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-1n 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

Baltimore, David

Besmer, Peter

Chu, Louise

Huang, Alice S.

English

PubMed

Huang, A S

Besmer, P

Chu, L

Baltimore, D

The Jackson Laboratory: The Mouseion at the JAXlibrary

Growth of pseudotypes of vesicular stomatitis virus with n-tropic murine leukemia virus coats in cells resistant to n-tropic viruses.

Caltech Authors - Main

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-1b) 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
0-210-~~~~~~~~~~
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, VSVmade 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 VSV(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
MuLV 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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Growth of Pseudotypes of Vesicular Stomatitis Virus with N-Tropic Murine Leukemia Virus Coats in Cells Resistant to N-Tropic Viruses

A comparative study of the growth of VSV in five tissue culture systems.

A major genetic locus affecting resistance to infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses.

A major genetic locus affecting resistance to infection with murine leukemia viruses. II. Apparent identity to a major locus described for resistance to Friend murine leukemia virus.

A major genetic locus affecting resistance to infection with murine leukemia viruses. III. Assignment of the Fu-i locus to linkage group VIII of the mouse.

A study of the basic aspects of neutralization of two animal viruses, Western equine encephalitis virus and poliomyelitis virus.

Absence of interference during high-multiplicity infecJ. VIROL. tion by clonally purified vesicular stomatitis virus.

Defective T particles of vesicular stomatitis virus. I. Preparation, morphology and some biologic properties.

Host-range restrictions of murine leukemia viruses in mouse embryo cell cultures.

Infection of an established mouse bone marrow cell line (,JLS-V9) with Rauscher and Moloney murine leukemia viruses. Cancer Res.

Mixed culture cytopathogenicity: a new test for growth of murine leukemia viruses in tissue culture.

Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells.

Phenotypic mixing of enveloped proteins of the parainfluenza SV5 and vesicular stomatitis virus.

Plaque assay techniques for murine leukemia viruses.

Plaque production by the polyoma virus.

Proteins of vesicular stomatitis virus and of phenotypically mixed vesicular stomatitis virus-simian virus 5 virions.

Pseudotypes of vesicular stomatitis virus with the coat of' murine leukemia and of avian myeloblastosis viruses.

Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines.

Quantitative studies ofthe growth of mouse embryo cells in culture and their development into established lines.

Reciprocal patterns of genetic resistance to avian tumor viruses in two lines of chickens.

Ribonucleic acid synthesis of vesicular stomatitis virus. I. Species of' ribonucleic acid found in Chinese hamster ovary cells infected with plaque-forming and defective particles.

Ribonucleic acid synthesis ofvesicular stomatitis virus. I. Species of' ribonucleic acid found in Chinese hamster ovary cells infected with plaque-forming and defective particles.

Spontaneous&quot; release of type C viruses from clonal lines of' &quot;spontaneously&quot; transformed BALB/3T3 cells.

Susceptibility of two strains of Friend leukemia virus in mice.

The influence of the partner cell on the production of L virus and the expression of viral surface antigen in hvbrid cells.

VSV pseudotype particles with the coat of avian myeloblastosis virus.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=356675

Growth of Pseudotypes of Vesicular Stomatitis Virus with N-Tropic Murine Leukemia Virus Coats in Cells Resistant to N-Tropic Viruses

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Caltech Authors - Main