JOURNAL
OF
VIROLOGY,
Sept.
1973,
p.
659-662
Copyright
0
1973
American
Society
for
Microbiology
Vol.
12,
No.
3
Printed
in
U.S.A.
NOTES
Growth
of
Pseudotypes
of
Vesicular
Stomatitis
Virus
with
N-Tropic
Murine
Leukemia
Virus
Coats
in
Cells
Resistant
to
N-Tropic
Viruses
ALICE
S.
HUANG,
PETER
BESMER,
LOUISE
CHU,
AND
DAVID
BALTIMORE
Department
of
Microbiology
and
Molecular
Genetics,
Harvard
Medical
School,
Boston,
Massachusetts
02115
and
Department
of
Biology,
Massachusetts
Institute
of
Technology,
Cambridge,
Massachusetts
02139
Received
for
publication
19
June
1973
Formation
of
pseudotypes
between
murine
RNA
tumor
viruses
and
vesicular
stomatitis
virus
(VSV)
has
been
confirmed.
Pseudotypes
of
VSV
genomes
coated
by
the
surface
envelope
from
an
N-tropic
tumor
virus
grew
equally
well
in
cells
homozygous
for
either
the
Fv-_n
or
Fv-1b
alleles.
Therefore,
the
product
of
the
Fv-1
locus,
which
restricts
growth
of
murine
RNA
tumor
viruses,
must
act
on
an
intracellular
aspect
of
tumor
virus
replication,
a
step
after
attachment
and
penetration.
Formation
of
mixed
viral
pseudotypes
among
the
enveloped
RNA
viruses
was
first
observed
between
a
rhabdovirus,
vesicular
stomatitis
virus
(VSV),
and
a
paramyxovirus,
simian
virus
5
(1).
This
exchange
involves
only
the
viral
glycoproteins
and
appears
to
occur
during
the
process
of
budding
out
from
the
plasma
mem-
brane
(1
1).
Recently,
Zavada
reported
the
occu-
rence
of
similar
pseudotype
formation
between
VSV
and
avian
or
murine
RNA
tumor
viruses
(22,
23).
These
mixed
particles
contain
the
VSV
genome
completely
coated
by
the
surface
glyco-
protein
antigens
of
RNA
tumor
viruses.
The
pseudotypes
were
identified
by
three
criteria:
(i)
their
resistance
to
neutralization
by
anti-YSV
antiserum;
(ii)
their
inability
to
initiate
VSV
plaques
in
cells
with
surface
restrictions
for
the
RNA
tumor
virus
which
donated
the
glyco-
protein;
and
(iii)
their
susceptibility
to
an-
tiserum
against
the
specific
RNA
tumor
viruses.
Our
objective
was
to
confirm
Zavada's
obser-
vations
and
then
to
use
VSV
pseudotypes
as
a
probe
to
study
the
major
genetic
locus
of
resistance
in
mice
to
RNA
tumor
viruses,
Fv-1
(5,
9).
Fv-1
maps
on
chromosome
4
(linkage
group
VIII)
(14).
Cells
of
mice
homozygous
for
one
of
the
two
known
alleles
at
this
locus,
designated
Fv-
In
and
Fv-lb,
are
permissive
for
N-tropic
or
B-tropic
RNA
tumor
vi-
ruses,
respectively
(12,
13).
B-tropic
virus
grows
poorly
in
cells
from
Fv-ln
/Fv
-_l
mice,
such
as
NIH
Swiss,
and
N-tropic
virus
grows
poorly
in
cells
from
Fv-lb/Fv_lb
mice,
such
as
BALB/c.
Heterozygotes
(FV-ln/Fv-1
b)
are
re-
sistant
to
infection
by
both
N-
and
B-tropic
viruses.
Because
VSV
grows
in
a
variety
of
cells
(10),
pseudotypes
of
VSV
with
coats
from
N-
tropic
RNA
tumor
viruses
can
be
used
to
determine
whether
the
product
of
the
Fv-1
locus
acts
as
a
barrier
at
the
surface
of
the
cell
or
if
it
mediates
restriction
directed
at
an
aspect
of
the
virus
which
is
inside
the
envelope.
If
restriction
is
a
surface
phenomenon,
then
the
VSV
pseudo-
type
of
an
N-tropic
tumor
virus
should
be
restricted
as
if
it
were
an
N-tropic
virus.
If
restriction
is
directed
at
the
core
of
the
virus,
and
is
therefore
presumably
an
intracellular
event,
then
the
VSV
pseudotype
should
be
insensitive
to
the
restriction
system.
Initially,
to
form
VSV
pseudotypes
we
used
a
line
of
BALB/c
murine
cells,
called
JLS-V11,
which
is
a
producer
of
Moloney
murine
leu-
kemia
virus
(M-MuLV)
(21).
Medium
from
these
cells
contained
M-MuLV
at
approxi-
659
NOTES
mately
5
x
105
PFU/ml
as
determined
by
the
XC
assay
(8,
15).
JLS-V11
cells
were
superin-
fected
with
purified
cloned
VSV
(17)
at
an
input
multiplicity
of
0.1
for
18
h
at
37
C.
The
medium
was
harvested
and
assayed
for
VSV
plaques
on
Chinese
hamster
ovary
(CHO)
cells
or
JLS-V9
cells.
The
latter
cells
are
identical
to
JLS-V11
cells
except
that
they
have
never
been
infected
with
M-MuLV
and
are
not
producing
detecta-
ble
murine
leukemia
virus
(MuLV)
(21).
VSV
from
JLS-V11
cells
had
a
titer
of
1.12
x
108
PFU/ml
on
JLS-V9
cells.
Neutralization
of
this
preparation
by
rabbit
antiserum
made
against
VSV
revealed
that
1/1,000
of
the
plaques
on
JLS-V9
cells
were
initiated
by
VSV
particles
which
were
resistant
to
neutralization
by
an-
tiserum
(Fig.
la).
A
control
VSV
preparation
grown
in
JLS-V9
cells
in
the
absence
of
any
M-MuLV
was
neutralized
by
antiserum
to
a
residual
fraction
of
1/20,000
(Fig.
la).
The
a.
JLS-V9
b.
CHO
0-2
10-~~~~~~~~~~
Yvo
1io3
*
4
-4_
~
~
~
~
~
~
~
~
~
~
-
0.33
1.0
10
0.33
1.0
1
Antiserum
Dilution
(x
10-2
)
FIG.
1.
Detection
of
pseudotypes
of
VSV(M-
MuLV).
Rabbit
anti-VSV
antiserum
was
prepared
against
purified
cloned
VSV
(17),
as
previously
de-
scribed
(16),
except
the
virus
was
not
inactivated.
JLS-V9
and
JLS-Vll
cells
were
grown
as
monolayers
in
the
same
medium
as
CHO
cells
in
suspension
(16).
VSV
grown
in
either
JLS-V1l
or
JLS-V9
cells
was
tested
against
antiserum
essentially
by
the
plaque-
reduction
technique
of
multiplicity
analysis
(3).
In
summary,
VSV
was
adjusted
to
a
titer
of
2
x
107
PFU/ml
and
mixed
with
an
equal
volume
of
medium
containing
rabbit
anti-VSV
antiserum
at
the
indi-
cated
dilutions.
Control
mixtures
were
made
contain-
ing
normal
rabbit
serum.
The
mixtures
were
in-
cubated
for
1
h
at
37
C
and
then
diluted
and
plated
on
either
JLS-V9
cell
monolayers
or
CHO
cell
monolay-
ers.
The
plaque
assay
on
both
cell
types
was
per-
formed
as
described
previously
for
CHO
cells
(16).
The
surviving
fraction
of
plaques,
V/V.,
was
plotted
versus
antiserum
concentration.
Symbols:
0,
VSV
made
in
JLS-Vl
I
cells;
0,
VSV
made
in
JLS-V9
cells.
difference
between
the
two
preparations
was
indicative
of
the
presence
of
VSV
pseudotypes
(VSV
[M-MuLV
]).
Assay
of
the
same
VSV
prep-
arations
on
CHO
cells
showed
that
all
of
the
plaques
on
these
cells
were
initiated
by
VSV
which
was
susceptible
to
anti-VSV
antiserum
(Fig.
lb).
These
results
indicate
that
VSV(M-
MuLV)
was
capable
of
forming
plaques
on
JLS-V9
cells
and
not
on
CHO
cells,
presumably
due
to
their
inability
to
enter
into
the
CHO
cells.
VSV(M-MuLV)
cannot
be
used
for
studying
the
Fv-1
locus
because
Moloney
virus
is
NB-
tropic,
that
is,
it
is
not
restricted
by
the
Fv-1
locus
(5).
Therefore,
to
obtain
pseudotypes
of
VSV
in
coats
of
N-tropic
MuLV
(VSV[MuLV-
N]),
NIH/3T3
cells
chronically
infected
with
N-tropic
MuLV
were
infected
with
VSV
at
a
multiplicity
of
0.5
to
1.
The
co-infected
NIH/3T3
cells
were
incubated
at
37
C
for
15
h.
(N-tropic
MuLV
was
kindly
supplied
by
Janet
Hartley
and
Wallace
P.
Rowe.
When
tested
in
the
XC
assay
in
our
laboratory,
this
virus
showed
a
100-fold
higher
titer
in
NIH/3T3
cells
than
in
BALB/3T3
cells.)
VSV
obtained
from
the
co-infected
cells
was
assayed
on
CHO,
NIH/3T3
(derived
from
NIH
Swiss
mice;
reference
7),
3T6
(derived
from
random-bred
Swiss
mice;
reference
19),
and
JLS-V9
cells.
3T6
cells
contain
the
Fv_lb
locus
(18)
as
do
JLS-V9
cells
which
derive
from
BALB/c
mice
(21).
Tests
in
our
laboratory
confirmed
the
selective
sensitivity
of
those
two
cell
lines
to
B-tropic
MuLV.
The
VSV
prepara-
tion
grown
on
co-infected
NIH/3T3
cells
had
a
titer
of
200
x
106
PFU/ml
of
CHO
cells,
6.75
x
10'
PFU/ml
on
NIH/3T3
cells,
1.78
x
106
PFU/ml
on
3T6
cells,
and
7.10
x
106
PFU/
ml
on
JLS-V9
cells.
Figure
2
shows
that
when
the
VSV
stock
from
co-infected
NIH/3T3
cells
was
neutralized
by
anti-VSV
antiserum
and
then
assayed
on
NIH/
3T3,
3T6,
and
JLS-V9
cells,
pseudotypes
of
VSV(VSV
[MuLV-N
])
were
detected.No
pseudo-
types
were
detected
on
CHO
cells,
which
gave
results
similar
to
that
shown
in
Fig.
lb.
VSV
genomes
coated
by
the
surface
proteins
of
an
N-tropic
MuLV
were
therefore
apparently
equally
capable
of
penetrating
and
replicating
in
murine
cells
with
either
the
Fv-1"
or
Fv_lb
locus.
Lack
of
infection
of
CHO
cells
indicates
that
the
VSV(MuLV-N)
contained
absorption
sites
specific
for
murine
cells.
The
slight
in-
crease
in
titer
of
VSV(MuLV-N)
on
NIH/3T3
and
JLS-V9
cells
compared
to
the
titer
on
3T6
cells
was
probably
not
significant
and
cor-
related
with
the
differences
in
plating
efficiency
of
VSV
on
the
three
types
of
cells.
This
report
confirms
Zavada's
findings
of
the
660
J
.
V
IROL.
VOL.
12,
1973
V/
/V
0.5
1.0
0.5
10
2.0
/'l
0.5
1.0
2.0
Antiserum
Dilution
(x
10-2)
FIG.
2.
Growth
of
VS
V(MuL
V-N)
on
NIH/3T3,
3T6
cells,
and
JLS-V9
cells.
NIH/3T3
and
3T6
cells
were
grown
as
monolayers
in
the
presence
of
Dulbecco
medium
(2)
plus
10%
calf
serum.
JLS-V9
cells
were
grown
as
described
in
Fig.
1.
VSV
from
NIH/3T3
cells
or
from
NIH/3T3
cells
co-infected
with
N-tropic
MuL
V
were
neutralized
exactly
as
described
for
Fig.
I
except
that
the
initial
virus
titer
was
approximately
2
x
106
to
4
x
106
PFU/ml.
Plaque
assays
were
done
on
NIH/3T3,
3T6,
and
JLS-V9
monolavers
by
the
proce-
dure
for
CHO
cells,
except
that
the
appropriate
culture
medium
was
used
for
each
type
of
cell.
Symbols:
0,
VSV
made
in
NIH/3T3
cells
co-infected
with
N-tropic
MuLV;
0,
VSV
made
in
uninfected
NIH/3T3
cells.
formation
of
pseudotypes
of
VSV
genomes
in
envelopes
of
RNA
tumor
viruses
(22,
23).
The
studies
also
indicate
that
VSV
pseudotypes
made
in
cells
co-infected
with
N-tropic
MuLV
will
grow
in
cells
homozygous
for
either
the
Fv-ln
or
Fv-1b
alleles.
Thus
FL-]
restriction
against
murine
tumor
viruses
apparently
is
not
due
to
lack
of
receptor
sites
on
the
cell
surface
but
rather
is
due
to
intracellular
restriction.
Such
an
interpretation
is
consistent
with
the
finding
that
the
Fv-1
alleles
show
genetic
domi-
nance
for
resistance
(12).
Whether
the
restric-
tion
acts
directly
on
the
nucleic
acid,
in
analogy
with
bacterial
restriction
systems,
or
on
another
target
remains
to
be
determined.
Feny6
et
al.
(4)
and
Yoshikura
(21a)
also
recently
suggested
that
the
Fv-1
product
can
have
an
intracellular
site
of
action.
The
inability
of
VSV(MuLV)
to
infect
Chi-
nese
hamster
cells,
and
Zavada's
earlier
demon-
stration
of
their
inability
to
infect
human
cells
and
their
inefficient
infection
of
Syrian
hamster
cells
(23),
demonstrates
that
across
species
barriers
a
true
surface
restriction
does
exist.
Also,
with
avian
tumor
viruses
surface
resist-
ance
is
evident
(20).
The
VSV
pseudotypes
thus
allow
one
to
readily
distinguish
two
types
of
cellular
resistance
to
RNA
tumor
viruses.
Further
experiments
using
a
variety
of
cell
genotypes
and
using
antiserum
directed
against
specific
MuLV
strains
are
in
progress.
Of
partic-
ular
interest
is
the
possibility
of
forming
not
only
pseudotypes
containing
VSV
genomes
but
also
of
forming
particles
containing
MuLV
ge-
nomes
coated
by
VSV
antigens.
We
are
pres-
ently
testing
for
the
existence
of
these
pseudo-
types
in
our
viral
preparations.
We
thank
Mort
Litt
and
S.
M.
Perlman
for
help
with
antiserum
and
viral
preparations
and
Tazwell
Wilson
for
excellent
technical
assistance.
We
are
especially
grateful
to
Janet
Hartley
for
supplying
N-tropic
MuLV.
This
work
was
supported
by
a
National
Science
Founda-
tion
research
grant
GB
34266,
American
Cancer
Society
research
grants
VC-63
and
VC-4D,
and
a
contract
from
the
Viral
Cancer
Program.
A.S.H.
is
a
Research
Career
Develop-
ment
Awardee
of
the
Public
Health
Service
(Al
70413).
P.
B.
was
supported
by
the
Schweizerische
Krebsliga.
D.
B.
is
an
American
Cancer
Society
Professor
of
Microbiology.
This
work
grew
out
of a
summer
sojourn
at
the
Salk
Institute
for
Biological
Studies.
ADDENDUM
IN
PROOF
Similar
experiments
and
results
using
VSV-N-
tropic
and
B-tropic
Friend
leukemia
virus
pseudo-
types
were
performed
by
T.
G.
Krontiris,
R.
Soeir6,
and
B.
N.
Fields
(personal
communication).
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NOTES
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