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JOURNAL
OF
VIROLOGY,
Oct.
1968,
p.
1064-1075
Copyright
r,
1968
Amiler-ican
Society
for
Micr-obiology
Vol.
2,
No.
10
Pri,iwted
in
U.S.A.
Virus-Receptor
Interaction
in
an
Adenovirus
System
LENNART
PHILIPSON,
KARL
LONBERG-HOLM,1
AND
ULF
PETTERSSON
Deplrtnent
oJ
Microbiology,
Tue
Waillenlberg
Laboratory,
Uppsala
University,
Uppsalca,
Sweledn
Received
for
publication
25
July
1968
HeLa
or
KB
cells
each
contain
around
104
receptor
sites
for
adenovirus
type
2.
These
are
inactivated
by
treatment
of
live
cells
with
subtilisin.
The
receptor
activity
of
the
enzyme-treated
cells
is
regained
after
4
to
8
hr
of
incubation
in
complete
medium.
A
technique
that
utilizes
the
difference
in
buoyant
density
between
free
virus
and
virus-receptor
complex
was
developed
to
demonstrate
receptor
activity.
Cellular
fractionation
revealed
that
receptors
were
confined
mainly
to
the
plasma
membrane
fraction
and
that
negligible
receptor
activity
could
be
demonstrated
in
enzyme-treated
cells.
Subtilisin
probably
did
not
penetrate
the
cell
membrane;
thus,
the
receptors
are
limited
to
the
cell
surface.
Purified
fiber
of
the
virion
com-
pletely
prevents
attachment
of
adenovirus
types
2
and
5
to
receptor
sites
at
a
ratio
of
105
protein
molecules
per
cell.
Adsorption
studies
indicate
that
105
to
106
receptor
sites
are
available
for
the
structural
protein.
The
fiber
does
not
affect
attachment
of
poliovirus
type
1.
Animal
viruses
generally
are
considered
to
be
taken
into
cells
by
pinocytosis
after
specific
interaction
of
the
virus
with
receptor
sites
at
the
cell
surface
(3,
11).
The
evidence
for
this
stems
mainly
from
electron
microscopy.
Such
studies
of
the
early
interaction
of
adenovirLtses
with
cells
suggest
that
intact
virions
are
taken
up
by
pinocytosis
and
subsequently
are
decoated
close
to
the
nuclear
membrane
(2).
Biochemical
tech-
niques
that
have
been
applied
recently
to
study
viral
entry
(9,
10, 15,
23,
26)
reveal
in
adenovirus
systems
a
rapid
uptake
of
virions
by
cells
(15,
23,
28).
Shortly
afterwards,
the
virion
is
converted
to
a
deoxyribonuclease-sensitive
structure
that
contains
viral
protein
and
all
of
the
nucleic
acid
of
the
virion.
This
structure
gains
rapid
entry
to
the
cell
nucleus
(Lonberg-Holm
and
Philipson,
in
preparation).
In
other
virus-cell
systems
the
initial
virus-
receptor
interaction
appears
to
involve
con-
siderable
specificity;
for
example,
HeLa
cell
receptors
for
picornaviruses
differ
in
suscepti-
bility
to
enzymatic
destruction
and
in
the
time
required
for
their
regeneration
(17,
25,
32).
The
receptors
for
poliovirus
appear
also
to
be
differ-
ent
from
those
of
coxsackievirus,
as
revealed
by
cross-saturation
experiments
(17).
This
study
aims
to
establish
the
specificity
of
adenovirus-receptor
interaction
and
demonstrate
that
receptors
are
preferentially
located
at
the
plasma
membrane
of
susceptible
cells.
The
num-
I
Recipient
of
special
National
Institutes
of
Health
Researchi
Fellowship
Award
5F3-AI-6864.
ber
of
active
sites
per
cell
and
the
characteristics
of
the
virus-receptor
complex
are
reported.
The
effect
of
purified
virus
capsid
proteins
on
the
virus-cell
interaction
have
also
been
studied.
A
preliminary
report
on
some
of
the
results
has
been
published
(24).
MATERIALS
AND
METHODS
Cells.
HeLa
and
KB
cells
obtained
from
Micro-
biological
Associates,
Inc.,
Bethesda,
Md.,
were
grown
in
spinner
cultures
in
Eagle's
spinner
medium
(4)
with
5%,1
horse
serum,
2%-
calf
serum,
penicillin,
streptomycin,
and
kanamycin.
KB
cells
that
were
used
for
virus
assay
were
grown
in
plastic
petri
dishes
with
Eagle's
minimal
essential
medium
(MEM)
with
15%',
calf
serum
and
4%(
tryptose
phosphate
broth.
Viri.s.
The
prototype
strains
of
adenovirus
types
2
and
5
were
obtained
from
R.
J.
Huebner
of
the
Na-
tional
Institutes
of
Health.
Nonradioactive
virus
pools
were
prepared
by
a
modification
of
the
method
of
Green
and
Piiia
(5)
similar
to
that
previously
described
for
2P-labeled
virus
(23).
However,
CsCI
was
used
for
both
isopycnic
banding
steps.
The
second
of
these
employed
a
shallow
preformed
gradient
of
1.30
to
1.41
g
cc
and
centrifugation
for
16
hr
at
about
33,000
X
g.
Virus
was
then
dialyzed
against
and
stored
in
0.25
M
sucrose
that
contained
10-3
M
MgCl2
and
0.02
M
tris(hydroxymethyl)aminomethane
(Tris)-
chloride
buffer
(pH
7.4)
at
I
to
2
X
10'3
particles
per
ml.
In
our
calculations,
one
optical
density
(OD)
unit
(A2:,7
to
A
,o)
is
equal
to
7
X
1oll
particles.
Preparation
f
rladioactive
viras.
32P-Iab-led
adeno-
virus
was
produced
in
KB
or
HeLa
cells.
These
were
growni
as
monolayers
in
32-oz
prescription
bottles
in
MEM
that
contained
15%'
calf
serum
and
4'-C
1064
ADENOVIRUS
RECEPTORS
tryptose
phosphate
broth.
Before
infection,
the
cells
were
washed
twice
with
MEM
and
were
then
covered
with
1
ml
of
phosphate-buffered
saline
(PBSA)
that
contained
about
2
X
108
fluorescent
foci
units
(FFU)
of
virus
(multiplicity
about
10).
They
were
thus-
incubated
for
1
hr
at
37
C.
MEM
(30
ml)
with
10'%(
calf
seruLm
was
then
added.
After
about
24
hr,
the
cells
were
washed
twice
with
Eagle's
MEM
in
which
the
phosphate
had
been
replaced
with
citrate;
they
were
then
covered
with
30
ml
of
the
same
medium
that
contained
10c%
calf
serum,
which
had
also
been
dialyzed
against
10
volumes
of
citrate-MEM.
To
each
bottle
was
added
0.2
to
0.25
mc
of
32p
high
spe-
cific
activity
orthophosphate
(The
Radiochemical
Centre,
Amersham,
England).
Two
or
more
days
later,
at
complete
cytopathic
effect,
the
cells
were
harvested
and
the
virus
was
purified
as
described
above.
The
product
usually
contained
less
than
10%
of
the
total
32p
in
a
form
that
could
be
rendered
acid-
soluble
by
deoxyribonuclease
treatment.
l4C-threonine-labeled
adenovirus
type
2
was
pre-
pared
in
a
similar
way.
The
regular
medium
was
changed
after
about
2
days
and
replaced
with
MEM
that
contained
10
,c
per
bottle
of
uniformly
labeled
14C-L-threonine
(International
Chemical
and
Nuclear
Corp.,
City
of
Industry,
Calif.)
with
3
,ug
of
carrier
per
ml.
Virus
intfectivity.
Infectivity
was
assayed
in
KB
cells
by
fluorescent
focus
formation
(22)
as
modified
by
Thiel
and
Smith
(29).
Virus
attachment
was
made
with
1-hr
continuous
rocking
at
room
temperature
and
incubation
was
for
72
hr.
Specific
antisera
to
adenovirus
type
2
hexon
was
employed
(20),
followed
by
fluorescein-conjugated
goat
antirabbit
globulin
(Microbiological
Associates,
Inc.,
Bethesda,
Mary-
land).
In
a
separate
communication,
evidence
will
be
presented
that
one
FFU
corresponds
to
approximately
30
physical
particles
(Lonberg-Holm
and
Philipson,
in
preparationI).
Attaclmenit
of
labeled
virus
to
cells.
Attachment
of
labeled
virus
to
cells
was
carried
out
in
1
ml
of
spinner
medium
with
2%,-c
calf
serum
in
round-bottom
tubes
shaken
vigorously
in
a
37
C
bath
at
a
cell
density
of
5
X
107
per
ml.
Samples
taken
out
at
different
times
were
centrifuged
and
the
supernate
was
assayed
for
radioactivity.
The
cells
were
assayed
after
washing
twice
with
the
spinner
calf
medium.
Entzymes
anid
entzyme
assay.
Three-times
crystallized
trypsin
and
chymotrypsin
were
purchased
from
Worthington
Biochemical
Corp.,
Freehold,
N.J.
Pronase
was
purified
from
Pronase
grade
B
(Calbio-
chem,
Los
Angeles,
Calif.)
by
chromatography
on
carboxymethyl
cellulose,
which
separates
the
pro-
teolytic
activity
from
peptidase
and
esterase
activity
(30).
Crystallized
subtilisin,
of
the
Novo
type,
was
supplied
by
Novo
Industries,
Copenhagen,
Denmark.
Lactate
dehydrogenase
was
assayed
by
a
modifica-
tion
of
the
method
of
Kubowitz
and
Ott
(14).
Di-
luted
cell
extracts
were
added,
at
zero-time,
to
a
mixture
containing
1
ymole
of
pyruvate,
0.2
pmole
of
NADH2,
and
150
,ymoles
of
Tris
buffer
(pH
7.4)
per
3
ml
of
final
volume.
The
decrease
in
A340
was
measured
at
15-sec
intervals
for
about
3
min.
One
unit
of
activity,
about
I
,Amole/min,
is
equivalent
to
an
initial
rate
of
decrease
in
A34,,
of
2.0
per
min.
Cell
Jractioniationi.
Cells
were
cooled
to
0
to
4
C
before
disruption
and
all
subsequent
work
was
in
the
cold.
The
pH
values
reported
are
measured,
however
at
room
temperature.
Cell
fractions
were
stored
at
-60
C
until
assayed.
The
experiments
in
this
report
used
cells
disrupted
by
either
sonic
treatment
or
the
method
referred
to
as
"intracellular
cavitation"
with
nitrogen
gas.
For
sonic
treatment,
a
predetermined
number
of
cells
were
washed
twice
in
Hank's
balanced
salt
solution
(HBSS)
and
the
final
pellet
was
taken
up
in
5
to
10
volumes
of
0.05
M
Tris
buffer
(pH
7.4)
and
then
disrupted
for
15
sec
with
the
1-cm
probe
of
the
MSE
(Measuring
&
Scientific
Equipment,
Ltd.,
London,
England)
100
watt
Ultrasonic
Disintegrator.
Larger
debris
was
removed
by
6-min
centrifugation
at
280
X
g
and
the
supernatant
fluid
was
then
cen-
trifuged
for
1
hr
at
80,000
X
g
(average).
The
par-
ticulates
were
resuspended
in
0.1
M
NaCl
(about
1
ml/108
cell
equivalents).
The
supernatant
fluid
was
also
sometimes
divided
into
clear
and
turbid
(lipid-
containing)
portions,
but
this
did
not
significantly
affect
the
results.
Frozen
samples
of
particulates
were
again
sonically
treated
before
use.
"Intracellular
cavitation"
was
also
employed,
but
only
with
HeLa
cells.
The
method
was
developed
by
Wallach
and
Kamat
(31)
for
ascites
tumor
cells.
Usually
about
4
X
l09
HeLa
cells
were
washed
twice
in
0.15
M
NaCl,
0.005
m
(pH
7.4)
Tris-chloride,
and
were
suspended
in
100
ml
of
0.25
M
sucrose
that
contained
0.005
M
Tris
buffer
(pH
7.4)
and
0.0002
M
MgSO4.
They
were
subjected
for
45
min,
with
occasional
vigorous
rocking,
to
70
kg/cm2
of
N2
in
a
silicone-coated
stainless
steel
pressure
bomb.
The
cells
were
discharged
and
disrupted,
ethylene-
diaminetetraacetate
(EDTA)
was
added
to
a
concen-
tration
of
0.001
M,
and
then
heavy
particles
including
nuclei
were
sedimented
at
280
X
g
for
12
min
("crude
nuclei").
"Crude
mitochondria"
were
sedimented
at
15,500
X
g
(average)
for
15
min,
and
"crude
microsomes"
at
104,000
X
g
(average)
for
45
min.
"Crude
microsomes",
which
include
the
plasma
membrane
fragments,
were
stored
at
-60
C
in
0.25
M
sucrose
that
contained
0.001
M
(pH
8.1
or
8.6)
Tris-
chloride.
In
eight
experiments,
the
average
yields
of
protein
per
108
cells
were
3.9,
3.1,
3.0
and
8.0
mg
for
crude
nuclei,
crude
mitochondria,
crude
microsomes,
and
the
supernatant
fluid,
respectively.
Crude
microsomes
were
thawed,
washed
in
0.01
M
Tris
buffer
and
lysed
in
0.001
M
Tris
buffer
according
to
the
original
method
(31),
with
the
exception
that
pH
8.1
Tris
was
substituted
for
pH
8.6.
The
protein
yield
in
the
washed and
lysed
microsomes
averaged
about
20%c
of
the
crude
microsome
fraction.
Washed
and
lysed
microsomes
were
further
purified
by
adding
magnesiuLm
and
banding
for
5
hr
on
a
Ficoll
barrier,
according
to
Wallach
and
Kamat
(31).
However,
the
pH
and
density
of
the
Ficoll
were
lowered
from
8.6
to
8.1
and
from
1.092
to
1.062,
respectively
(Wallach,
private
comnmioiicationI).
When
samples
were
centri-
fuged
for
longer
than
5
hr,
the
membranes
tended
to
aggregate.
The
yield
of
Ficoll-purified
membranes,
which
should
be
rich
in
plasma
membrane
fragments,
was
about
0.2
mg/108
cells.
The
protein
contents
of
the
cell
fractions
were
VOL
2
,
1968
1065
PHILIPSON,
LONBERG-HOLM,
AND
PETTERSSON
determined
by
the
method
of
Lowry
(18)
on
samples
dialyzed
in
the
cold
against
0.1
M
NaOH.
However,
samples
of
membranes
mixed
with
Ficoll
were
diluted
with
saline
and
pelleted
in
the
ultracentrifuge
to
remove
the
Ficoll;
they
were
then
assayed
directly.
Bovine
serum
albumin
was
used
as
a
standard.
In
vitro
microtube
assay
for
receptor
activity.
In
the
microtube
assay,
samples
of
receptor
are
incubated
with
32P-virus,
and
the
reaction
mixture
is
separated
on
a
small
CsCl
step-gradient
by
ultracentrifugation
into
"free"
and
"bound"
fractions.
The
percentage
of
the
total
32P
which
is
bound
is
multiplied
by
the
input
number
of
virus
particles
to
give
the
number
bound.
The
details
of
the
microtube
assay
were
varied
somewhat
during
the
course
of
the
work.
In
general,
about
1
X
1010
particles
of
adenovirus
that
contain
about
104
counts/min
of
32p
are
incubated
with
sufficient
receptor
to
bind
10
to
30%
(often
about
0.01
mg
of
protein)
for
30
min
at
37
C
in
200
gliters
of
0.1
M
NaCl
that
contains
150
,Ag
of
BSA,
0.4
,mole
of
EDTA,
1
mg
Tween
40,
and
5
,umoles
(pH
7.4)
of
Tris
buffer.
After
incubation,
the
samples
are
chilled
and
300
,uliters
of
1.2
g
per
cc
of
CsCl
solution
is
added
as
a
diluent.
Of
this
solution,
200
,uliters
are
layered
in
a
0.5
X
4.2-cm
cellulose
tube
(Spinco
305528)
that
already
contains
layers
of
200
,uliters
of
1.41
g
and
220
Mliters
of
1.30
g
per
cc
of
CsCl
solutions
that
contains
0.02
M
(pH
7.4)
Tris
buffer.
Seven
tubes
are
held
in
each
of
three
nylon
adapters
made
espe-
cially
for
the
MSE
"superspeed"
type
30,
swingout,
ultracentrifuge
rotor.
In
this
way,
21
samples
are
centrifuged
at
45,000
X
g
for
16 hr
at
6
C.
The
lower
virus
band
is
removed,
together
with
a
total
of
350
,uliters
of
CsCl
solution,
from
the
bottom
of
the
tube
("free"
virus
fraction).
The
virus
that
has
associated
with
the
membrane-bound
receptor
is
located
at
the
top
of
the
tube
(in
the
remaining
270
,uliters),
or
has
stuck
to
the
tube
walls,
so
that
the
entire
remaining
solution,
together
with
the
tube
itself
(cut
in
three
parts),
is
counted
as
the
"bound"
virus.
The
samples
are
counted
directly
(1)
in
10
ml
of
0.1
M
NaOH
(by
Cerenkov
radiation)
with
the
3H-setting
in
the
Tri-Carb
liquid
scintillation
spectrometer
(Packard
Instrument
Co.,
Inc.,
Downers
Grove,
Ill.)
Blanks
with
no
receptor
are
always
relatively
high,
but
the
use
of
BSA
in
the
incubation
helps
lower
these.
Also,
a
mixture
that
contains
a
10-fold
excess
of
nonradioactive
virus
that
has
been
prein-
cubated
with
about
100
,yg
of
crude
microsomal
material
of
low
receptor
activity
from
subtilisin-
treated
cells
is
usually
added
at
the
end
of
the
incu-
bation
to
further
reduce
the
blank.
This
is
particu-
larly
useful
when
low
levels
of
receptor
protein
are
used,
in
which
case
there
is
increased
sticking
of
32P-virus
to
the
top
of
the
tubes.
With
all
precautions,
the
blanks
fall
in
the
range
of
6
to
10%
apparent
binding.
In
each
experiment,
this
must
be
subtracted
to
give
the
"net"
binding.
Structural
proteins
of
adenovirus.
The
purification
and
characterization
of
hexon
and
fiber
of
adenovirus
type
2
has
been
described
in
previous
reports
(20,
21).
14C-labeled
purified
structural
proteins
were
obtained
from
the
production
of
14C-labeled
virus.
'311-labeling
of
structural
proteins.
High
specific
activity
1311-labeled
purified
fiber
and
hexon
were
made
according
to
the
technique
of
Hunter
and
Greenwood
(8)
by
the
use
of
chloramine-T
to
oxidize
iodide
to
iodine
and
sodium
metabisulfite
to
reduce
unused
iodine.
The
1131-labeled
fiber
and
hexon
pro-
teins
were
purified
by
gel-filtration
on
Sephadex
G-100.
A
specific
activity
of
1
and
5
,uc/,ug
was
obtained
for
the
hexon
and
fiber
preparations,
respectively,
when
20
to
100
j,g
was
labeled
with
1
mc
of
13'
in
0.1
ml.
Cell
cloniing.
KB
or
HeLa
cells
were
diluted
in
serial
10-fold
dilutions
in
Eagle's
MEM,
with
double
concentrations
of
amino
acids
and
15%
fetal
calf
serum.
They
were
plated
(5
ml)
on
plastic
petri
dishes
(60
X
15
mm)
and
incubated
for
10
days
in
a
humidi-
fied
atmosphere
of
5%
CO2
in
air.
The
cells
were
then
stained
with
0.005%
neutral
red
in
MEM
for
3
hr,
and
the
clones
were
counted.
Hemagglutination
and
hemagglutinationl-inhibitioni
was
carried
out
as
described
in
a
previous
report
(20).
Radial
immunodiffusion
was
used
to
determine
low
concentrations
of
fiber
and
hexon
proteins.
The
modi-
fied
Mancini
method
(19)
was
used
as
previously
described
(21).
Concentrated
preparations
were
determined
by
dry
weight.
RESULTS
Enzymatic
susceptibility
of
adenovirus
re-
ceptors.
Suspensions
that
contained
5
x
107
KB
or
HeLa
cells
per
ml
were
treated
with
1
mg
of
different
proteolytic
enzymes
per
ml
for
30
min
at
37
C
in
Hank's
balanced
salt
solution
(HBSS).
The
cells
were
then
washed
three
times
in
ten
volumes
of
Eagle's
spinner
medium
with
2%
calf
serum.
Finally,
they
were
resuspended
in
1
ml
of
the
same
medium.
The
attachment
of
32P-labeled
adenovirus
to
these
cells
was
deter-
mined
at
different
times
as
described
in
Materials
and
Methods.
Figure
1
shows
that
60%
of
the
32p_
labeled
virus
is
attached
in
the
untreated
cells
at
30
min,
but
that
both
trypsin-
and
chymotrypsin-
treated
cells
attach
adenovirus
at
a
faster
rate.
Pronase-
and
subtilisin-treated
cells
attach
adeno-
virus
at
a
slower
rate,
and
it
appears
in
the
latter
case
that
no
receptor
sites
are
available.
The
enzymatic
treatments
did
not
influence
the
gross
morphology
of
the
cells.
Subtilisin
concentration
and
removal
of
re-
ceptors.
The
apparent
susceptibility
of
adeno-
virus
receptors
to
subtilisin
could
have
been
due
to
destruction
of
or
irreversible
changes
in
the
cells.
The
effect
of
different
concentrations
of
subtilisin
on
cell
number,
cell
cloning
efficiency,
and
their
ability
to
attach
adenovirus
was
studied.
HeLa
cells
(6
X
107/ml)
were
treated
in
HBSS
at
37
C
for
30
min
with
different
concentrations
of
subtilisin,
varying
from
50
to
1,000
,g/ml.
The
1066
J.
VIROL.
ADENOVIRUS
RECEPTORS
cells
(17,
32).
HeLa
cells
at
5
X
107/ml
were
*
*
.
.
treated
with
400
Ag
of
subtilisin
per
ml
for
30
min
at
37
C.
The
cells
were
washed
twice
with
Eagle's
0&3
spinner
medium
that
contained
2%
calf
serum.
They
were
resuspended
in
ordinary
spinner
medium
at
a
density
of
1
X
106
per
ml.
Puromycin
and
cycloheximide
were
used
at
10
and
5
,g/ml,
respectively,
and
were
included
in
the
washing
step.
At
different
time
intervals
5
x
107
cells
were
harvested,
resuspended
in
1
ml,
and
assayed
for
the
30-min
attachment
of
32P-labeled
virus.
0
Figure
3
shows
that
subtilisin
treatment
reduces
adenovirus
attachment
from
85
to
40%.
At
4
to
8
hr
after
treatment,
the
cells
begin
to
regain
receptor
activity,
and
this
approaches
untreated
*
\
;
values
at
8
hr.
That
resynthesis
of
receptors
occurs
after
a
certain
lag
period
is
supported
by
the
fact
that
receptor
activity
does
not
regenerate
10
20
30 40
50
60
in
the
presence
of
the
inhibitors
of
protein
syn-
thesis,
puromycin
and
cycloheximide.
TIME
IN
MIN.
FIG.
1.
Attachment
of
32P-labeled
adenovirus
type
2
to
KB
cells
treated
by
different
enzymes.
Cells
(5
X
107)
in
I
ml
of
HBSS
were
treated
at
1
mg/ml
for
60
miii
at
37
C.
The
cells
subsequently
were
washed
three
times
in
HBSS
and
resuspended
inl
Eagle's
spinner
medium
with
2%O
calf
serum;
they
were
attachmentt
assayed
as
described
in
Materials
and
Methods.
(1)
Coontrol
cell.
(2)
Trypsiii.
(3)
Clhymotrypsin.
(4)
Pronase.
(5)
Subtilisini.
cells
were
washed
three
times
and
counted.
They
were
plated
for
cloning
efficiency
and
tested
for
the
attachment
of
32P-labeled
adenovirus
to
5
X
107
cells
at
a
multiplicity
of
about
50.
Figure
2
shows
that
with
HeLa
cells,
200
to
400
Mg
of
subtilisin
are
required
to
prevent
adenovirus
attachment,
but
that
1
mg/ml
does
not
signifi-
cantly
reduce
the
cell
number
or
the
number
of
cell
clones.
The
cloning
efficiency
is
around
50%O
with
the
technique
that
was
used.
Similar
results
were
obtained
with
KB
cells,
although
the
cell
numbers
and
the
cloning
effi-
ciency
were
reduced
about
twofold
at
the
highest
two
concentrations
of
subtilisin.
KB
cells
thus
appear
more
susceptible
to
subtilisin
injury
than
HeLa
cells,
but
in
both
systems
there
is
a
significant
difference
between
the
effect
on
cell
number
and
cloning
efficiency
on
the
one
hand
and
adenovirus
attachment
on
the
other.
Regeneration
of
receptors.
Since
both
HeLa
and
KB
cells
showed
unimpaired
cloning
effi-
ciency
after
treatment
with
200
to
600
MAg
of
subtilisin
per
ml,
it
was
of
interest
to
determine
whether
adenovirus
receptors
could
be
regen-
erated
after
enzymatic
removal.
Similar
experi-
ments
have
been
carried
out
by
Crowell
and
collaborators
for
picornavirus
receptors
on
HeLa
s
to
o1
80
C
60
L-
,r
20
I
07
03
v
ozzz
01
4
7i-
0
1
2
3
4
5
6
7
8
9
10
Concern,ratior,
of
subt,lison
X
lOOq/ornl
x
0.7
U
t
E
03
FIG.
2.
Tlhe
effect
of
different
concentrations
of
subtilisin
ulpoIn
attachment
of
adenovirus,
cell
number,
antd
cloning
efficiency
of
HeLa
cells.
The
procedure
for
attachment
of
virus
is
described
in
Materials
and
Meth-
ods,
and
the
procedure
for
enzymatic
treatment
in
the
legenid
of
Fig.
1.
X
100
0
Z
60
40
z
u
20
tL
0.30
1
2
3
4
5
6
7
8
9
10
24
48
TIME
IN
HOURS
FIG.
3.
Regeneration
of
adenoviruis
receptors
on
HeLa
cells
treated
for
30
miii
at
37
C
with
subtilisin
at
0.4
mg/ml.
The
cells
were
washed
three
times
after
subtilisin
treatmenit
in
HBSS
and
incubated
in
spinner
medium.
Samples
of
cells
incubated
without
(0)
and
with
5
uAg
of
puromycin
per
ml
(0)
were
taken
at
various
times,
and
attachment
of
32P-labeled
adenovirus
was
assayed.
1.0
0
w
CE
0.5
0
0
z
CE
0.1
LL
1067
VOL.
2,2
1968
0
9
0
0
-------
W-
o
PHILIPSON,
LONBERG-HOLM,
AND
PETTERSSON
Assay
of
virus-receptor
complexes.
An
in
vitro
assay
for
virus-receptor
interaction
is
needed
for
the
study
of
the
subcellular
localization
of
receptor.
At
first,
it
was
hoped
that
the
virus-
receptor
complex
could
be
assayed
by
decreased
infectivity.
HeLa
cells
were
disrupted
by
various
methods
and
tested
for
inhibition
of
infectivity.
Table
1
shows
the
results
of
an
experiment
that
used
both
total
cell
sonic-treated
material
and
unwashed
microsomal
membranes
that
were
obtained
after
intracellular
cavitation.
Although
whole
sonically
treated
cells
or
crude
microsomes
are
rich
in
adenovirus
receptor,
they
were
unable
to
reduce
infectivity
by
as
much
as
one
log
unit,
even
when
receptor
material
was
in
great
excess.
There
was
only
about
a
70%
reduction
in
9
x
105
FFU
after
incubation
with
microsomes
from
1.7
X
107
cells.
Cell
fractions
were
also
tested
for
inhibition
of
the
complete
adenovirus
type
2
hemagglutination
of
rat
erythrocytes.
In
an
experiment
paralleling
Table
1,
microsomal
membranes
at
3.5
X
107
cell
equivalents
per
0.4
ml
were
shown
to
inhibit
just
four
hemagglutinin
units
of
adenovirus
2
(about
2
x
108
FFU).
Thus
neither
hemagglutination-
inhibition
or
infectivity
tests
were
of
practical
use
to
measure
virus-receptor
interaction
because
of
the
amounts
of
receptor
required
and
the
low
sensitivity,
respectively.
TABLE
1.
Inhibition
of
adenovirus
type
2
infectivity
by
cell
fractions
in
vitroa
Total
Per
cent
protein
No.
of
cell
of
Cell
fractions
(jsg/0.4
equivalents
control
ml)
activity
Total
cell
sonic
1,700
8.6
X
106
44
extractb
Crude
microsomesc
590
1.7
X
107
33
295
8.7
X
106
64
148
4.4
X
108
126
74
2.2
X
106
105
Cell
material
was
incubated
with
9
X
105
FFU
of
purified
adenovirus
type
2
in
0.4
ml
of
PBSA
for
30
min
at
37
C.
The
mixture
was
then
diluted
25-fold
and
assayed
for
remaining
FFU.
(The
presence
of
small
amounts
of
cell
material
seems
reproducibly
to
preserve
some
infectivity
lost
during
incubation
in
PBSA.)
b
Cells
sonically
treated
for
15
sec
in
PBSA
and
spun
at
low
speed
to
remove
any
unbroken
cells.
c
Crude
microsomes
prepared
by
cavitation.
The
preparation
contains
about
3000
receptors
per
cell
equivalent,
as
determined
by
the
microtube
assay.
The
same
preparation
required,
in
a
paral-
lel
experiment,
3.5
X
107
cell
equivalents
per
0.4
ml
to
give
a
complete
HI
of
4
units,
as
assayed
with
pure
adenovirus
type
2
and
rat
erythrocytes.
An
alternative
method
was
developed
based
upon
the
large
difference
in
buoyant
density
of
the
free
virus
and
the
virus-receptor
complex.
Figure
4
shows
a
CsCl
isopycnic
banding
of
adenovirus
after
incubation
with
the
particulate
fraction
of
sonically
treated
HeLa
cells.
Most
of
the
virus
has
been
complexed
and
has
decreased
its
density
from
1.34
to
1.15
to
1.25
g/cc.
The
receptor
complex
can
be
disrupted
by
0.2%
deoxycholate,
0.5%
Triton
X-100,
or
0.5%
Nonidet
P-40,
and
it
is
partly
destroyed
by
sonic
treatment.
The
complex
is
not
formed
if
0.4
mg
of
subtilisin
per
ml
is
present
during
the
incubation,
but
it
is
not
blocked
by
0.1
mg
of
deoxyribonuclease
or
0.4
mg
of
trypsin
per ml.
None
of
these
treatments
appreciably
changes
the
buoyant
density
of
the
free
virus.
Since
analysis
by
gradient
centrifugation
is
laborious,
a
simplified
microtube
assay
procedure,
previously
described,
was
developed.
Figure
5
shows
the
separation
of
the
free
32P-virus
and
the
virus-receptor
complex,
as
obtained
in
the
micro-
tube
assay.
The
virus
actually
bands
sharply;
however,
for
the
figure,
single-drop
fractions
were
collected
and
the
band
appears
broad.
Radioactive
virus
is
not
displaced
from
the
complex
by
the
addition
of
excess
nonradioactive
virus
at
the
end
of
the
incubation,
and
a
10-fold
excess,
as
previously
described,
is
usually
em-
cprn
6000
1.40
1.32
1.24
1.16
1.08
gm/cc
FIG.
4.
Buoyant
density
of
adenovirus-receptor
complex.
3.2
X
1010
32Padeno-2
virus
particles
were
incubated
for
30
min
at
37
C
with
the
particulates
derived
from
8.9
X
106
sonically
treated
HeLa
cells
(about
I
mg
of
protein)
in
0.43
ml
of
0.1
M
NaCl.
The
mixture
was
diluted
to
I
ml
with
0.1
M
NaCI,
layered
on
top
of
a
1.20
to
1.41
g/cc
linear
CsCI
gradient
of
3.6
ml,
and
spun
for
3.5
hr
at
165,000
X
g
in
the
Spinco
SW-S0
rotor.
Fractions
(3-drop)
were
collected.
Density
was
determined
with
a
micropycnom-
eter
on
every
fifth
sample.
The
small
peak
to
the
left
is
at
the
position
offree
virus;
the
large
peak
to
the
right
is
complexed
with
receptor.
1068
J.
VIROL.
ADENOVIRUS
RECEPTORS
ployed
to
reduce
the
blank.
This
gives
the
addi-
tional
advantage
that
the
location
of
the
virus
band
is
then
visible
in
each
gradient
after
cen-
trifugation.
It
is
not
practical,
or
even
possible,
to
make
an
adsorption
isotherm
on
each
receptor
prepara-
tion.
Instead,
we
usually
have
worked
with
a
fixed
level
of
input
virus
(about
1
x
1010
particles
per
0.2
ml).
There
is
the
additional
difficulty
that
the
binding
is
not
a
strictly
linear
function
of
the
amount
of
receptor.
We
usually
have
calculated
the
content
of
receptor
sites
at
receptor
levels
which
bind
less
than
30%
(net)
of
the
input
virus,
and
we
estimate
that
in
general
the
error
from
nonlinearity
is
less
than
20%.
Although
pH
7.4
is
used
routinely
for
complex
formation,
control
experiments
have
shown
that
binding
has
a
broad
pH
maximum
between
6
and
8.
Number
of
receptor
sites
per
cell.
The
attach-
ment
of
adenovirus
type
2
to
HeLa
and
KB
cells
cpm
was
studied
at
multiplicities
of
500
to
20,000.
In
Fig.
6A,
the
multiplicity
attached
to
HeLa
cells
is
plotted
linearly
against
input
multiplicity,
and
the
cells
are
saturated
with
about
104
particles
each,
assuming
that
each
receptor
can
only
be
used
once.
A
similar
capacity
was
found
for
KB
cells.
Experiments
were
made
also
with
fractions
of
disintegrated
cells.
Figure
6B
shows
one
experi-
ment
in
which
the
particulate
fraction
of
sonically
treated
HeLa
cells
is
able
to
bind
around
5000
particles
per
cell
equivalent.
The
binding
capacity
is
sometimes
greater,
especially
with
the
total
unfractionated
sonically-treated
material,
and
values
of
almost
104
virus
particles
per
cell
equiva-
lent
have
been
observed
for
both
HeLa
and
KB
cells
(Table
2).
Subcellular
localization
of
viral
receptors.
When
cells
were
disrupted
by
sonic
treatment,
much
of
the
receptor
activity
could
be
sedimented
at
80,000
X
g
(Table
2).
Furthermore,
the
specific
activity
of
the
particulate
fraction
(receptors
per
pg
of
protein)
was
about
twice
that
of
the
total
homogenate,
or
about
3
to
4
times
that
of
the
aoo0
600
400k
200
0
B00
[
600
-
F.)
°
10
I,
Ii
5
z~
\
m
B
4
00~
200
1.n
2
4
6
8
10
12
14
16
drop
FIG.
5.
Isopycnic
banding
of
32P-Ad
2
and
32P-Ad
2-receptor
complex
in
microtubes.
The
tops
of
the
gradients
are
to
the
right;
one
drop
fractions
are
counted
directly
int
0.1
molar
NaOH.
"T"
represen2ts
radio-
activity
adhered
to
the
tube.
A.
Virus
particles
(1.1
X
1010
were
incubated
and
diluted;
38%
of
the
total
was
separated.
B.
Ficoll-purified
membrane
(4.2
X
106
cell
equivalenits)
are
added
as
receptor.
A
B
5
10
15
20
0
b~~~~~~~
I
Z
5
10
15
Input
multip/icity
x103
32.4
62
FIG.
6.
Thle
relationship
between
input
multiplicity
and
attached
multiplicity
(A)
in
vivo
awd
(B)
in
vitro.
A.
32P-labeled
virus
of
different
specific
activity
was
attached
to
S
X
107
intact
HeLa
cells
in
I
ml,
and
the
virus
attached
was
determined
after
incubation
for
60
min
at
37
C.
B.
1.02
X
10'°
32P-adenovirus
type
2
particles
were
diluted
with
increasing
amounts
of
nonradioactive
virus
and
incubated
with
160
,ug
of
protein
particulates
obtained
from
3.64
X
106
sonically
treated
HeLa
cells
in
0.2
ml.
Net
virus
bound
per
cell
equivalent
was
plotted
against
total
virus
per
cell
equivalent.
VOL.
2,
1968
1069
A
PHILIPSON,
LONBERG-HOLM,
AND
PETTERSSON
TABLE
2.
Receptors
for
adelnoviruts
type
2
of
KB
alid
HeLa
cells
wit/i
or
witholit
slibtilisinl
treatment
prior
to
disintegration.
Fractions
Total
sonicate,
Particulatesa
Total
sonicatea
Particulates-
Particulatesb
Supernatant
fluidb
Particulatesc
Supernatant
fluidc
Receptors
per
cell
equivalent
Control
9180
5960
9730
7880
6490
1390
4680
2160
Subtilisin
1160
570
910
150
230
<720
Receptors
per
pg
of
protein
Control
Subtilisin
56
116
42
96
52
13
64
24
29
10
I1
2
4
<8
a,
b,
c
These
are
separate
experiments.
KB
or
HeLa
cells
were
washed
with
HBSS
and
suspended
in
HBSS
to
about
5
X
10'/ml.
To
one
portion
was
added
0.5
mg
of
subtilisin
per
ml
and
the
cells
were
shaken
for
30
min
at
37
C.
Cells
were
then
washed
twice
in
the
cold
with
Spinner
medium
that
contained
serum,
and
twice
with
HBSS.
They
were
then
sonically
treated
and
fractionated
as
described.
Receptor
activity
was
determined
by
the
microtube
assay.
supernatant
fraction.
Pretreatment
of
the
cells
with
subtilisin
prior
to
disintegration
greatly
reduced
the
receptor
sites
in
either
the
particulate
or
supernatant
fractions.
The
distribution
of
receptors
was
investigated
in
various
fractions
of
HeLa
cells
that
were
disrupted
by
"intracellular
cavitation"
with
nitrogen
gas
(7).
The
broken
cells
were
frac-
tionated
according
to
Wallach
and
Kamat
(31).
Two
typical
experiments
are
given
in
Table
3,
which
shows
that
the
crude
microsomal
fraction,
which
should
contain
plasma
membrane
frag-
ments,
has
more
total
and
specific
recwotor
activity
than
the
other
crude
fractions.
The
specific
activity
of
the
microsomes
is
increased
further
by
a
procedure
of
washing,
lysis,
and
then
centrifugation
on
a
cushion
of
Ficoll
in
the
pres-
ence
of
0.001
M
Mg++,
a
series
of
steps
designed
for
enrichment
of
plasma
membrane
vesicles
(31).
In
nine
experiments,
the
final
activity
of
the
Ficoll-purified
membranes
ranged
between
710
and
2220
receptors
per
pg
of
protein,
and
it
averaged
1270
per
pg.
There
appears
to
be
some
receptor
activity
in
every
cell
fraction
(Tables
2
and
3)
and
it
is
important
to
decide
whether
this
is
a
result
of
contamination
by
plasma
membranes
or
of
the
existence
of
internal
virus
receptors.
Pretreatment
with
subtilisin
strongly
reduces
the
activity
in
each
fraction,
and
experiments
were
made
to
test
whether
subtilisin
enters
the
cells.
It
was
found
that
LDH,
(lactate
dehydrogenase,
EC
1.1.1.27),
is
particularly
sensitive
to
subtilisin
inactivation.
KB
cells
in
PBSA
(5
X
107/ml)
were
sonically
treated
and
an
undiluted
post-
TABLE
3.
Receptors
for
adenioviras
type
2
in
HeLa
cells
brokelz
by
inltracel//i/ar
cavitationi
wit/
or
vithout
siubtilisiui
pretreatmenit
Receptor
per
Receptors
per
cell
equivalent
pg
of
protein
Extracts
Control
Sub-
Control
Sub-
tilisin
tilisin
a
Crude
nuclei
730
140
27
4
Crude
mitochondria
1220
<40
62
<7
Crude
microsomes
3380
170
122
8
Supernatant
fluid
1100
40
17
7
Crude
microsomes
3930
130
Washed
and
lysed
2930
410
microsomes
Ficoll
purified
mem-
2530
1360
branes
a,
b
These
are
separate
experiments.
HeLa
cells
were
treated
with
subtilisin,
as
in
Table
2,
and
disrupted
and
fractionated
as
described
in
Ma-
terials
and
Methods.
microsomal
supernatant
fluid
was
prepared
in
the
ultracentrifuge.
Small
volumes
of
subtilisin
solution
were
added
to
samples
and
these
were
incubated
for
30
min
at
37
C.
They
were
then
diluted
and
assayed
for
LDH.
It
was
found
that
20
,ug
of
subtilisin
per
ml,
a
level
of
only
4%c
of
that
used
with
the
intact
cells,
destroyed
50%,c
of
the
enzyme
activity.
The
supernatant
fraction,
and
in
one
case
the
particulates,
of
the
subtilisin
pretreated
cells
described
in
Table
2
were
assayed
for
LDH.
The
results
are
shown
in
Table
4.
There
is
no
signifi-
Cel!
type
KB
cells
HeLa
cells
1070
J.
VIROL.
ADENOVIRUS
RECEPTORS
cant
decrease
in
intracellular
LDH,
and
it
is
concluded
that
subtilisin
probably
does
not
enter
the
cell.
Therefore,
the
decrease
in
receptors
in
the
various
cell
fractions
of
Tables
2
and
3
is
probably
due
to
the
destruction
of
receptor
at
the
cell
surface.
Hence,
all,
or
almost
all,
of
the
cell-associated
receptor
must
be
at
the
plasma
membrane.
Substructure
of
the
virion
engaged
in
receptor
interaction.
The
effect
of
viral
structural
com-
ponents
on
receptor
interaction
was
studied
by
preincubation
of
cells
with
highiy
purified
hexon
and
fiber
proteins
of
adenovirus
type
2,
and
then
by
assaying
for
attachment
of
32P-labeled
adeno-
virus.
The
purified
fiber
and
hexon
antigens
have
been
described
(20,
21).
HeLa
and
KB
cells
at
a
concentration
of
5
X
107
cells/ml
were
incubated
with
different
concentrations
of
hexon
and
fiber
antigens
for
30
min
at
37
C.
Subsequently,
the
cells
were
washed
and
assayed
for
the
attachment
of
32P-labeled
adenovirus
during
30-
and
60-min
incubation
at
37
C.
Figure
7
shows
that
as
little
as
0.1
,ug
of
fiber
per
5
X
107
cells
can
significantly
reduce
the
amount
of
adenovirus
attached,
and
that
a
linear
relationship
is
evident
at
low
con-
centrations
of
fiber
between
fiber
concentration
and
impairment
of
attachment.
The
hexon
protein
does
not
affect
virus
attachment
(Fig.
7).
If
the
fiber
has
a
molecular
weight
of
70,000
(21)
and
there
are
10,000
receptor
sites
per
cell,
0.1
Mg
of
fiber
corresponds
to
2
molecules
per
receptor,
or
20,000
molecules
per
cell.
The
same
calculation
for
hexon,
assuming
a
molecular
weight
of
410,000
(20),
indicates
that
a
multiplicity
of
9
x
105
hexon
molecules
per
cell
will
not
interfere
with
virus
attachment.
To
study
further
the
specificity
involved
in
the
block
of
attachment
by
fiber,
adenovirus
infec-
tivity
and
adenovirus
type
5
were
assayed
in
the
same
system.
The
block
of
uptake
of
poliovirus
type
1
was
investigated
also.
Table
5
shows
that
cells
pretreated
with
5
Mg
of
fiber
are
blocked
for
attachment
of
adenovirus
type
2
infectivity
and
adenovirus
type
5
uptake
assayed
by
radioactivity.
Poliovirus
type
1
was
taken
up,
however,
to
the
same
extent
by
fiber-treated
and
control
cells.
A
specific
interference
of
adeno-2
fiber
with
adenovirus-receptor
interaction
was
also
ob-
TABLE
4.
Lack
of
effect
of
subtilisin
treatmenzt
of
initact
cells
oni
cellular
lactic
dehydrogenzase
LDH
(units/108
cells)
Cells
Fractions
Control
tilisin
KB
cells
Supernatant
fluida
64
77
HeLa
cells
Supernatant
fluida
71
57
Supernatant
fluidb
30
26
Particulatesc
2.6
2.2
Supernatant
fluidc
65
64
a,b,c
These
are
the
same
experiments
shown
in
Table
2.
LDH
was
assayed
as
described
in
Ma-
terials
and
Methods.
'ci
I
Q.
100
50
0
0.5
Fiber
uglml
-
1.00
10
20
30
Hexon
/Jgm/
FIG.
7.
The
effect
of
differen2t
conicenitrationis
of
purified
fiber
or
hexoni
proteins
onz
adentovirus
attach-
menit.
HeLa
cells
(5
X
107)
were
incubated
with
indi-
cated
amounzts
of
hexon
or
fiber
for
30
miii
at
37
C.
Attachmenzt
of
32P-labeled
adenzovirus,
at
a
multi-
plicity
of
about
50,
was
subsequenitly
assayed
at
30
min
(0)
and
60
min
(0)
at
37
C.
TABLE
5.
Attachment
of
differenit
adentoviruses
and
poliovirus
type
I
to
adenlovirus
type 2
fiber-HeLa
cell
complexes.
Per
cent
attached
virusb
V'irus
Multiplicitya
Assay
Normal
HeLa
cells
Fiber-treated
cells
Adenovirus
type
2
1
Infectivity
98.7
12
Adenovirus
type
5
5
Radioactivity
73
9
Poliovirus
type
1
1
Infectivity
99.5
99.5
aMultiplicity
refers
to
FFU
per
cell
for
adenovirus
and
PFU
per
cell
for
poliovirus.
b
HeLa
cells
(5
X
107)
were
incubated
for
30
min
at
37
C
with
and
without
fiber
at
a
concentration
of
5
,g
in
1
ml.
Attachment
of
adenovirus
was
determined
after
an
additional
60-min
period.
Poliovirus
attachment
was
determined
by
the
plaque
assay
(25).
VOL.
2,
1968
1071
PHILIPSON,
LONBERG-HOLM,
AND
PETTERSSON
served
in
the
in
vitro
system.
Figure
8
shows
the
dose
response
curve
with
crude
microsomes,
as
determined
in
the
microtube
assay.
In
this
assay,
an
inhibition
is
detected
at
about
0.1
,g
of
fiber
per
ml
(or
per
2
X
107
cell
equivalents),
about
the
same
concentration
seen
with
whole
cells.
Adenovirus
types
2
and
5
are
equally
susceptible.
Attachment
of
fiber
and
hexon.
Because
the
block
of
virus
attachment
by
fiber
was
concentra-
tion-dependent
even
with
high
multiplicities,
the
kinetics
of
fiber
and
hexon
attachment
to
cells
was
studied
in
more
detail.
Around
5,000
counts
;'min
of
purified
hexon
or
fiber
labeled
with
'4C-threonine
at
a
specific
activity
of
85
and
140
counts/min
per
,g,
respectively,
was
incubated
with
5
x
107
HeLa
or
KB
cells
(in
1
ml),
and
at
different
time
intervals,
samples
were
removed
and
the
cell
and
medium
phases
were
assayed
for
radioactivity.
Separate
samples
of
cells
were
assayed
for
attachment
of
32P-labeled
adenovirus
at
a
multiplicity
of
about
50
as
a
control.
Figure
9A
shows
that
no
measureable
attachment
of
fiber
or
hexon
could
be
demonstrated
in
60
min
at
37
C,
although
adenovirus
attached
to
80%,
in
the
same
period.
In
this
experiment,
however,
59
and
35
,ug
of
hexon
and
fiber,
respectively,
were
used,
which
oo
r
L.1
0z
qj
(.
qJ
0.
qj
-cl
(.
r3
II
aoF
i\
60
F
A
.
A
40F
\
A
O
\
20F
0
0.1
1.0
10.0
Fiber
p2g/m/
FIG.
8.
In
vitro
block
of
adenioviruls
types
2
anid
5
attachment
to
receptors
by
adeliovirius
type
2
fiber.
Crudcle
HeLa
microsomes
(2.08
X
107
cell
eqiuivalenits
per
n7l)
w1ere
inlicubated
with
vcaryintg
amoluts
o
fiber
for
15
mniii
at
37
C.
Their
abihity
to
attach
virlus
was
theni
determiined
by
the
miiicrotumbe
assay
uisilug
either
.2P-adenioviruus
type
2
or
5.
Opeln
anidfilled
symbols
are
separacte
experimentts;
A,
type
2
viruts,
0,
type
5
virts.
0.5
qj
L,
U1)
1:3
L.
u
U
U
0.21
A
B
10
30
60
Time
in
min.
FIG.
9.
A.
Lack
of
apparelit
attachmenit
of
14C-
labeled
fiber
(0)
anid
hexon
(0)
at
specific
activities
of
140
anid
85
couniits/miti
of
protein2,
per
jig,
respectively,
to
5
X
107
HeLa
cells
in
I
ml.
Attachmetit
of
32p_
labeled
adenioviruts
(A)
at
a
mu/ltiplicity
of
aroiun1d
50
as
a
conztrol.
B.
Attachmeiit
rate
of
13II-labeled
fiber
(0)
antd
hexonz
(0)
with
specific
activities
of
S
anid
I
colnlts/mili
of
proteini
per
pg,
respectively.
Attach-
meiit
of
32P-labeled
adelnoviruis
(A)
at
a
nmiultiplicity
of
abouit
50
as
a
conltrol.
correspond
to
multiplicities
of
1
and
6
x
106
proteins
per
cell.
Higher
specific
activities
clearly
were
required.
Therefore,
purified
structural
proteins
were
labeled
with
'31I
and
preparations
with
1.0
counts/
min
per
pg
for
hexon
and
5
counts/min
per
pg
for
fiber
were
obtained.
These
products
were
incubated
in
1
ml
with
5
x
107
cells
at
a
multi-
plicity
of
about
1
X
104
protein
molecules/cell.
The
kinetics
of
attachment
of
the
13'l
label
are
shown
in
Fig.
9B.
Seventy
per
cent
of
the
fiber
is
attached,
but
no
hexon.
To
test
that
the
1311
of
the
fiber
was
mainly
confined
to
functionally
intact
molecules,
an
adsorption
isotherm
was
made
with
fiber
of
varying
specific
activity.
To
0.1
,ug
of
'31I-labeled
fiber
was
added
varying
amounts
of
cold
fiber,
and
the
attachment
of
the
iodine
label
to
5
x
107
1072
J
V
IROL.
L\
I
\\
ADENOVIRUS
RECEPTORS
i
1.0
U)
0.5
.0
.0.
(I.1
0.1
0
10
30
60
Tlme
In
rn/n.
FIG.
10.
Attachmelnt
of
'311-labeled
fiber
to
HeLa
cells.
1311-labeled
fiber
(0.1
jig,
50,000
counlts/mi,i)
was
incubated
with
S
X
107
HeLa
cells
in
I
ml
at
37
C.
In
separate
tubes,
cold
fiber
was
added
in
varyinzg
conicenitrations.
1311-labeled
fiber
treated
with
anitifiber
anitiseruim
diluted
100-fold
for
30
miii
at
37
C
was
also
tested
for
attachmenit.
Samples
were
takeni
out
at
differeiit
time
initervals
an2d
radioactivity
was
determinied
in
the
cell
and
superncatanat
fractionis.
Symbols:
0,
'311-labeledfiber;
0,
'311-fiber
+
I
,ug
of
cold
fiber
per
ml;
A,
"3II-fiber
+
10
jig
of
cold
fiber
per
ml;
7,
1311-fiber
+
antifiber
antiserum.
cells
in
1
ml
was
determined
at
different
times.
An
experiment
where
70%
of
the
control
131I-
fiber
attaches
to
cells
in
30
min
is
shown
in
Fig.
10.
(This
corresponds
to
2
X
104
protein
molecules/
cell.)
At
concentrations
corresponding
to
2
x
105
and
2
x
106
protein
molecules
per
cell,
the
attach-
ment
is
60
and
<5%,
respectively.
Inhibition
of
1311-fiber
attachment
by
pretreatment
with
specific
antifiber
antibody
is
also
shown
in
Fig.
10.
The
antifiber
serum
was
produced
as
described
previ-
ously
(21)
and
it
was
incubated
at
1
:100%
with
0.1
,ug
of
'311-fiber
for
30
min
at
37
C
prior
to
mixing
with
5
X
107
cells
in
1
ml.
The
attachment
of
control
preparations
incubated
with
normal
serum
is
also
shown.
Antifiber
antibodies
at
low
concentrations
completely
prevent
attachment
of
the
'311-fiber.
These
results
indicate
that
the
31I-label
is
confined
mainly
to
functionally
intact
fiber,
and
that
the
saturation
density
for
fiber
per
cell
is
higher
than
for
intact
virus,
around
105
protein
molecules
per
cell
compared
to
104
virus
particles
per
cell.
DISCUSSION
The
evidence
indicates
that
adenovirus
type
2
receptors
are
located
at
the
plasma
membrane
of
HeLa
and
KB
cells.
Although
some
receptor
activity
can
be
recovered
in
other
subcellular
fractions,
the
control
experiments
that
utilize
subtilisin
susceptibility
of
intracellular
lactate
dehydrogenase
(Table
4)
seem
to
rule
out
that
treatment
with
proteolytic
enzyme
prior
to
disintegration
can
digest
internal
receptor
com-
ponents.
The
location
of
receptors
at
the
plasma
mem-
brane
has
been
established
for
picornavirus
(17,
32).
Treatment
of
intact
cells
with
trypsin
and
chymotrypsin,
which
destroy
the
receptors
for
polio-
and
coxsackievirus
(17,
25),
enhances
the
attachment
rate
for
adenovirus
(Fig.
1).
However,
it
does
not
change
the
adsorption
isotherm
(un-
published
data),
indicating
that
mild
proteolytic
digestion
does
not
expose
additional
sites
not
readily
available
for
the
virion.
Proteolytic
en-
zymes
with
broad
activity,
such
as
Pronase
and
subtilisin,
were
required
to
remove
the
receptor
activity
for
adenovirus,
from
which
it
can
be
inferred
that
picorna-
and
adenovirus
receptors
are
probably
not
the
same.
A
clear
difference
is
also
shown
by
the
fact
that
adenovirus
fiber
can
block adenovirus
uptake
but
does
not
affect
polio-
virus
attachment
(Table
5).
In
contrast
to
previ-
ous
studies
on
virus-receptor
interaction
(17,
25,
32),
neither
the
inhibition
of
virus
infectivity
nor
hemagglutination-inhibition
was
sensitive
enough
to
use
as
an
assay
system.
It
was
possible
to
develop
a
sensitive
and
specific
in
vitro
assay
for
adenovirus
receptors
based
upon
the
stability
of
the
virus-receptor
complex
in
CsCl
solutions
in
which
the
complex
can
be
banded
at
about
1.2
g/cc.
The
low
buoyant
density
and
sensitivity
of
the
complex
to
ionic
detergents,
together
with
the
method
of
partial
purification
of
the
receptor,
clearly
suggests
that
the
in
vitro
receptor
is
either
attached
to
or
composed
of
lipoprotein
membrane
fragments.
Membrane
fragments
prepared
by
the
method
of
Wallach
and
Kamat
(31),
which
should
be
rich
in
plasma
membrane
fragments,
bind
about
1000
or
more
virus
particles
per
pg
of
pro-
tein.
Because
there
are
about
4000
virus
particles
per
pg
of
virus
protein
with
pure
adenovirus
type
2
(6),
it
does
not
seem
likely
that
much
further
receptor
purification
can
be
made
without
breaking
the
membrane
fragments
into
soluble
components
(not
sedimented
at
104,000
x
g).
When
the
receptors
were
removed
from
intact
HeLa
or
KB
cells
with
subtilisin,
the
cells
were
still
intact
and
could
be
cloned
with
an
efficiency
comparable
to
untreated
cells.
The
receptor
activity
was
regenerated
in
a
period
of
4
to
8
hr,
a
process
which
might
require
protein
synthesis
because
cycloheximide
and
puromycin
inhibit
regeneration.
Because
of
the
extended
period
necessary
for
regeneration,
it
is
difficult,
however,
to
assess
the
requirement
for
protein
synthesis.
1073
VOL.
2,
1968
Z3
--L.
PHILIPSON,
LONBERG-HOLM,
AND
PETTERSSON
In
similar
experiments,
Crowell
and
his
asso-
ciates
(17,
32)
recorded
regeneration
times
of
1
to
4
hr
for
picornavirus
receptors.
The
differ-
ence
in
time
interval
required
may
reflect
only
the
broadness
in
specificity
of
the
proteolytic
enzymes,
because
trypsin-sensitive
polio
receptors
require
1
to
3
hours,
chymotrypsin-sensitive
coxsackie
B3
receptors
require
2
to
4
hours
(17,
32),
and
the
subtilisin-sensitive
adenovirus
re-
ceptors
require
4
to
8
hours.
The
broader
the
specificity
of
the
enzyme,
the
longer
the
time
required
for
resynthesis
of
the
membrane
unit
removed.
HeLa
and
KB
cells
in
suspension
cultures
are
saturated
with
adenoviruses
at
a
density
of
around
104
particles
per
intact
cell,
and
a
saturation
density
at
about
the
same
level
was
observed
in
the
in
vitro
assay
with
disintegrated
cells.
The
kinetics
of
virus-cell
attachment
at
high
multi-
plicities
indicate
that
receptor
sites
are
only
used
once.
The
fact
that
the
in vitro
and
in
vivo
satura-
tion
is
of
the
same
order
indicates
that
most
of
the
cell
receptors
are
on
the
plasma
membrane,
provided
that
no
reduction
in
receptor
activity
occurs
at
cell
disintegration.
The
saturation
capacity
of
the
cell
for
fiber
protein
appears
to
be
one
order
of
magnitude
higher
than
that
for
virus;
this
may
imply
either
that
the
virus
cannot
reach
some
of
the
receptors
or
that
re-
ceptors
are
grouped
in
patches
that
are
small
in
relation
to
the
virus.
A
third
possibility
is
that
the
fiber
penetrates
the
plasma
membrane.
Our
results
cannot
differentiate
among
these
alterna-
tives.
The
fiber
prevents
adenovirus
attachment
without
interfering
with
poliovirus
attachment,
as
assayed
by
infectivity.
Type
2
fiber
also
pre-
vents
attachment
of
adenovirus
type
5,
which
indicates
common
receptor
structures
for
sub-
group
III
of
adenovirus.
The
hexon
protein
did
not
affect
adenovirus
attachment.
Similar
results
have
been
obtained
by
Levine
and
Ginsberg
(16),
although
higher
concentrations
of
fiber
were
used
in
their
experiments.
At
a
ratio
of
104
to
105
fiber
molecules
per
cell
(0.02
to
0.2
,ug/107
cells)
we
observed
a
significant
reduc-
tion
in
adenovirus
attachment
both
in
the
in
vitro
and
in
the
in
vivo
assay.
At
the
same
fiber
to
cell
ratio,
it
was
possible
also
to
demonstrate
a
rapid
association
of
'3II-labeled
fiber
with
intact
cells.
This
was
prevented
by
cold
fiber
or
antibodies,
or
both.
Cell-associated
fiber
could
not
be
directly
demonstrated
at
higher
concentrations
of
fiber
because
the
cells
are
saturated
at
around
105
to
106
molecules
each.
Levine
and
Ginsberg
(16)
found
only
0.3
to
0.4%
of
the
fiber
associated
with
cells
at
a
concentration
of
240
Ag
per
1.5
x
105
cells.
Thus,
it
is
important
to
consider
the
number
of
receptor
sites
that
are
available
in
order
to
detect
the
cell
association
of
proteins
which
adhere
by
specific
mechanisms.
Similar
considerations
may
apply
to
interferon
uptake,
which
has
been
difficult
to
measure
in
cell
cultures
(27).
The
variance
of
our
results
with
those
of
Levine
and
Ginsberg,
who
used
adenovirus
type
5
(16),
may
reside
either
in
the
fact
that
we
em-
ployed
the
type
2
fiber,
which
has
been
suggested
to
contain
the
erythrocyte
receptor
modifying
factor
(12,
13)
not
present
in
type
5,
or
that
a
different
purification
method
has
been
used
for
the
fiber,
which
includes
trypsin
digestion.
Fur-
ther
studies
are
required
to
delineate
a
possible
difference
between
fibers
of
different
adenovirus
types.
ACKNOWLEDGMENTS
This
investigation
was
supported
by
grants
from
the
Damon
Runyan
Memorial
Fund,
the
Swedish
Medical
Research
Council,
and
the
Swedish
Cancer
Society.
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1075
