Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus

VIROLOGY

82, 221-231 (1977)

Pseudotypes of Vesicular Stomatitis Virus with Envelope Antigens Provided by Murine Mammary Tumor Virus J. ZAVADA,’ Imperial

C. DICKSON,2

Cancer Research Fund Laboratories,

AND

R. WEISS

P.O. Box 123, Lincoln’s England

Inn Fields,

London,

WC2.4 3PX.

Accepted May 26,1977 Infection of two mouse mammary carcinoma cell lines with vesicular stomatitis virus (VSV) resulted in the formation of at least two types of particles containing the VSV genome but expressing different envelope characteristics (VSV pseudotypes). One of these VSV pseudotypes was infectious for a cell line derived from normal mouse mammary epithelial cells and mouse embryo cells but noninfectious for 3T3 cells, mink lung cells, and Vero cells. If mouse mammary tumor cells were treated with dexamethasone some days prior to infection with VSV, the titer of this pseudotype was significantly increased. In contrast, the second pseudotype was infectious for mink cells, but not for the other cell lines tested, and the titer of this second pseudotype was unaffected by the presence of dexamethasone. The first pseudotype was found to be almost completely neutralized by anti-murine mammary tumor virus (MuMTV) serum whereas the second pseudotype was only partially neutralized at a higher antiserum concentration. Neither pseudotype showed the neutralization, host range, or interference properties of either ecotropic or xenotropic murine C-type viruses. These results suggest that the first pseudotype is VSV(MuMTV). The other pseudotype is less well defined but conceivably may represent a xenotropic MuMTV. In the course of these studies, a filterable agent was observed in GR mammary carcinoma cultures that reactivated the infectivity of VSV neutralized by antiserum. This agent was transmissible to mink cells. INTRODUCTION

a low efficiency (Vaidya et al., 1976; Lasfargues et al ., 1976). When cells producing oncornaviruses are infected with vesicular stomatitis virus (VSV), the resulting mixed infection yields a proportion of particles which contain the VSV genome and the envelope glycoproteins of the oncornaviruses (VSV pseudotypes). Consequently, the VSV pseudotype possesses several properties specified by the oncornavirus glycoprotein, such as neutralization, host range, and characteristics (Zgvada, interference 1972a, b; Huang et al., 1973; Krontiris et al., 1973; Love and Weiss, 1974; Boettiger et al., 1975, Ztivada, 1976). In this paper we describe the infection of two cell lines derived from mouse mammary carcinomas with VSV which result in the formation of at least two different pseudotypes. One pseudotype appears to be VSV(MuMTV), while the other may be

Carcinogenesis induced by mouse mammary tumor viruses (MuMTV), provides a model system for studying the interactions of genetic, hormonal, and viral functions in the progress of malignant disease (Bentvelzen, 1972; Nandi and McGrath, 1973). In regard to the viral function examinations of the species and tissue tropism and of neutralization by homologous or heterologous antisera have been retarded by the lack of an in vitro assay for MuMTV. Only recently has in vitro infection been successfully performed, albeit at ’ Permanent address: Institute of Virology, Slovak Academy of Sciences, Bratislava, Czechoslovakia. Recipient of a Fellowship under the British Royal Society-Czechoslovak Academy of Sciences Exchange Agreement. 2 Author to whom reprint requests should be addressed. 221 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0042-6822

222

ZAVADA,

DICKSON,

related to MuMTV, on the basis of neutralization studies, but demonstrates a different host range. MATERIALS

AND

METHODS

Viruses and Cells VSV. A mutant tlB17 of VSV (Indiana

strain) which has a thermolabile glycoprotein was used for all experiments (Zavada, 197213). Stock virus was recloned and grown up in Vero cells. MuMTV. Two cell lines, GR and MmEiMT/cl (Mm5) derived from spontaneous mouse mammary tumors of the GR and C3H mouse strains, respectively, were used as a source of MuMTV. The GR cell line was described by Ringold et al., (1975) and was kindly supplied by Dr. K. Yamamoto, San Francisco Medical Center, and the MmSMT/cl is a clone of the line derived by Owens and Hackett (1972) and was kindly supplied by Dr. L. 0. Arthur, Frederick Cancer Research Center, Maryland. Production of MuMTV by both cell lines was monitored by reverse transcriptase assays using the exogenous template/primer poly(rC:dG),,-,s (Dickson et al., 1974). The enzyme activity showed a preference for magnesium as the divalent cation characteristic of MuMTV and other B-type viruses (Dion et al., 1974). Neither cell line produced detectable amounts of Ctype viruses as assayed by Mn2+-dependent reverse transcriptase, nor were C-type viral proteins detected in highly concentrated virus preparations by radiolabeling and concomitant analysis on polyacrylamide gels. Murine (ecotropic) leukemia virus. The Moloney strain of murine leukemia virus (MuLV-M) was grown as a chronic infection of NIH/3T3 cells. Murine xenotropic C-type virus (MuLVX) . Two different isolates of MuLV-X were

used: One was isolated from BALB/c mice (N. Teich, unpublished) and grown in rabbit SIRC cells (Leerhoy, 1965); the other was obtained by cocultivation of bone marrow cells from NZB mice with a line of mink cells (CCL 64) (Weiss and Wong, 1977). Normal mammary gland cell line (NMG). This cell line was isolated fi-om

AND

WEISS

the NAMRU mouse strain by Owens et al. (1974) and is maintained in culture as a continuous cell line. NIHi3T3 cells. These are a fibroblastoid cell line isolated from the NIH/Swiss mouse strain (Jainchill et al., 1969). BALBi3T3 cells. These are a similar line derived from BALB/c mouse embryos (Aaronson and Todaro, 1968). Mink lung cells (CCL 64). These were derived from mink embryo lung cell cultures (Henderson et al., 1974). The Vero cell line. This was established from an African green monkey kidney culture (Earley et al., 1967). NRK cells. These are a line derived from normal rat kidney (Due-Nguyen et al., 1966). Bat Tb-1 -Lu cells. These are a line (CCL 88) derived from an adult bat lung. PYS cells. These are a cell line of endodermal character derived from a teratocarcinema (Lehman et al., 1974). Primary cultures of mouse embryos. The BALB/c, C57BL, CBA, and TO strains were supplied by the cell production laboratory, Imperial Cancer Research Fund. Media

The normal and transformed mammary gland cell lines were maintained in Dulbecco’s modified Eagle’s medium containing antibiotics and supplemented with heat-inactivated (56” for 30 min) 10% fetal calf serum. All other cell lines were maintained in the same medium supplemented with heat-inactivated 10% calf serum. Antiserum

and y-Globulin

Preparations

Antiserum against VSV was prepared in sheep as previously described (Zavada, 1972b). Antisera against MuMTV and MuLV-M were prepared in rabbits using gradientpurified virus, which was disrupted with 0.5% Triton X-100 and 0.5% sodium deoxycholate. The MuMTV was prepared from the tissue culture fluids of primary spontaneous mammary tumors of the BR6 mouse strain grown in the presence of dexamethasone to stimulate virus production (Dickson et al., 1974). MuLV-M was grown in NIHl3T3 cells as described above. The rab- _

VSV(MuMTV)

bits were inoculated intramuscularly at four sites, initially with 0.5 mg of viral protein in Freund’s complete adjuvant and subsequently at intervals of 2 weeks with the same disrupted virus in Freund’s incomplete adjuvant. Rabbits were bled 1 week following each booster injection and the serum was stored at -70”. The serum obtained before immunization with MuMTV was used as a supply of normal rabbit serum. y-Globulin (IgG) fractions from the above normal and immune sera were prepared by ammonium sulfate precipitation, once with 50% saturated and twice with 40% saturated solutions. The precipitated globulins were dissolved in phosphatebuffered saline, pH 7.4 (PBS), to a volume equivalent to the original serum, dialyzed against the same buffered saline, and filtered through 0.22-pm Millipore filters. Preparation

of VSV Pseudotypes

Initially, VSV pseudotype stocks were prepared by growing VSV tlB17 in the oncornavirus-producing cell lines at 32” and neutralizing the parent-like VSV with antiserum (Zavada, 1972a) or by heating the mutant at 45” for 1 hr as previously described (Zavada, 1972b). These preliminary experiments revealed that the VSV pseudotypes obtained in the mammary tumor cell lines were easily inactivated nonspecifically. Consequently several precautions were taken to protect the integrity of the VSV pseudotypes formed. Unidentified entities in serum were found to inhibit pseudotype stability, and therefore the medium was always supplemented with heat-inactivated fetal calf serum. Maximum titers of the pseudotypes were obtained when cultures were infected at an m.o.i. of 1-5 PFU per cell and culture media were harvested after 12-16 hr incubation at 32”. The harvested fluids were carefully adjusted with 1% acetic acid to pH 7.2-7.4. Following pH adjustment the infectious culture fluids were clarified by centrifugation at 4000 r-pm for 10 min and stored at -70”. Infectivity of the VSV pseudotypes was lost in a few days at 4”, but stored well at -70”. Neutralization of the parent VSV was car-

223

PSEUDOTYPES

ried out overnight at 4”. In experiments where specific neutralizations were performed, normal and various immune IgGs (anti-MuLV or anti-MuMTV) at several dilutions were also incorporated with the anti-VSV IgG. Dilution of the pseudotype stocks was performed in complete PBS containing heat-inactivated 1% fetal calf serum. The titer of VSV pseudotypes was determined in a plaque assay using several cell lines grown to confluence in 30mm-diameter petri dishes (Nunc). The virus was allowed to adsorb at room temperature for 2 hr in the presence of Polybrene (20 pglml). After adsorption the residual inoculum was removed and the cells were rinsed with PBS and overlaid with agar medium. In some experiments the harvest fluids at a 1:lO dilution in complete PBS were heated at 45” for 1 hr by immersion of a vial in a water bath. This treatment completely inactivates the thermolabile envelope antigen of VSV tlB17 so that only pseudotype particles survive as plaqueforming units (Zavada, 197213).The presence of pseudotypes was then assayed as described above, except that the cells were not rinsed with PBS following incubation at room temperature for 2 hr. RESULTS

Host Range of VSV Pseudotypes Obtained from Mouse Mammary Tumor Cells and Effect of Dexamethasone

When VSV tlB17 was grown through control or oncornavirus-producing cells, a titer of about log PFU/ml of virus was obtained, except for the Mm5 cell line which yielded about 10’ PFU/ml (Fig. 1). The plaque titers were similar when assayed on Vero, mink, or NMG cell lines but showed a lo-fold lower efficiency on NIH/3T3 cells. The VSV stocks were also treated with neutralizing antiserum or IgG specific to VSV and assayed for their ability to form plaques on the test cell lines. After antiserum treatment, the virus stocks derived from uninfected cell lines showed no residual infectivity, whereas those grown through oncornavirus-producing cells all showed a residual

224

ZAVADA, GR

DICKSON, GR+DEX

AND

Mm5

WEISS Mm5 + DEX

SIRC MuLV-X

d

NIH/3T3 MuLV-M

1 :d

FIG. 1. Host range specificities of VSV pseudotypes obtained from cells producing MuMTV, MuLV-M, and MuLV-X. VSV was propagated in control and oncornavirus-infected cells. The mammary tumor lines were maintained in media with or without dexamethasone(DEX1 as indicated. Infectivity of the harvested VSV was determined directly (open columns) and after treatment with anti-V?%’ IgG (shaded columns) at a 150 dilution overnight at 4”. The plaque assays were performed on (a) mink cells, (b) Vero cells, (cl NMG cells, and (d) NIH/3T3 cells.

as would be expected if pseudotypes were present. Figure 1 also shows that the pseudotypes obtained from cells producing ecotropic and xenotropic MuLV possessed the expected host range. The ecotropic VSV(MuLV-M) formed plaques on both mouse cell types used in the plaque assay, namely, NIH/3T3 cells and NMG cells, but did not form plaques on mink or Vero cells; the xenotropic VSV(muLV-X) formed plaques on the mink and Vero cells but not on mouse cells. The pseudotypes obtained from both of the mouse mammary tumor cell lines showed a different host range from either of the VSV(MuLV) pseudotype stocks, in that they plated on NMG cells and mink cells but not on NIH/3T3 cells or Vero cells. Previous studies have shown that, following dexamethasone stimulation, these mammary tumor cell lines produced greatly enhanced amounts of MuMTV (McGrath, 1971; Parks et al., 1974; Dickson et al., 1974; Fine et al., 1974; Ringold et al., 1975). Consistent with these reports, cultures grown in the presence of dexa-

infectivity,

methasone yielded a lo- to 20-fold higher titer of VSV pseudotypes when assayed on NMG cells (Fig. 1). However, dexamethasone had no effect on the pseudotype titer when the same stock was assayed on mink cells. This finding indicates that the VSV pseudotype stock obtained from either GR or Mm5 cells may be comprised of two different pseudotypes; one plates on NMG cells and is stimulated by the presence of dexamethasone in the cultures during VSV infection Yecotropic”), and the other plates on mink cells and is unaffected by dexamethasone (“xenotropic”). The use of these terms “ecotropic” and “xenotropic” refers to the ability of the derived envelopes of the VSV pseudotypes to adsorb to and penetrate the cells mentioned and does not imply that the virus providing the envelope can itself infect and replicate in these same cells. Further evidence to support the idea of two different pseudotypes is that the xenotropic component appears to be more labile than the ecotropic component when stored at 4” and that the former is more sensitive to inhibitors in nonimmune rabbit serum (Table 1).

VSV(MuMTV)

225

PSEUDOTYPES

Several other cell lines and secondary ble to infection. The pseudotypes showed a mouse embryo cell cultures were tested for lower plating efficiency on embryo cell cultheir susceptibility to infection by the VSV tures of the C57BL and TO strains than pseudotypes obtained from the mammary on cells of the BALB/c and CBA strains. tumor cell lines (Table 2). Apart from the The titers of the pseudotype were enmink cell line, all the nonmurine cell hanced by dexamethasone in a similar lines, bat, rat, monkey, and human, and manner to that found for pseudotypes plating on NMG cells, thus indicating that the mouse fibroblastic cell lines, BALB/ 3T3 and NIH/3T3, were resistant to infec- only the ecotropic MuMTV pseudotype tion. In contrast a cell line derived from a was infectious in all these murine culmouse teratocarcinoma of endodermal tures . character (PYS cells) and secondary Specificity of VSV Pseudomouse embryo cell cultures were suscepti- Neutralization twes TABLE 1 EFFECT OF UNHEATED NONIMMUNE RABBIT SERUM AND STORAGE AT 4” ON THE INFECTIVITY OF VSV PSEUD~TYPES OBTAINED FROM MOUSE MAMMARY TUMOR CELLS (GR) AND PLAQUE ASSAYED IN MOUSE (NMG) AND MINK CELLS

Infectivity (log PFU/ml) of pseudotypes= assayed on

Treatment

Untreated control Normal rabbit serum (1:16) Harvest stored at 4” for 0 Days 3 Days

NMG

Mink

3.82 3.28

3.72 2.18

3.36 3.09

3.88 2.37

(2Pseudotypes determined as infectivity to anti-VSV IgG 1:30.

resistant

To characterize the VSV pseudotypes derived from cultures infected with MuLV-M and MuLV-X further, and to compare them with the VSV pseudotypes derived from the mammary tumor cell lines, the pseudotype stocks were treated with control or immune IgG prepared against MuMTV or MuLV-M as described in Materials and Methods (Table 3). Antibodies to MuLV-M are very effective at neutralizing the VSV(MuLV-Ml, but do not affect either of the pseudotypes derived from the mammary tumor cell lines. By contrast, the antibodies to MuMTV effectively neutralize the ecotropic component of the mammary tumor cell line and, to a lesser extent, the xenotropic component.

TABLE 2 Host RANGE SPECIFICITY OF THE PUTATIVE VSV(MuMTV) PSEUD~TYPES OBTAINED FROM MAMMARY TUMOR CELL LINES

Assay cells (Species: Line or Strain) Monkey:Vero Mink:CCL 64 Rat:NRK Bat:Tb .l. lu Human:HeLa Mouse:NMG PYS NIH/3T3 BALB/3T3 C57BLb CBAb TO* BALB/c*

Host cell for VSV propagation: Progeny assayed with (+) and without (-1 anti-VSV IgG GR plus Dex

GR

Mm5 plus Dex

Mm5

-

+

-

+

-

+

-

+

8.25” 8.69 7.15 7.07 7.00 8.38 8.42 6.85 7.12 6.30 6.80 7.03 7.25

Cl.00 2.95 Cl.00 Cl.00 Cl.00 2.76 2.54 Cl.00
8.30 8.52 7.23 6.80 7.10 8.45 8.48 6.85 6.98 6.26 6.95 6.80 7.30

‘cl.00 3.12 Cl.00 Cl.00 Cl.00 3.52 3.69

6.80 6.95 5.38 5.42 5.35 6.79 6.92 6.00 6.10 5.29 5.85 5.74 5.99

Cl.00 2.21 Cl.00 Cl.00 Cl.00 2.58 2.72 Cl.00 Cl.00 1.48 2.52 1.75 2.82

7.10 7.10 5.52 5.48 5.61 6.87 7.00 6.12 5.99 5.25 5.90 5.88 5.85

Cl.00 2.03 Cl.00 Cl.00 Cl.00 3.48 3.65 Cl.00 Cl.00 2.30 3.98 2.55 3.85

(1Infectivity expressed as log PFU per milliliter. * Secondary cultures of mouse embryo cells.

Cl.00

Cl.00 2.73 3.73 2.78 3.85

226

ZAVADA,

DICKSON, TABLE

SPECIFIC

%?

1

NEUTRALIZATION

OF VSV PSEUDOTYPES

Source of VSV pseudotype”

GR plus Dex’ Mm5 plus Dex MuLV-M MuLV-X(BALB/c)

2

GR plus Dex

Mm5 plus Dex

GR plus Dex (45”)

Mm5 plus Dex (45”)

MuLV-M MuLV-X

*giYa;”

AND

WEISS

3

WITH IgG ISOLATED SERA

FROM NONIMMUNE

OR IMMUNE

RABBIT

Preparation of IgG Untreated

NRSb 1:20

NMG Mink NMG Mink NMG Mink

3.73d 3.67 3.85 2.40 5.22 3.60

3.81 3.66 3.71 2.45 5.08 3.56

NMG BALBlc Mink NMG BALB/c Mink NMG BALB/c Mink NMG BALB/c Mink NMG Mink

3.39 3.88 2.85 3.60 4.26 1.78 3.10 3.65 2.30 3.55 4.20
Anti-MuMTV 1:20

I:50

1:20

2.68 3.67

1.00 2.45

NT’ NT NT NT 2.48 2.92

3.70 3.73 3.97 2.48

2.15

1.20 5.12 3.63

NT NT NT NT NT NT NT NT NT NT NT

1.30 1.58 2.62 Cl.00 1.60 1.60 1.78 1.30 2.52 1.78 1.30

Cl.00 1.30 1.58 Cl.00 1.00 1.30 1.60 Cl.00 1.60 Cl.00 1.00

NT

Cl.00

Cl.00

NT

NT

4.29

NT

NT

a Pseudotypes were defined as the infectious fraction following for the two designated by (459, which were prepared by heating b Normal rabbit serum. c Dexamethasone (5 Fg/mll. d Infectivity expressed as log PFU per milliliter. v Not tested.

Heat Stability of VW Pseudotypes Derived from Mammary Tumor Cell Lines

An alternate procedure for preparing VSV pseudotypes is to utilize the heat lability of the glycoprotein of the mutant VSV tlB17 (Zavada, 1972b). The glycoprotein loses its biological function when heated at 45”, whereas the glycoprotein of the oncornavirus in the pseudotype may not be as sensitive. The results of this type

Cl.00

2.32 NT NT

4.75

Neutralization of the xenotropic component was only demonstrated at a higher concentration of anti-MuMTV IgG. This would indicate that the xenotropic component may be related to MuMTV, possibly being VSV(MuMTV-X). However, other explanations for this xenotropic component are also tenable.

Anti-MuLV-M

I:50

4.68 4.33

NT NT NT NT NT NT NT NT NT NT NT NT 2.30 4.12

neutralization with anti-VSV at 45” for 60 min.

Cl.00

2.24 3.10 4.10 2.96 3.60 4.30 2.00 3.25 3.92 2.48 3.72 4.00 Cl.00 Cl.00

2.35 IgG, except

of experiment are shown in Fig. 2, and demonstrate that when the mutant tlB17 is grown in the mammary tumor cell lines both the ecotropic and zenotropic pseudotypes formed are more stable at 45” than the parental VSV. After neutralizing the heat-inactivated pseudotype stock with anti-MuMTV, no surviving pseudotype particles were observed (Table 3), conflrming the identity of heat-stable fraction as VSV(MuMTV) . Specific Interference to VSV Pseudotype Infection by Oncornaviruses

Interference is the third property of VSV pseudotypes which reflects the envelope properties of the contributing oncornavir-us. The results described in the previous sections on host range and neutralization were sufficient to identify a pseudotype

VSV(MuMTV)

FIG. 2. Thermal inactivation of VSV tlB17 grown in virus-negative and in two MuMTV-producing mouse cell lines, VSV was grown in (A) NIH/ 3T3, (B) GR, and (C) Mm5 cells at 32”. Prior to infection with VSV, both the mammary tumor cell lines were maintained for 2 days in medium containing 5 pg/ml of dexamethasone. Infectious tissue culture fluids were diluted 1:lO in Tris-buffered saline, pH 7.7, and heated at 45” for intervals up to 60 min. The surviving infectivity was determined in (+) Vero cells, (A) mink cells, (0) NMG cells, and (a) BALB/c secondary embryo cells. The arrows ( J ) indicate complete inactivation.

obtained from MuLV-M-producing cells as VSV(MuLV-M) and one of the pseudotypes from the mammary tumor cell lines as VW (ecotropic MuMTV). To characterize the xenotropic pseudotypes further, VSV was propagated in SIRC cells infected with two different isolates of MuLV-X and in the mammary tumor cell lines. Pseudotype stocks were assayed both in control mink cells and mink cells preinfected with both isolates of MuLV-X. Furthermore, infection of mink cells with the xenotropic component of GR cells was also attempted as follows: A g-cm plate was seeded with 2 x lo6 mink cells and 2 x lo6 GR cells or MM5 cells. ARer 2 days the cultures were split 1:4 and incubated for a further 2 days. Tissue culture medium from the cocultivation was filtered through 0.45-pm Millipore filters and used for infection of cultures of subconfluent mink cells. After a few days the cells were passaged again before being used as assay cells in the interference plaque assays (mink/GR and mink/Mm5 cells). The results of the interference experiment are shown in Fig. 3 and can be summarized as follows.

227

PSEUDOTYPES

(1) VSV(MuLV-X) pseudotypes harvested from cells producing xenotropic MuLV were not able to plate on mink cells preinfected with either strain of MuLV-X, whereas they plated on control mink cells. (2) The pseudotypes obtained from the mammary tumor line shows no interference when plated on mink cell preinfected with MuLV-X, indicating that neither pseudotype is related to MuLV-X. (3) Surprisingly, all the pseudotypes mentioned above gave a titer of approximately loj PFU/ml when assayed on the mink/GR cells. This was also true of otherwise neutralized parental VSV grown through uninfected mink cells. The size of the plaques on these assay cells was heterogeneous, varying from a normal size (>2 mm) to “pinpoint,” in contrast to the normal plaque assay. This suggests that an Mink

SIRC MuLV-X

GR+DEX

9-

a-l-i 3‘ k s ?

65-

4-

3-

2-

l-

:

3ccl

Assay

d cell

,.bcd

FIG. 3. Specific interference of VSV pseudotypes and reactivation of antibody-neutralized VSV. VSV was grown in control or oncornavirus-producing cells. Total infectivity (open columns) and pseudotype infectivity (shaded columns) (virus treated with anti-VSV IgG as in Fig. 1) were determined by plaque assay in (a) control mink cells, (b) mink cells preinfected with MuLV-X (BALB/c), (c) mink cells preinfected with MuLV-X (NZB), and (d) mink cells preinfected with a filtrate of culture medium from GR mammary tumor cells (mink/GR).

228

ZAVADA,

DICKSON,

agent present in the GR cell line is infectious fnr mink cells and imparts a capacity of these cultures to reactivate antibodyneutralized VSV or its pseudotypes. Presumably this reactivation could take place soon after infection (large plaques) or after a delay resulting in smaller plaques. Reactivation

of Neutralized

VW

Because the mink cells infected with an agent derived from GR cells possessed a capacity for reactivating antibody-neutralized VSV, this property was examined in the parent GR line and the other mammary tumor line, Mm5. Table 4 shows that the GR line indeed reactivated neutralized VSV but that this activity was not so pronounced in the Mm5 line. The “reactivating” agent is not neutralized by antiMuMTV IgG (1:lO dilution) in an endpoint dilution assay of the agent on mink cells. Furthermore, the reactivation was restricted to antibody-neutralized VSV as the heat-inactivated mutant VSV tlB17 was not reactivated. The filterable nature of the reactivating agent implies that it might be a mycoplasma or virus. Attempts to detect mycoplasma in the GR cell line and the mink/ GR line by thymidine autoradiography and thin-section electron microscopy were negative (data not shown). Growth of the mink/GR cells in gentamycin (50 pg/ml) had no effect on the ability of these cultures to reactivate neutralized VSV. Consequently the agent does not appear to be a TABLE

4

INFECTIVITY OF VFV tlB17 PROPAGATED IN THE CONTROL MINK CELL LINE AND AMAYED UNTREATED OR FOLLOWING TREATMENT WITH ANTI-VW IgG OR HEATING AT 45” FOR 1 HR Infectivity

(log PFU/ml)

%,’

Mink Mink/GR Mink/Mm5 NIH/3T3 GR Mm5 ’ Not tested.

Untreated control

Anti-VSV IgG 1:30

8.19 ‘7.80 8.14 8.15 8.38 8.32

Cl.00 4.48 Cl.00
Heating 45” Cl.00 Cl.00 Cl.00 NT” NT NT

at

AND

WEISS

mycoplasma. VSV was propagated in mink, mink/GR, and mink/Mm5 cells, and the harvested VSV was treated with antiVSV serum and plated on the test cell lines for the detection of pseudotypes. The results indicate that no detectable pseudotypes of VSV were formed in any of these cell cultures. This suggests that the agents originally revealed by the VSV pseudotype technique were not capable of replicating to high titer in these cell lines. Furthermore, the agent present in the mink/GR cultures that reactivates neutralized VSV was not itself able to make a detectable VSV pseudotype, as might be expected of a paramyxovirus present in sufficient titer to induce fusion. DISCUSSION

When cells infected with paramyxoviruses (Choppin and Compans, 19701, myxoviruses (Zgvada and Rosenbergova, 19721, oncornaviruses (Ztivada 1972a), togaviruses (Zgvadova et al., 1977), and herpesviruses (Huang et al., 1975) are doubly infected with VSV the progeny virus contains both the parent and, usually, the VSV pseudotypes. The pseudotypes containing the VSV genome are enveloped in the glycoproteins of the second virus and express the neutralization, host range, and interference characteristics of the donor virus. In some instances the reciprocal pseudotypes have also been detected (Weiss et al., 1975,1977; Livingstone et al., 1976). VSV pseudotypes have been utilized for studying mechanisms of host restriction to oncornaviruses in birds and mammals (Huang et al., 1973; Krontiris et al., 1973; Love and Weiss; 1974, Boettiger et al., 1975; Weisset al., 1975). Complete replication of oncornaviruses is not required for the formation of pseudotypes, for the expression of the viral glycoprotein in the host plasma membrane is sufficient for formation of VSV pseudotypes, as demonstrated in the “chicken helper factor” system (Love and Weiss, 1974) and in the SSPE/measles virus system (Wild et al., 1976). The ability of VSV pseudotypes to pick up envelope antigens of a partially expressed or defective oncornavirus, opens the attractive prospect of employing these

VSV(MuMTV)

PSEUDOTYPES

229

methods of VSV pseudotype formation to murine C-type viruses (Levy, 1973; Hardetect unknown viruses, hopefully includtley and Rowe, 1976; Rasheed et al., 1976). ing those putatively connected with some Alternatively, the xenotropic pseudohuman tumors. type produced in GR and Mm5 cells may However, until recently, we failed to represent a mosaic particle containing glyproduce firm evidence of VSV pseudotype coprotein determinants both of an unparticles in cultures shedding completely known virus which defines the host range infectious virions of some known oncorna- and of ecotropic MuMTV. viruses, such as MuMTV, and of woolly The ability to produce VSV(MuMTV) monkey virus (Love, Teich, and Weiss un- pseudotypes may prove useful as it reduces published observations; Weiss and Wong, the duration of neutralization tests from 1977). One of these viruses, MuMTV, has the present 1 year or so for completion of been shown in this report to produce pseu- an in uiuo tumor induction assay to an in dotypes. Their detection depends on using vitro plaque assay which takes 2 days. The care to avoid exposure to nonspecific se- ability to make VSV(MuMTV) pseudorum inhibitors, to fluctuations in pH, and types will also allow an examination of the to several other factors which taken to- cell surface receptor restrictions for gether can reduce the titer below con% MuMTV among cells derived from differdence levels. Furthermore, a suitable indi- ent tissues or different genotypes. In fact cator cell must be used, as in the case of our results already indicate that the diffiVSV(MuMTV) which did not plate on the culty to obtain in vitro infections with MuMTV is not a result of an inability of mouse cell lines NIH/3T3 and BALB/3T3 but did plate on secondary cultures of the virus to penetrate host cells, and indimouse embryo cells which appear to be cate which cells should be useful for furprimarily fibroblastic in morphology. This ther studies. The finding of a filterable agent able to property of plating only on freshly derived fibroblastoid cells did not apply to other reactivate serum-neutralized VSV was types of mouse cells because the certainly a surprise. At this moment, VSV(MuMTV) plated efficiently on nor- nothing indicates that the effect is related mal mammary gland (NMG) cells and an to MuMTV synthesis as it was not neutralendodermal cell line (PYS) derived from a ized with antibodies directed towards teratocarcinoma, both of which are estab- MuMTV. It might represent an unknown contaminating virus; although the nature lished cell lines. During these experiments several unex- and effects of this agent will be interesting pected factors emerged which deserve dis- per se, we will not speculate further until cussion. The first of these was the detec- more information can be obtained. Finally, the results suggest that under tion of a putative xenotropic MuMTV pseudotype, which is dexamethasone inde- suitable conditions probably all oncornavipendent and infectious for mink cells. This ruses will form envelope pseudotypes with pseudotype is clearly different from both VSV even when the molecular weight of the ecotropic MuLV-M and the two iso- the glycoprotein differs significantly from VSV G protein. We have already successlates of MuLV-X. A partial neutralization fully applied the procedure described here of the xenotropic MuMTV pseudotype with anti-MuMTV antibodies indicates that it for the preparation of VSV pseudotypes may share a surface antigen related to with primate oncornaviruses (Schnitzer, MuMTV. In fact it may be this component Zavada, and Weiss, unpublished results). as opposed to the ecotropic MuMTV that Consequently this procedure represents a potential new tool for attempting to detect has recently been reported to infect mink and cat cells (Vaidya et al., 1976; Las- viral antigens of putative human tumor fargues et al., 1976). At this stage we may viruses. only speculate that several MuMTVs may ACKNOWLEDGMENTS exist with ecotropic, xenotropic, and amWe thank Maureen At&will for skillful techniphotropic host specificities similar to the

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cal assistance and Dr. N. Teich for critically the manuscript.

DICK SON, reading

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