTED

I 1

FIGURF 1.

Respiratory

activity

mitochondrial

ing

an initial

tissues.

continued

to exhibit

infected

tissue

activity,

but

largely

unaltered.

coupling

process

is

Normally, energy their

coupling energy

activity

energy

is

coupling

activity

results

suggest

Chang,

of mitochondrial

mitochondria process. dependent

possess

accumulation

with

that

the

a variety

of other

of calcium

dition

system.

607

In Fig.

ion

(Ca++),

potential

exhibit a greatly ++ to the reaction of Ca

membrane

of neoplastic

decreased

(24).

3 (+ADP)

by the

membranes

mitochondria

(H+)

state

2, it

ADP:O ratio associated

is energy

In this marked

context alterations

cells.

pumping with

the

as reflected

the

by

stoichiometric

REV infected

H+ ejection is

in

respiratory

ways of utilizing

that for

mitochondria

alteration

(15) have observed

via

developexhibit

control

of REV infection.

ways is

tissues

mitochondria

The major

as measured

2 illustrates

of protons

1 mM

rapidly

11, while

the

Figure

ejection

with

(p.i.), (Fig.

et al.

One of these

2 mM Tris,

and REV infected

associated

as a consequence that

with

10 mg. of

1 mM K Cl,

of activity.

associated

was initiated

contained

control

activity

is

impaired

to note

3 activity

system

usually

level

The oxidizable

Infection.

from

succinoxidase

These

content

PI - Post

an increasing

the

mitochondria.

5 days post-infection

mitochondria

of interest

in protein

in

after

decreased

liver

8

fl Ilannitol,

mitochondria

increase

However,

a strikingly

it

that

7

and state

0.25

M EIYTA.

1 demonstrates

6

The 3 ml assay

protein,

25

m2p04,

exhibit

of chicken

ADP (+ADP).

125A(E4

5 4 PI. OAV

3

was 6 mM succinate

substrate

Figure

2

shown that

liver

after

ad-

in response

BIOCHEMICAL

Vol. 48, No. 3, 1972

I

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

II’ ElECTION 200 n mToles Ca”

Fig. -260 c-0 290

2r

m”‘eS

2. -260

%, 1.4200

% 1.4200 1.4100 1.4000 1.3900 1.3900 1.3700

v

FRACTION

II+ ejection

were

to a medium containing

by control

pH 72. II+ ejection

was monitored

were

arrow.

added

FIGURE 3.

at each

Sedimentation

2.5 ml with

buffer profile

by differential fig.

0.25

0.001

!.I Mannitol, a recording

of REV infected

and ten

Sedimentation

as in

mitochondria.

and velocity

to equilibrium

purified

and REV infected

with

profile

differential

FIGURE 4.

10

I 30

FRACTION

FIGURE 2. added

! 20

I 30

and the

fractions O.D.

of control

608

and 1 mM Kl12P04, 200 nMoles

mitochondria

purified

The gradient were

measured

collected,

of calcium

by

was centrifuged diluted

to

at 260 and 280 nm.

(non-infected)

and velocity

3.

XKCl,

pH meter.

sedimentation. drop

Mitochondria

sedimentation.

chick

liver

mitochondria

Other

conditions

BIOCHEMICAL

Vol. 48, No. 3, 1972

to calcium

additions,

while

REV infected

again

suggesting

separate

tissue

experiments

(not parallels

and is

day p.i.,

changes

section

Control

tissue control

tissue

Figs.

exhibited

mitochondria

3 is

were

negatively

observed

objects

stained,

which

contained

results

in size suggested

mitochondria.

elements

It

required

neoplasia

pathology

appears

on the

5 and 7 days p.i. with

with

re5th or

with

it

a significantly

The significance

(26)

mitochondria that

buoyant 1.167

matrix.

conformation

was observed

193 g,m/cc vs r=

(25).

translucent

tissue

greater

gross

reports

an orthodox

centrifugation,

showed

previous

an electron

and infected

was

infected

density

gm/cc)

of the band

into

for

viral

previously

were

isolated

(17)

protein)

were

injected

(i.p.)

symptoms

similar

tissue

and exsmined

were

then

as shown in

atp=l.182

and shape that

gm/cc

unchallenged from chick into to those

occurred

and would

40,

chicks. livers

7 days p.i.

one-day

induced

609

with

old

in

association

contain

all

be effective To test

of the

in transmitting this

proposal,

Mitochondria

chicks. REV.

These

viruses.

such mitochondria

replication

mitochondria

particles.

RNA C-type

replication

that

by the preceding

microscopy, located

unenveloped

viral

purified

by electron

up to 16 internally

was proposed

chondria

exhibited

symptoms

of control

from REV infected

approximate These

in Ca++ in-

at the moment undetermined.

When mitochondria method,

swollen

(f =l.

3 and 4, respectively.

in Fig.

appeared

density.

mitochondria

decrease

In

at 6 and 8 days p.i.,

in agreement

in the matrix

gradient

the

processes.

reticuloendotheliosis

excised

mitochondria

by density

H+X mg-1 ,

7 to 11 days p.i.

from tissue

density

coupling

that

capacity

exhibited

When the buoyant exsmined

of viral

morphology,

tissues

in energy

was observed

onset

of H+X mg-1 ,

up to 160 nMoles

a maximum of 100 Moles

of the neoplastic

prepared

and no alterations

it

after

in situ,

chick

can eject

in H+ejection

lethal

in mitochondrial

The mitochondria,

the

the

can eject

alteration

shown),

The development

Liver

with

mitochondria

an 18% and 36$ decrease

spectively. 6th

mitochondria

an REV associated

duced Ii+ ejection with

control

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

After

Dead chicks

mito(3.1

mg

8 days chicks were

autopsied

BIOCHEMICAL

Vol. 48, No. 3, 1972

and found

to contain

the

36 of the

14 days,

liver

40 chicks

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lesions

typical

had died

Within

of reticuloendotheliosis.

of reticuloendotheliosis.

DISCUSSIOIJ The results

presented

Oda (13) has described sity)

of hepatoma

clusions

in nucleic

be a target

reports,

The importance

subsequent

for

of these

event

needs

and clarify

the

acid

doses

in the

elaboration

observations

viral

in-

shown that

of AH-130

cells.

hepatoma

of the

at least elucidation

and experiments

reported

den-

challenged

to suggest

further

presumably

of hyperimmunizing

context

modification, in

(and

Oda (13) has also

lethal

observations

investigators.

(16) have reported

capable

seem reasonable

of other

content

et al.

are

when viewed

oncogenic

little

by results

cells.

membranes

make it

orgsnelle

Kara,

of Rous Sarcoma

against

investigation,

neoplastic

while

or mitochondrial

The preceding

extend

alterations

mitochondria,

and protecting

present

are supported

in mitochondria

mitochondria rats

here

that

results

of the

mitochondria

in the

case of REV.

of the viral are

may

induced

in progress

to

here.

ACKNOWLEDGMENTS This study was supported under Contract Virus Cancer Program of the National Cancer of Chemistry, Indiana State University.

No. 69-2048 within the Special Institute. NIR. PHS. and the Dent.

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