Pseudotypes of avian sarcoma viruses with the envelope properties of vesicular stomatitis virus

VIROLOGY 76, 8084325 (1977)

Pseudotypes of Avian Sarcoma Viruses with the Envelope Properties of Vesicular Stomatitis Virus ROBIN A. WEISS,’ DAVID BOETTIGER,2 Imperial

AND

HELEN M. MURPHY

Cancer Research Fund Laboratories, P.O. Box 123, Lincoln’s WC2A 3PX, England Accepted September

Inn Fields,

London,

17,1976

Upon superinfection of cells producing Rous sarcoma virus (RSV) with temperaturesensitive mutants of vesicular stomatitis virus (VSV), two kinds of pseudotype viruses are produced: VSV genomes within particles bearing the envelope antigens of RSV, denoted VSV(RSV), and RSV genomes within particles bearing the envelope antigens of VSV, denoted RSV(VSV). The VSV(RSV) pseudotypes are recognized as the fraction of plaque-forming units resistant to neutralization by antiserum to VSV or, in the case of thermolabile envelope mutants of VSV, resistant to heat inactivation; they possess the host range restrictions of RSV and are neutralized by antisera specific to the RSV subgroup. The RSV(VSV1 pseudotypes are recognized as the fraction of focus-forming units which transforms chick cells resistant to infection with the strain of RSV used. Both kinds of pseudotypes are produced concomitantly with VSV synthesis. VSV(RSV) particles comprise up to 12% of the VSV progeny titer and RSV(VSV) up to 1% of the RSV titer, but pseudotype fractions varied according to the VSV mutant used for superinfection. The proportions of pseudotypes in harvests of mixed infections are not reduced by filtration through 0.2-pm pore size filters to eliminate large aggregates of virus particles, and pseudotypes are not formed by mixing pure-grown RSV and VSV particles in vitro. VSV acts as a helper virus for BH-RSV(-1, which is defective in envelope antigen, but not for aBH-RSV(-), which is also defective in RNA-directed DNA polymerase activity. The titer of BH-RSV(VSV) is enhanced by the presence of the avian leukosis helper virus, RAV-1, and more than 90% of this mixed pseudotype stock is neutralized by antiserum to either VSV or RAV-1, indicating that the RSV particles bear a mosaic of both VSV and RAV-1 envelope antigens. RSV(VSV) pseudotypes transform cells of four out of five mammalian species tested. Like RSV of subgroup D and B77, the focus-forming titer of RSV(VSV) assayed on mammalian cells is lOOO-fold lower than on chick cells. INTRODUCTION

When cells producing avian RNA tumor viruses are superinfected with vesicular stomatitis virus (VSV), phenotypically mixed particles of VSV are produced (Zavada, 1972a, b). These phenotypically mixed particles, or pseudotypes, of VSV bear the subgroup-specific envelope antigens of the RNA tumor virus, according to host range, interference, and neutralization tests (Love and Weiss, 1974; Boettiger ’ Address reprint requests to Dr. Weiss. 2 Present address: Department of Microbiology, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19174.

et al., 1975). Previously published experiments indicated that mixed infections of VSV and RNA tumor viruses also yield reciprocal pseudotypes, i.e., RNA tumor virus particles which bear the envelope specificity of VSV (Weiss et al., 1975; Livingston et al., 1976). In this paper we present analysis of the formation and properties of Rous sarcoma virus (RSV) pseudotypes determined by VSV. The formation of VSV pseudotypes bearing the envelope antigens of unrelated animal viruses is a widespread phenomenon, yet reciprocal phenotypic mixing has only recently been reported. VSV was first shown to form pseudotypes with a para-

808 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0042-6822

RSV(VSV)

PSEUDOTYPES

809

resistant to infection with subgroup E virus but susceptible to other subgroups) were derived from embryos obtained from our own Brown Leghorn flock or from SPAFAS Inc., Norwich, Connecticut; C/AE cells (chick cells resistant to subgroups A and E) were recovered from frozen cell stocks originally derived from Reaseheath C-line embryos obtained from Houghton Poultry Research Station, Houghton, Cambridgeshire, England; and C/ABE and C/C cells were similarly derived from Line 7 and Line 15 embryos respectively, kindly provided as frozen cultures by L. B. Crittenden. Japanese quail eggs (Coturnix coturnix var. japonica) were obtained from Houghton Poultry Research Station and turkey eggs (Meleagris gaZZoparvo) from British United Turkeys Ltd., High Wycombe, Buckinghamshire, England. Mammalian cells of the following established cell lines were used for RSV transformation assays: rat NRK cells (DucNguyen et al., 1966), kindly provided by J. Wyke; mouse NIH-3T3 cells (Jainchill et MATERIALS AND METHODS al., 19691, kindly provided by N. Teich; All experiments involving VSV or phe- mink lung cells (CCL64 of the American notypically mixed virus stocks were con- Type Tissue Culture Collection), kindly ducted under conditions suitable for han- provided by R. Gallagher; and 2T human dling potentially hazardous infectious or- sarcoma cells (Ponten and Saksela, 19671, ganisms and were contained within the kindly provided by J. Zavada. In addition, institute’s virus isolation laboratory. tertiary cultures of bovine lens epithelium Origin of cells. Fibroblastic cultures cells were used, kindly provided by J. Taywere derived from lo- to 11-day chick, lor-Papadimitriou. quail, or turkey embryos by standard trypThe establishment of long-term cultures sinization procedures. The cultures were of quail and turkey cells transformed by grown in Dulbecco’s modification of Ea- the defective Bryan high-titer strain of gle’s medium supplemented with 10% RSV (BH-RSV) and its cy mutant is detryptose phosphate broth, 1% heat-inactiscribed in detail elsewhere (Murphy, vated chick serum, and 1% fetal calf serum 1977). These cultures released cloned BHand were maintained at 37-38” until used RSV (- 1 particles and were maintained in for experiments or assays as indicated. Dulbecco-Eagle’s medium with 10% trypThe cells were used for virus growth and tose phosphate broth, 1% heat-inactivated assay either as secondary or tertiary cul- chick serum, 4% fetal calf serum, and 1% tures or up to seven passages (without sig- DMSO. The 16Q clone represents a quail nificant changes in the plating efficiency cell transformed by and producing clone 16 of RSV or VSV) following recovery of pri- BH-RSV(- ), which is a polymerase-posimary stocks stored above liquid nitrogen tive recombinant of aBH-RSV and RAVin a medium containing 25% fetal calf se- 49. These cells were used between the 35th rum and 10% dimethylsulfoxide (DMSO). and 40th passage. The 3T cells are turkey Avian embryo cells were screened for cells transformed by clone 3 BH-RSV( -) susceptibility to the different host range (Ogura and Friis, 19751,which was introsubgroups of RSV. C/E cells (chick cells duced into the turkey cells by means of myxovirus (Choppin and Compans, 1970; McSharry et al., 1971) and has since been reported for myxovirus (Zavada and Rosenbergova, 1972), mammalian and avian RNA tumor viruses (Zavada, 1972a, b; Huang et al., 1973; Krontiris et al., 1973; Love and Weiss, 1974), herpes simplex virus (Huang et al., 1974) and Sindbis virus (Zavadova and Zavada, personal communication). While phenotypic mixing of envelope markers occurred with most stocks and mutants of VSV, Zavada and Zavodska (1973/1974)have shown that functional complementation of VSV by RNA tumor viruses was found only with mutants belonging to complementation group V (Indiana strain VSV) which is believed to represent the envelope antigen (G protein). Therefore phenotypic mixing appeared to involve the envelope antigens only. Similarly, in the reciprocal phenotypic mixing of RSV and VSV analyzed here, we have observed no evidence of complementation beyond that of envelope defects.

810

WEISS,

BOETTIGER,

inactivated Sendai virus. The a40T cells are turkey cells transformed by aBH-RSV introduced as a RAV-49 pseudotype. The 3T and a40T cells were used between the 10th and 14th passage. All three transformed cell lines, lSQ, 3T, and (~40T, released substantial quantities of noninfectious BH-RSV(-) particles that were defective in the major envelope glycoprotein, gp85. The (Y mutant is also defective for RNA-directed DNA polymerase activity (Hanafusa and Hanafusa, 1971; Murphy, 1977). The presence, if any, of chick helper factor (chf) coded for by the endogenous virus genome of chick cells (Weiss, 1969a; Hanafusa et al ., 1970) was detected by the appearance of RSV(chf) pseudotypes infective for quail or turkey cells following cocultivation of the cells under test with 16Q cells producing BH-RSV( -). Chf-positive cells were not used for the propagation of virus and pseudotype stocks but were used where appropriate as assay cells.

AND

MURPHY

Avian fibroblasts treated with 0.05% trypsin in Tris-EDTA were suspended in a medium containing 5% calf serum and were seeded in Dulbecco-Eagle’s medium diluted to contain
RSVWSV)

virus stocks. The following mutants of VSV were kindly provided by the people indicated in parentheses: Indiana serotype tlB1’7 (J. Zavada); tsTl026 fC. P. Stanners); tsG114, tsG31, tsG41, tsO45, and Cocal serotype Sl (C. Pringle). The nomenclature used here is that recommended by Cormack et al. (1973). All VSV mutants were plaque-puri~ed twice sequentially, and the virus stocks used for experiments were passaged only twice after cloning. VSV stocks were propagated at 31-33”. Except where otherwise stated, mixed infections were carried out at a VSV multiplicity of 2 by allowing VSV to adsorb onto confluently transformed, TV-producing cells for 1 hr at room temperature, washing twice with growth meditmr, and incubating for 12-18 hr at 33”. At harvest, media were frozen-thawed twice, and cell debris was removed by centrifugation at 2500 ‘pm. DMSO was added to 5% volume, and stocks were stored at -70” or above liquid nitrogen. For VSV plaque assays, approximately 10” cells were seeded per 30-mm Linbro tissue culture well 18-24 hr before infection. Virus was diluted in complete PBS with 1% fetal calf serum, and 0.1 ml aliquots were adsorbed onto the cells for 1 hr at room temperature. The assay wells were overlaid with Dulbecco-Eagle’s medium supplemented with 2% fetal calf serum and 1.1% agar (final concentration) and were incubated at 33” to allow replication of all ts mutants. Plaques were counted 2-3 days later, 2-4 hr after overlaying with agar medium containing 0.07% neutral red diluted in PBS. Viral ~~ter~rence. Chick cells were infected with approxima~ly 10” infectious units of RAV and after three passages were challenged with RSV or VSV. Interference (Vogt and Ishizaki, 1966) was assayed by estimating the relative reduction of plating efficiency on RAV-infected cells to mock-infected cells of RSV strains or pseudotypes or of VSV pseudotypes. Challenge with a noninterfering pseudotype was included in the assay as a check for the subgroup specificity. Infection inacti~uted

811

PSEUDOTYPES

of RSV and VSV mediated by Sendai virus. Cells were

seeded as for standard focus or plaque assays and washed once in serum-free medium, and approxima~ly 800 hemagglutinin units of ~-propiolac~ne-inactiva~d Sendai virus diluted in Dulbecco-Eagle’s medium were added (Enders et al., 1967). The Sendai virus was adsorbed onto the cells for approximately 30 min at 4” without CO, supplementation so that the diluent attained an approximate pH of 7.9. The cells were then washed once in serumfree medium, 0.1 ml of virus was added and adsorbed for 15 min at 4” and 45 min at 37”; the cells were overlaid with appropriate agar medium, and foci or plaques were counted as for standard assays. Antisera. Neutralizing antisera to RAV-1 and RAV-49 were obtained from infected Brown Leghorn fowl (Weiss, 1969b). Neutralizing antiserum to VSV was obtained from a hyperi~une sheep and was generously provided by J. Zavada (1972b). Each antiserum was used at a final dilution of 5% in reaction mixtures. Undiluted or 10-l dilutions of virus samples were neutralized for 40-60 min at 37” in a water bath and were then diluted for virus assay. At an ~tise~m concentration of 5%, no surviving fractions were observed in neutralization tests of homologous RSV pseudotypes for anti-RAV-1 and anti-RAV-49 sera or of pure-grown Indiana serotype VSV mutants for anti-VSV serum. Biochemical

assays of RSV

particles.

The physical particle assay by [:“Hluridine incorporation into viral RNA and the assay of RNA-directed DNA polymerase activity in avian tumor virus particles were conducted as described by Murphy (1977). The polymerase reaction included salmon sperm DNA as the exogenous template and [“HITTP as the labeled precursor. RESULTS

Expansion of RSV Host Range following Mixed Infection with VSV

To ascertain whether RSV can assemble VSV envelope antigens to form RSV(VSV) pseudotypes, conditions were sought in which both viruses could function simultaneously in the same cell and yet might be selectively titrated after harvest. Superin-

812

WEISS,

BOE’lTIGER,

fection of RSV-producing cells by nonleaky, temperature-sensitive mutants of VSV appeared to be the most promising procedure for this purpose. After growth at the permissive temperature, VSV may be titrated by routine plaque assay and RSV by a focus assay incubated at 41” so that the VSV present in the mixed stock would not replicate in and kill the focus assay cultures. The susceptibility of chick cells to infection by RSV is governed by interaction of the viral envelope antigens with genetically determined cell surface receptors (Payne and Biggs, 1964; Rubin, 1965; Vogt and Ishizaki, 1965; Weiss, 1976). Since VSV can infect all chicks cells tested, the assembly of VSV envelope antigens in RSV particles should permit the RSV to infect resistant cells. Once a cell becomes infected with RSV, a focus of Rous cells will appear in the assay cell monolayer by cell proliferation alone, not requiring further cycles of viral infection (Temin and Rubin, 1958; Weiss, 1971). Thus RSV(VSV) pseudotypes may be titrated as focus-forming units (FFU) on resistant cells. High multiplicity infection of cells by VSV frequently causes an inhibition of host cell syntheses, in which case RSV synthesis might be similarly shut off. Farmilo and Stanners (1972) reported that the VSV mutant tsT1026 is inefficient in shutting off host cell functions and that when incubated at the “semipermissive” temperature of 38.5”, viral glycoprotein was synthesized without significant impairment of cell division and other host cell functions. For this reason we chose first to use VSV tsT1026 for superinfection of cells producing RSV on the assumption that RSV production would be maintained, at least at the “semipermissive” temperature for VSV synthesis, and that phenotypic mixing might occur. Cultures that were confluently transformed by PR-RSV-A were therefore mock-infected or infected with tsT1026 at 2 multiplicities of infection (m.0.i.) and were incubated at a range of temperatures for 16 hr when media were harvested for the preparation of virus stocks. The focus-forming units present in the stocks were assayed on susceptible and

AND

MURPHY

resistant cells incubated at 41”, and the results of this assay are shown in Table 1. There was little reduction in the titer of RSV produced following infection of VSV tsT1026 at a multiplicity of 2 whether incubated at permissive (33”), semipermissive (37-38.5”), or nonpermissive (40”) temperatures. However, a significant reduction of RSV titer was observed in cells infected at the higher m.o.i. and incubated at permissive temperatures, presumably as a result of inhibition of host cell and RSV synthesis. Whereas pure-grown PR-RSV-A was not infectious on genetically resistant Reaseheath Line C (C/AE) cells, foci in the resistant cell cultures were detected in harvests of mixed infections. Since the highest titer of RSV with the expanded host range (about 1% of total RSV) as observed after VSV infection at the fully permissive temperature of 33”, at an m.o.i. of 2, this temperature and approximate m.o.i. were used for subsequent experiments involving VSV superinfection. Mixed virus harvests also showed focus-forming activity on genetically resistant Line 7 C/ABE cells and on C/E cells rendered resistant by RAV-1 interference. Successful infection of the same assay cell types with PR-RSV-C showed that resistance and interference are subgroup specific. It appears, therefore, that superinfection of RSV-producing cells with VSV enables a fraction of the RSV progeny to infect and transform otherwise resistant cells. Reciprocal

Phenotypic

Mixing

In Table 2, the results are shown of an assay of all the viral phenotypes likely to occur following superinfection of cells producing PR-RSV-A with VSV tsT1026 at 33” and an m.o.i. of 2. The total VSV titer was asayed by plaque titraton on C/E cells, whereas the titer of VSV(RSV-A) pseudotype was assayed as the resistant fraction of plaque-forming units (PFU) after neutralization with VSV antiserum at a concentration (5%) sufficient to neutralize all pure-grown VSV. The absence of plaques on C/AE cells indicated that the antiserum-resistant fraction of VSV was specified by RSV envelope antigens. The total

RSV(VSV)

TABLE HOST RANGE

AND TITERS

OF PR-RSV-A

813

PSEUDOTYPES 1

AFTER SUPERINFECTION

OF RSV-PRODUCING

CELLS WITH WV

tsT1026 RSV

Superinfecting

Titer

VSV

(log FFU/ml)

on

m.0.i.”

Temperatureb (“C)

Brown Leghorn C/E cells

Line C C/AE cells

Line 7 C/ABE cells

PR-RSV-A

None

33 40

7.0 7.2

11.0 11.0

1.5 1.8

2.3 NT”

PR-RSV-A

2

33 37 38.5 40

6.8 6.8 6.8 6.9

4.7 3.8 2.2 Cl.0

4.8 4.0 2.6 1.3

4.7 NT 2.8 NT

PR-RSV-A

17

33 37 38.5 40

5.2 5.3 5.6 6.2

2.0 2.3 1.4 Cl.0

NT NT NT NT

NT NT NT NT

7.1

7.3

6.6

6.9

PR-RSV-C’

RAV-1 C/E cells’

II Multiplicity of infection. b Temperature of incubation after superinfection; all stocks were harvested 16 hr after superinfection. ’ Brown Leghorn C/E cells infected with lo4 infective units of RAV-I 7 days and two transfers before focus assay. ” NT, not tested. ” Laboratory stock. TABLE ASSAY

Mixed

virus stock

PR-RSV-A and VSV tsT1026 mixed before assay”

MIXED

Method of assay Plaques or foci

PR-RSV-A-producing cells superinfected with VSV tsT1026

2

OF PHENOTYPICALLY

Plaques Plaques Plaques Foci Foci Plaques Plaques Plaques Foci Foci

VIRUSES

Expected phenotype

vsv antiserum

Assay cells

Temperature (“C)

C/E C/E C/AE C/E CIAE C/AE

33 33 33 41 41 41

vsv

+ I’ + -

C/E C/E C/E CIAE

33 33 41 41

vsv

+

+ -

VSV(RSV) RSV RSVWSV)

VSV(RSV) RSV RSVWSV)

I’ The virus stock was incubated with neutralizing antiserum before infection. h Pure-grown RSV and VSV were mixed and incubated at 37” for 1 hr before dilution

RSV in the mixed harvest was assayed by titration of foci on C/E cells, whereas the titer of RSV(VSV) was asayed by a focus titration on C/AE cells. Neutralization by VSV antiserum indicated that the RSV with an expanded host range was indeed dependent on VSV envelope antigens.

Observed titer (log PFU/ml or FFU/ml)

8.1 6.2 Cl.0 6.8 4.7 Cl.0 8.4 11.0 7.2 Cl.0 and assay.

It was possible that the capacity of RSV grown with VSV to infect resistant cells resulted from a nonspecific effect of the VSV on the assay cell surface comparable to the effect on cells of inactivated Sendai virus (Enders et al., 1967; Weiss, 1969a1, thereby permitting PR-RSV-A to enter

814

WEISS, BOETTIGER,

otherwise resistant cells. However, when pure-grown PR-RSV-A and VSV were mixed together and incubated for 60 min at 37” before assay, no expansion of RSV host range and no surviving fraction of VSV neutralization was observed (Table 2). In further tests of this type, a low titer ([email protected] PFU or FFU/ml) of modified VSV or RSV was observed in two of five experiments, but these titers represented only a small fraction of the phenotypically modified viruses detectable following mixed infection. We conclude, therefore, that reciprocal phenotypic mixing occurs on mixed infection of RSV and VSV, giving rise to VSV(RSV) and RSV(VSV) pseudotypes, each representing about 1% of the total population of infectious VSV and RSV. In order to study the kinetics of phenotypic mixing, VSV, RSV, and their reciprocal pseudotypes were titrated in harvests taken at different times following super-infection of RSV-producing cells with VSV. Replicate cultures of RSV-transformed cells were infected with VSV tsT1026 at an m.o.i. of 2, and duplicate cultures were harvested at 2- or 4-hr intervals during incubation at 33”. In each harvest, the titers of total and phenotypically mixed VSV and RSV were assayed by the methods listed in Table 2. The results are shown in Fig. 1. The synthesis of nonneutralizable VSV(RSV) pseudotypes increased in parallel to total VSV synthesis and represented about 2% of the total VSV. The synthesis of RSV(VSV) was first clearly detected 6 hr after VSV infection, 2 hr earlier than progeny VSV synthesis could be definitively observed, and it continued to increase in titer until 16 hr postinfection. Between 12 and 20 hr after VSV infection, the titer of total RSV dropped approximately fivefold, and during this time the cytopathic effect of VSV on the transformed cells became marked. Thus the synthesis of phenotypically mixed VSV and RSV stocks appears to be greatest when the parental viruses are each producing near maximum titers. Identification of Phenotypically Modified Viruses as Pseudotype Particles

The detection of focus-forming or plaque-forming viruses with the apparent

AND MURPHY

/

I 4 CPE

6 12 Hours after VSV infectii t +

16

I 20

+

FIG. 1. Appearance of phenotypically mixed%ruses following superinfection of PR-RSV-A-producing cells with VSV tsT1026. Culture medium was harvested from parallel cultures of doubly infected cells at eight time points during the 20-hr period. The cytopathic effect (CPE) was judged at the time of medium harvest. VSV, RSV, and their reciprocal pseudotypes were assayed as described in Table 2.

envelope properties of an unrelated virus does not prove that the infective units assayed represent single virus particles with phenotypically substituted envelope antigens. On the one hand, the modified virus might represent particles of unsubstituted RSV and VSV that are clumped together, perhaps mutually aiding penetration into host cells; on the other hand, the modified virus might represent genetically recombinant forms rather than transient, phenotypic modifications. To test whether the phenotypically mixed viruses merely represented virus aggregates, the proportion of apparent VSV(RSV) and RSV(VSV) in a mixed stock was titrated after filtration through a 0.22-pm pore size Millipore filter or after disruption by ultrasound (Table 3). Titration of total and phenotypically modified plaque-forming or focus-forming units was carried out as described in Table 2. The results indicate that although filtration reduced the overall titers of both VSV and RSV approximately lo-fold, the proportion of infective units that appeared to be phenotypically mixed was not significantly altered in either case. Neither did sonic disruption of viral aggregates reduce the proportion of phenotypically mixed particles; indeed, both the total RSV titer and the

RSV(VSV)

815

PSEUDOTYPES TABLE

3

PROPORTION OF PHENOTYPICALLY MIXED VSV AND RSV BEFORE AND AFTER FILTRATION OR SONICATION Mixed virus harvest Standard” Filtered* Sonicated’

Titer vsv 7.7 6.6

7.9

(log PFU/ml) VSV(RSV)

VSV(RSV) (%)

5.8 4.6 5.8

1.2

1.0 0.8

Titer

(log FFU/ml)

RSV(VSV) (%f

RSV(VSV)

RSV

4.5 3.4 5.1

6.7 5.5 7.0

0.6 0.8 1.3

fl Medium harvested after superinfection of PR-RSV-A-producing cells with VSV tsT1026 was processed in the standard way described in Materials and Methods. Total virus and phenotypically mixed fractions were assayed as described in Table 2. ’ The virus stock was passed through a 0.22-pm pore size Millipore filter immediately before assay. r The virus stock sealed in a vial was treated with ultrasonic vibration (Branson 12 sonic cleaner) for 30 set immediately before assay.

fraction of RSV(VSV) showing an expanded host range increased by a factor of 2, possibly due to the disaggregation of cytopathic VSV particles from the RSV particles. It seems reasonable to conclude, therefore, that the modified biological properties of the VSV and RSV stocks result from the substitution of the alternative envelope antigens during the assembly of individual virus particles. This conclusion is supported by immuno-electron microscopic observations (unpublished) and by the observation that VSV forms pseudotypes with the endogenous viral “helper factor” of chick cells in the absence of complete, C-type particle synthesis (Love and Weiss, 1974). The progeny of the RSV possessing an expanded host range were studied in order to determine whether the RSV(VSV) in mixed virus harvests represented pseudotypes or stable recombinants. RSV was harvested from a number of foci of transformed cells that appeared in C/AE assay plates infected with a mixed virus stock (Table 4). No RSV infective for C/AE cells was detectable in viruses harvested directly from foci appearing in “resistant” cells. Cells of eight foci appearing in resistant C/AE cells were aspirated and seeded onto C/AE and C/E monolayers to allow proliferation of the transformed cells and propagation of RSV. Growth among C/AE cells should select for recombinant RSV and growth on C/E cells should support the growth of both parental and recombinant RSV. In neither case was RSV infective for C/AE cells released by these cultures after 10 days of incubation. Thus none of the foci

TABLE

4

REVEIWON OF PR-RSV-A(VSV) PSEUD~TYPES TO GENOTYPE HOST RANGE Source of RSV

Number of foci tested

lean Titer (log FFU/ml) on C/E cells

RSVWSV) stock Disrupted cells aspirated from individual foci initiated by RSV(VSV) on ClAE cells Cells aspirated from individual foci initiated by RSVWSV) on CIAE cells and grown for 10 days with C/AE cells and grown for 10 days with C/E cells

tj

20

7 6.5 2.8

CIAE cells 4.4

Cl.0

8

tested bred true for VSV envelope properties, although they were initially infected and transformed by RSV(VSV). It has been reported previously that VSV tZB17 mixed with avian leukemia virus strains did not breed true for the envelope properties of the leukemia virus (Zavada 1972a; Love and Weiss, 1974). Similar tests were carried out with VSV tsT1026 propagated in cells producing PR-RSV-A. Plaques initiated by pseudotypes were identified in the usual way as survivors of neutralization by VSV antiserum, and the phenotypes of 12 plaque-purified stocks were studied. Each of the 12 cloned stocks after propagation in uninfected chick cells was fully neutralized by VSV antiserum.

816

WEISS,

BOE’ITIGER,

Uncloned VSV pseudotype virus also did not maintain the tumor virus envelope specificity when propagated in uninfected chick cells at three different multiplicities. Thus the reciprocal exchange of envelope antigens between RSV and VW did not result in heritable modifications in either case. Phenotypic Mixing between Various Strains of RSV and VSV The formation of RSVWSV) pseudotypes of strains other than PR-RSV-A was examined. Chf-negative chicken or quail cells were infected with four different strains of avian sarcoma virus belonging to subgroups A, C, and E. The defective BH-RSV strain was propagated with RAV1 or RAV-0 as “helper” viruses and therefore already constituted pseudotypes, though of host range restricted to that of the helper virus. One or two passages after RSV infection, when the cultures appeared to be completely transformed, one subculture of each strain was superinfected with VSV tsT1026 and another was mock-infected. The harvested media were assayed for focus-forming activity on susceptible and resistant cells, and RSV(VSV) pseudotypes were detected in all mixed stocks (Table 5). The pseudotype fraction did not differ markedly for the TABLE RSV(VSV)

AND

MURPHY

different avian sarcoma virus strains, although it was somewhat lower for SRRSV-A and SR-RSV-E. The ability of different VSV mutants to form RSVWSV) and VSV(RAV) pseudotypes was examined by infecting a series of cultures confluently transformed by BHRSV(RAV-1). VSV ts mutants of the Indiana serotype belonging to different complementation groups and VSV t&l of the Coca1 serotype were used at an m.o.i. of approximately 2. A cytopathic effect (CPE) became apparent at different times from 12 to 24 hr after VSV infection according to the mutant used, and culture medium was harvested when the CPE was equivalent to the stage for optimal harvest of pseudotypes with tsT1026. Table 6 shows the titers of total viruses and of pseudotypes in the mixed stocks. The formation of RSV(VSV) pseudotypes was detected with all the VSV mutants, and the highest titer was obtained with tsT1026. Despite incubation of focus assays at 41”, some cytopathic effects were noted in undiluted or 10-l dilutions of mixed stocks containing tsG31, ts045, and t&l so that the RSV(VSV) titers of these stocks may be The formation of underestimated. VSV(RAV-1) pseudotypes was also detected for all VSV mutants of the Indiana serotype, with mutants tZB17, tsG41, and 5

PSEUDOTYPES OF DIFFERENT STRAINS OF AVIAN SARCOMA VIRUS AFTER SUPERINFECTION OF VIRUS-PRODUCING CELLS WITH VSV tsT1026” Titer

Sarcoma virus

(log FFU/ml)

on

RSV(VSV) (o/o)

Subgroup

Strain

Host

A

BH-RSVCRAV-1) SR-RSV PR-RSV

Spafas

6.9 5.3 6.8

4.6 2.5 4.7

0.5 0.2 0.8

C

PR-RSV B77

C-line

6.3 7.0

4.0 4.8

0.5 0.6

E

BH-RSV(RAV-01 SR-RSV

Quail

5.7 5.6

3.3 2.7

0.4 0.1

Susceptible cells”

Resistant cells’

(I VSV titers in all mixed stocks varied between 107.’ and lo*.” PFU/ml. b Susceptible cells used were Brown Leghorn for subgroups A and C and East Lansing Line 15 for subgroup E. r Resistant cells used were Reaseheath Line C for subgroups A and E and East Lansing Line 15 for subgroup C. No foci were observed on resistant cells infected with 0.1 ml undiluted and IO-’ dilution of the avian sarcoma viruses propagated without superinfection by VSV.

RSV(VSV)

TABLE RSV AND VSV PSEUDOTYPESFORMED Superinfecting

VSV

Mutant

Complementation group0

tsT1026 tsGll4 tlB17 tsG31 tsG41 ts045 tsi?1 None

I I I&V III IV V s

817

PSEUDOTYPES 6

SUPERINFECTION OF CELLS PRODUCING BH-RSVCRAV-1) SEVEN VSV MUTANTS

AFTER

Titer RSV(RAV-1) 7.3 6.9 6.5 6.5 7.2 6.7 6.8 7.5

(log FFU/ml) RSV(VSV)” 4.7 3.9 3.5 3.2 2.0 2.6 1.8 Cl.0

Titer vsv 8.0 8.6 8.8 7.9 7.8 9.0 8.2

WITH

(log PFUlml) VSVCRAV-I)’ 6.3 7.0 7.7 6.3 6.9 8.2

I’ According to the designation of Cormack et al. (1973). b Assayed as FFU on C/AE cells, as described in Table 2. c Assayed as antiserum-resistant PFU, as described in Table 2. Propagation of each of the VSV mutants in uninfected chick cells yielded VSV stocks with antiserum-resistant fractions of less than lo-% (with the exception of Coca1 61, which was not markedly neutralized by the Indiania antiserum and for which a pseudotype fraction could not therefore be estimated).

tsO45 yielding the highest pseudotype fractions. Thus there was no correlation between the RSV(VSV) fractions and the VSV(RAV-1) fractions obtained with different VSV mutants. VSV Acts as a Helper Virus for Defective RSV Since VSV will expand the host range of infectious RSV by phenotypic mixing, it was of interest to determine whether VSV will serve as a helper virus in conferring infectivity on noninfectious BH-RSV( -) particles. These particles lack the envelope antigens (Scheele and Hanafusa, 1971; Ogura and Friis, 1975) believed to be gp85 and, possibly, gp37 (Leamnson and Halpern, 1976), which determine the subgroup specificity of avian RNA tumor viruses. The (Y mutant of BH-RSV further lacks RNA-directed DNA polymerase (Hanafusa and Hanafusa, 1971), and in this case VSV would not be expected to act as a helper virus. In order to test complementation of defective RSV strains, quail cells transformed by clone 16 BH-RSV (16Q cells) and turkey cells transformed by clone 3 BH-RSV (3T cells) were used as sources of envelope-defective viruses, while turkey cells transformed by BHRSV ((~40T cells) represented cells releasing virus that was defective both for envelope antigens and for RNA-directed DNA

polymerase (Murphy, 1977). Turkey cells transformed by and releasing nondefective PR-RSV-A were used as a positive control. Table 7 shows that each of the four kinds of RSV-transformed cells before VSV infection released substantial quantities of C-type particles into the culture medium, as measured by incorporation of [“Hluridine into particle-associated RNA. Polymerase assays also indicated substantial virus particle production with the exception, as expected, of a40T cells. Focus assays showed that only the cells transformed by PR-RSV-A released infectious RSV and that this virus carried the expected host range restriction for C/AE cells. However, when C/AE cells were pretreated with inactivated Sendai virus, a low but significant focus titer was observed for PRRSV-A, 16Q virus, and 3T virus. The a40T virus was not infectious in Sendai-treated cells because, in the absence of polymerase, no provirus could be synthesized even if viral penetration is effected by the Sendai treatment (Hanafusa and Hanafusa, 1971). Super-infection of the 16Q and 3T cells with VSV tsT1026 yielded RSV infectious for C/E and C/AE cells, and VSV infection of PR-RSV-A-producing cells extended the host range of the RSV to C/AE cells. However, no infectious RSV was rescued from a40T cells upon VSV superinfection, despite the replication of

818

WEISS,

BOETTIGER,

AND

TABLE COMPLEMENTATION

MURPHY

7

BY VSV OF RSV DEFECTIVE

IN ENVELOPE

ANTIGE N

7

Polymer, ase activity (cpmjb

Assay cells’

PR-RSV-A turkey

60,380

38,000

C/E CIAE CIAE-Sendai

7.2 Cl.0 2.9

6.8)

-

6.5

3.3 3.5

BH-RSV( -) clone 16Q

22,000

35,320

C/E CIAE CIAE-Sendai

Cl.0 Cl.0 2.4

3.2 3.1 3.5

7.1
8.1

Cl.0

BH-RSV(-1 clone 3T

13,740

6,940

C/E CIAE CIAE-Sendai

Cl.0 Cl.0 1.6

2.5 2.3 2.5

6.4 Cl.0 2.3

6.3

Cl.0

(YBH-RSV( - 1 clone a40T

--I-

C/E CIAE CIAE-Sendai

Cl.0 Cl.0 Cl.0

7.2 Cl.0 2.8

6.6

Cl.0

Virus-produc ing cells

I:aH]uri-

dine1!abeled PIarticles (cpm)” I

44,660

240

T

Titer

(log FFU/ml)

RSV

13SV(VSV)” f I ESV(RAV-1)’

Cl.0 Cl.0 Cl.0

.’

1 !

II Xter (log PFU/ml)

T

vsv

VTSV(RSV)’

4.2

” Incorporation of [3H]uridine into RNA of virus particles per milliliter of supernatant medium, labeled over a 12-hr period and banded in a sucrose gradient. * Incorporation of [3H]TTP into DNA by RNA-directed DNA polymerase of virus particles harvested from supernatant medium, using exogenous template, expressed as counts per minute per milliliter of culture medium. r C/AE cells were treated where indicated with inactivated Sendai virus before RSV infection. d Titers of RSV harvested 16-20 hr after superinfection with VSV tsT1026 at an m.o.i. of 2. p Titers of RSV harvested 7 days after superinfection with RAV-1 at an m.o.i. of 0.01. ’ Nonneutralizable fraction of VSV tsT1026, as described in Table 2.

VSV in these cells. Superinfection of cells releasing defective RSV with RAV-1 rescued high titers of RSV(RAV-1) in each case. Thus the VSV acted as a helper virus for RSV defective in envelope antigens but not for RSV defective in RNAdirected DNA polymerase. VSV tsT1026 produced significantly lower progeny titers in transformed turkey cells than in quail or chick cells, and VSV propagated in cells releasing envelope-defective RSV did not yield an antiserumresistant fraction (Table 7). Mosaic RSV Particles Bearing both VSV and RAV-1 Envelope Antigens Having demonstrated that VSV will functionally complement defective BHRSV(-1, we investigated whether there was competition against or enhancement of the assembly of VSV envelope antigens into the RSV envelope when a helper RNA tumor virus was also able to provide envelope antigens. A culture of 16Q cells

producing defective BH-RSV(-1 particles was infected with RAV-1 and was maintained in parallel to an uninfected culture. Six days later, subcultures of 16Q-RAV-1 cells and 16Q cells were superinfected with VSV mutants tZB17 or tsT1026, and virus was harvested 16 hr later. The results of the virus assays are presented in Table 8. As expected, the focus assays showed that the BH-RSV(-1 particles were not infectious and that the BH-RSV(RAV-1) particles plated at high titer on C/E cells, but were not infectious for C/AE cells. With both VSV mutants, a substantial pseudotype PFU titer was found after growth in 16Q-RAV-1 cells, but no antiserum-resistant fraction was discernible after growth in 16Q cells. A comparative titration on C/AE cells was made of BH-RSV(VSV) produced by 16Q cells and 16Q-RAV-1 cells. It can be seen that the titers of BHRSV(VSV) were enhanced by the presence of RAV-1. This effect was more marked with tZB17 superinfection, when the presence of RAV-1 enhanced the RSV(VSV)

RSV(VSV1

TABLE FORMATION

OF BH-RSVWSV)

ISQ cells releasing BHRSV( - ) superinfected with RAV-1”

-

PSEUDOTYPES

8 IN THE PRESENCE

RSV (log FFU/ml)

vsv*

C/E

CIAE

-

Cl.0

Cl.0
+

7.1

819

PSEUDOTYPES

AND

ABSENCE

assayed on CIAE-Sendai’

OF RAV-I

VSV (log PFU/ml) Total

Pseudotype”

2.6 2.8

+

tlB17 tlB17

1.9 6.4

1.7 3.6

7.9 8.0

Cl.0 7.1

+

tsT1026 tsT1026

4.3 6.8

4.2 4.9

8.4 8.2

Cl.0 6.4

(1Infected with RAV-1 (m.o.i. of 0.01) 1 week before harvest for assay. h Infected with VSV (m.0.i. of 2) 16 hr before harvest for assay. ( C/AE cells were treated with inactivated Sendai virus before RSV infection. ‘l Antiserum-resistant titer, as described in Table 2.

titer almost loo-fold, than with tsT1026 sayed on C/AE cells. The anti-VSV sesuper-infection, when the presence of RAV- rum did not significantly neutralize pureas assayed on 1 enhanced the RSV(VSV) titer only five- grown BH-RSV(RAV-l), fold. However, both the overall titer and C/E cells, whereas anti-RAV-1 serum comthe relative pseudotype fraction of pletely neutralized this pseudotype. We RSV(VSV) formed with tsT1026 in the checked that the neutralizing effect of presence of RAV-1 were greater than the our tumor virus antisera were subgroup specific, insofar as anti-RAV-49 serum equivalent stocks formed with tZB17. neutralize BHThus there was no competition by RAV- did not significantly 1 envelope antigens for the assembly of RSV(RAV-1) and anti-RAV-1 serum did not neutralize BH-RSV(RAV-49) (results VSV antigens into RSV envelopes; rather, the presence of RAV-1 facilitated the for- not shown). mation of BH-RSV(VSV). This enhanceTable 9 shows that when cells producing ment of BH-RSV(VSV) titer was appar- the defective BH-RSV(-1 were superinently not due to enhanced BH-RSV release fected with VSV tsT1026, the resulting because the titer of BH-RSV( - ) and of BH- RSV(VSV) pseudotypes were neutralized RSV(RAV-1) on C/AE cells treated with by anti-VSV serum but not by anti-RAV-1 inactivated Sendai virus was approxi- serum. In contrast, super-infection of cells mately the same (Table 8). It is possible producing BH-RSV(RAV-1) with either that more VSV G-protein was synthesized VSV tsT1026 or tZB17 yielded RSV(VSV) in the cells infected with RAV-1; yet the pseudotypes which, when assayed on C/E titers of VSV progeny were not affected by cells, were partially neutralized by antithe presence or absence of RAV-1. It RAV-1 serum and were completely neuseemed probable, therefore, that RAV-1 tralized by a combination of the two antienhanced the RSV(VSV) titer by promot- sera. When assayed on C/AE cells, the ing the assembly of phenotypically mixed neutralization of the RSV(VSV) pseudoparticles bearing both RAV-1 and VSV an- types by anti-VSV serum was virtually tigens. To test this assumption, the effect complete; yet significant neutralization, of neutralizing antibodies on the different particularly in the case of the RSV(VSV BH-RSV(VSV) pseudotypes was examined tZB17) pseudotypes, was also obtained with (Table 9). Results presented in Table 2 anti-RAV-1 serum. This result suggests indicated that the expanded host range of that these pseudotypes possess mosaic envelopes, since the presence of the VSV glyPR-RSV-A(VSV) was completely neutralized by anti-VSV serum. This was con- coprotein is essential for infection of the firmed for al1 BH-RSV(VSV) stocks as- C/AE cells, and the presence of the RAV-1

WEISS,

BOETI’IGER,

AND

TABLE NEUTRALIZATION

Source of BH-RSV”

16Q-RAV-1

cells

16Q-RAV-1 cells superinfected with VSV tlB17

16Q-RAV-1 cells superinfected with VSV tsT1026

16Q cells super-infected with VSV tsT1026

OF BH-RSV(VSV)

Neutralizing

9

BY ANTIBODIES

serum

MURPHY

TO VSV OR TO RAV-I

SPECIFIC

Log FFU/ml

titer

on

C/E cells

C/AE cells

6.9 Cl.0 6.9

Cl.0

Nonimmune Anti-RAV-1 Anti-VSV Nonimmune Anti-RAV-1 Anti-VSV Anti-RAV-1 + anti-VSV

6.3 1.5 6.1 Cl.0

3.6 1.3 Cl.0 Cl.0

Cl.0

Cl.0

Nonimmune Anti-RAV-1 Anti-VSV

4.2 4.3 Cl.0

4.0 4.1 Cl.0

a 16Q cells releasing noninfectious described for Table 8. -

BH-RSV(-)

particles

glycoprotein is essential for neutralization by anti-RAV-1 serum. It also implies that RSV can be neutralized by the binding of antibody to envelope glycoproteins that are used for viral penetration of host cells lacking the appropriate receptors. The fraction of FFU observed in C/E and C/AE cells which is resistant to anti-RAV-1 but is neutralized by a combination of antiRAV-1 and anti-VSV presumably represents a population of pure RSV(VSV) particles. Thus the mixed virus harvest appears to contain a major fraction of pure RSV(RAV-11, a large minor fraction of mosaic, doubly neutralizable RSV(RAV-l/ VSV), and a smaller fraction of pure RSV(VSV) particles. Assays based on expansion of host range alone do not distinguish between pure RSV(VSV) particles and particles with mosaic envelopes of VSV and tumor virus antigens. Previous studies with VSV(AMV) and VSV(RAV-1) pseudotypes (Zavada, 197213; Love and Weiss, 1974)‘indicated that a substantial fraction of VSV pseudotypes are not neutralizable by anti-VSV serum, suggesting that those pseudotypes may represent particles in which the envelope anti-

RSVCRAV-1)

neutralized RSV(VSV)

h5.9 0

Nonimmune Anti-RAV-1 Anti-VSV Anti-RAV-1 + anti-VSV

6.7 3.3 6.6

Log fraction

4.7 3.0 Cl.0

were superinfected

4.8 0.2

2.3 >2.6

>5.3

>2.6

3.4 0.1 >5.7

1.7 >3.5 >3.5

-0.1 >3.2 with RAV-1 and/or VSV, as

gens are wholly substituted by the avian leukemia virus. Hence the assay of VSV(RSV) and VSV(RAV-1) titers in this study is based on nonneutralizable fractions. Without an assay for mixed particles comparable to the expansion of host range of RSV(VSV), it is difficult to determine whether the VSV(RAV-1) fraction may be even higher than that measured (Table 8) if mosaic particles were accounted for. Transformation RSVCVSV)

of Mammalian

Cells by

Some strains of avian sarcoma virus are oncogenic in uiuo and transform mammalian cells in culture. Previous studies on plating the relative efficiency of VSV(RSV) pseudotypes on avian and mammalian cells indicated that the specificity of the viral envelope plays an important role in the infection of mammalian cells by RSV (Boettiger et al., 1975). In addition to restrictions on viral penetration, the majority of mammalian cells infected by RSV do not express transformed properties (Boettiger, 1974). Since VSV is infectious for mammalian cells, attempts were made to transform cells derived from

RSV(VSV)

an accurate titer on account of the cytopathic effect of excess VSV present in the mixed stocks. Advantage was taken of the VSV mutant tsG114 which has a thermolabile polymerase. After heat inactivation at 45” for 1 hr, the PFU titer of VSV tsG114 in the mixed virus stock was reduced lo-“.’ with only a twofold drop in RSV titrated on C/E cells. Using the heat-inactivated stock, therefore, the cytopathic effect in focus assays was greatly reduced, so that it was possible to enumerate more accurately the number of RSV foci in mammalian cells. The focus titers shown in Table 10 indicate that, for the mammalian-tropic viruses, B77, BH-RSV(CZAV), and PR-RSVA(CZAV), the relative transformation frequency of mammalian cells was lo-;’ or less, compared to chick cells. Transformation of mammalian cells by RSVWSV) can be similarly expressed as the ratio of the focus titer on mammalian cells to that on chick C/AE cells, and this was also lo-:’ or less. Thus RSVWSV) pseudotypes appear to have approximately the same relative transformation frequencies as B77 and RSV(CZAV).

with mammalian species several RSV(VSV). Some difficulty was experienced in accomplishing successful transformation assays because it was not possible to incubate the mammalian cells above 39.9, at which temperature the cytopathic effect of the VSV was noted (at low virus dilutions) to be greater than in the chick cell assays carried out at 41”. The results of the most successful experiment are shown in Table 10. Strain B77 virus was used as an example of one avian sarcoma virus which has transforming activity for mammalian cells. The mammalian cell types most susceptible to transformation, NRK and bovine lens, gave a transformation frequency about lo-” times lower than that of chick cells infected with the same virus stock. Virus stocks from cells transformed by PR-RSV-A or BHRSV(-1 which had been superinfected with the subgroup D helper virus, CZAV, also transformed the mammalian cells, whereas PR-RSV-A and BH-RSV(RAV-1) without CZAV did not do so, presumably because subgroup A viruses cannot penetrate mammalian cells (Boettiger et al., 1975). Therefore, transformation by BHRSV(RAV-l/VSV) and PR-RSV-AWSV) pseudotypes could be assayed simply by plating onto mammalian cells. Pseudotypes of PR-RSV-A and of BH-RSV(RAV1) prepared with VSV tsT1026 yielded such low titers of focus-forming virus on mammalian cells that it was difficult to obtain

DISCUSSION

Superinfection of RSV-producing cells with VSV leads to the formation not only of VSV particles bearing RSV envelope components but also of RSV particles bearing VSV envelope components. The anti-

TABLE TRANSFORMATION

Virus

821

PSEUDOTYPES

OF MAMMALIAN

stock

10 CELLS

WITH

RSV titer Chick C/E

Chick CIAE

Rat NRK

RSVWSV)

(log FFU/ml) Mouse NIH-

on

Mink CCL64

Bovine lens

Human 2T

1.2

3.3

1.5

XT3

Bl7

7.0

6.8

3.4

PR-RSV-A PR-RSV-A(CZAV) PR-RSV-A(VSVtsT1026)

7.2 6.8 6.7


<0.4 2.0

‘co.4

1.0

<0.8

BH-RSV(CZAV) BH-RSV(RAV-11 BH-RSV(RAV-l/VSV BH-RSVCRAV-l/VSV

6.7 7.3

3.2 <0.4 1.7

<0.4

co.4

Cl.2

1.3

<0.4

Cl.2 1.0

tsT1026) tsG114)”

7.1 6.8

6.1 4.1 6.5


0 The virus stock was heated at 45” for 60 min prior to infection.

1.0

0.8

1.2

‘co.4

1.3 ~0.8

1.5

10.4 2.4

co.4

1.2

~0.8

3.6 co.4

1.5

1.2 co.4 Cl.2

1.9 1.5___~.--.- 1.2

822

WEISS.

BOETTIGER,

genie specificity and host range of the pseudotype particles were characteristic of the virus providing the envelope antigens. Each of the seven temperature-sensitive mutants of VSV tested formed RSV(VSV) pseudotypes, although the mutants of the Indiana serotype belonging to complementation group I yielded the highest titers, in particular tsT1026. Little variation was observed in the RSV(VSV) pseudotype fraction when VSV tsT1026 was grown in cells producing a variety of RSV strains belonging to three different envelope subgroups. The highest titers of RSV(VSV) were obtained after VSV superinfection at a permissive temperature, provided that the multiplicity of VSV was not so high that host cell functions and RSV synthesis were significantly shut down. It was not feasible to test the formation of RSV(VSV) using wild-type VSV because of the excess VSV present in the mixed stocks. The formation of VSV(RSV) and RSV(VSV) pseudotypes appeared to require double infection of the host cells. On propagation, the envelope specificity of the pseudotypes reverted to that of their focusforming or plaque-forming parent viruses, indicating that neither recombinant nor hybrid virus particles appeared in significant amounts in the pseudotype stocks. Little or no pseudotype fraction was detectable when RSV and VSV were mixed together irz vitro before assay, and the proportion of pseudotypes in harvests of mixed infections was not significantly reduced by removal of large virus aggregates by filtration or sonication. It seems reasonable, therefore, to view pseudotype formation as the assembly of the RSV subgroup-specific antigens (probably gp85) into the envelope of VSV virions and of VSV antigens (probably G-protein) into the envelope of RSV virions. This view is consistent with the formation of VSV(chf) pseudotypes in normal chf+ cells which express envelope antigens of the endogenous avian tumor virus on the cell surface without releasing Ctype virus particles (Love and Weiss, 1974) and with biochemical studies of VSV(SV5) pseudotypes (McSharry et al., 1971). Although pseudotypes of VSV have been prepared with a wide variety of enveloped

AND

MURPHY

viruses, RSV pseudotypes specified by unrelated viruses had not been reported until recently. Hanafusa (1964) did not observe any rescue of infectious BH-RSV with Newcastle disease virus, and Halpern et al. (1973) were unable to detect an expansion of RSV host range on mixed infection with avian reticuloendotheliosis virus. However, the formation of RSV(VSV) pseudotypes does not appear to be a unique example. Dyadkova and Kuznetsov (1970) published evidence suggestive of reciprocal phenotypic mixing between RSV and mumps virus, and Livingston et al. (1976) have recently reported the formation of murine sarcoma virus pseudotypes bearing VSV envelope antigens. In addition, phenotypic mixing between avian and mammalian RNA tumor viruses has recently been observed following mixed infection of dually permissive cells (Levy, 1977; Weiss and Wong, 1977). On mixed infection of BH-RSV(RAV-1) and VSV, the RSV(VSV) pseudotype plating on C/AE cells was completely neutralized by antiserum specific to VSV. However, partial neutralization of RSV(VSV) was also observed with antiserum specific to RAV-1, whereas RSV(VSV) formed after VSV infection of BH-RSV(-)-producing cells in the absence of RAV-1 was not neutralized by antiRAV-1 serum. This result suggests that the RSV(VSV) pseudotypes formed in RAV-l-producing cells carry RAV-1 envelope determinants in addition to VSV envelope antigens, because the neutralizing effect of the RAV-1 antiserum was specific to subgroup A envelope antigens. The observation that more BH-RSV(VSV) is produced when RAV-1 is present than when VSV is the sole “helper” virus suggests, together with the double neutralization data, that the RSV with an expanded host range represents particles bearing a mosaic of VSV and RAV-1 envelope antigens. Zavada et al. (1975) have proposed that the “doubly neutralizable” VSV particles formed in mixed infection with certain mammalian RNA tumor viruses carry mosaic envelopes, whereas the antiserumresistant fraction represents pseudotypes in which all or most of the envelope anti-

RSV(VSV)

PSEUDOTYPES

gens are provided by the alternate virus. Vogt (1967) has also suggested that phenotypic mixtures of nondefective RSV with RAV of a different envelope subgroup form mosaic particles, but these were not apparently susceptible to neutralization by antiserum specific to either subgroup alone. There is little indication of doubly neutralizable particles of VSV(RAV-11, though the addition of complement to mixtures of antiVSV serum and VSV(RAV-1) significantly decreased the surviving fraction (Weiss et al., 1975). Since there is no assay of VSV(RAV-1) equivalent to the host range expansion of RSV(VSV) pseudotypes, it would be difficult to distinguish a doubly neutralizable VSV(RAV-1) fraction from nonpseudotype VSV present in the same stock. Experiments are in progress to attempt to distinguish these two types of particle by selective immunoadsorption. The complete neutralization of VSV tsT1026 and tZB17 propagated in two independently derived clones of BH-RSV(-) cells confirms previous findings based on the thermolability of tZB17 (Ogura and Friis, 1975; Weiss, 1976) that BH-RSV does not provide functional envelope antigens to the VSV particle. VSV pseudotypes may also prove useful for characterizing thermolabile RSV mutants. It would be unwise, however, to assume that a failure to form pseudotypes provides definitive evidence for envelope defects. We have not been able to discern VSV pseudotypes with the envelope antigens of primate C-type viruses (Weiss and Wong, 1977) and even among the avian C-type viruses of a single subgroup, e.g., CZAV and RAV-50, large differences in VSV pseudotype fractions were obtained (Boettiger et al., 1975). Considering the reciprocal pseudotypes, we have shown that VSV envelope antigens will functionally complement BH-RSV but not CXBH-RSV,which also lacks polymerase. Furthermore, we have observed (not presented here) that the RSV(VSV) pseudotypes prepared with the thermolabile VSV mutants, tZB17 and ts045, are themselves thermolabile. Thus one can use phenotypic mixing as a means of defining “complementation groups” between unrelated viruses (Zavada and Zavodska, 1973/ 1974).

823

VSV is infectious for cells of a wide variety of vertebrate and invertebrate species, including man. Since RSV(VSV) pseudotypes have probably gained as broad a host range for viral penetration as VSV itself, these pseudotypes might be regarded as more hazardous than the parental RSV. On the other hand, previous studies of VSV(B77) and VSV(CZAV) pseudotypes indicated that viruses bearing these envelope antigens also have a high efficiency of penetration into mammalian cells, although the transformation frequency of mammalian cells is much lower than that of chick cells (Boettiger et al., 1975). It appears that many more mammalian cells are infected by and carry stable RSV proviruses than become phenotypically transformed (Boettiger 1974; Varmus et al., 1973). Our studies indicate that the transformation rate of RSV(VSV) in mammalian cells is similarly much lower than the expected penetration rate, which should be approximately the same in chick and mammalian cells. Since the absolute titers of RSV(VSV) are much lower than B77 titers and since many potentially infected cells at temperatures lower than 39.5” may be killed by excess VSV, it appears that RSV(VSV) pseudotypes represent less of a potential oncogenic hazard to people than do RSV strains of subgroup D and avian sarcoma virus B77. However, we feel that experiments on phenotypic mixing between unrelated viruses should be restricted to a suitable safety facility because the formation of recombinants or hybrid proviral elements has not been rigorously excluded. Since VSV appears to broaden the host range of RSV by providing envelope antigens only, the use of pseudotypes (for purposes other than their intrinsic interest) is limited to the infection of “resistant” cells, but this may be effected by alternative means, such as agents which promote membrane fusion. Finally, it should be borne in mind that many cell lines (e.g., L cells) in which VSV and other enveloped, lytic viruses are propagated may be chronically infected with C-type viruses of endogenous or exogenous origin, so that phenotypically mixed viruses might frequently be generated unwittingly.

824

WEISS,

BOETTIGE :R, AND

ACKNOWLEDGMENTS We are grateful to Alan Tucker and Gloria Charters for technical assistance and to Steven Martin, Natalie Teich, John Wyke, and Jan Zavada for critically reviewing the manuscript. REFERENCES BOETTIGER, D. (1974). Virogenic, non-transformed cells isolated following infection of normal rat kidney cells with B77 strain Rous sarcoma virus. Cell 3, 71-76. BOETTIGER, D., LOVE, D. N., and WEISS, R. A. (1975). Virus envelope markers in mammalian tropism of avian RNA tumor viruses. J. Virol. 15, 108-114. CHOPPIN, P. W., and COMPANS, R. W. (1970). Phenotypic mixing of envelope proteins of the parainfluenza virus SV5 and vesicular stomatitis virus. J. Virol. 5, 609-616. CORMACK, D. V., HOLLOWAY, A. F., and PRINGLE, C. R. (1973). Temperature-sensitive mutants of vesicular stomatitis virus: Homology and nomenclature. J. Gen. Virol. 19, 295-300. DUC-NGUYEN, H., ROSENBLUM, E. N. and ZIEGEL, R. F. (1966). Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bacterial. 92, 1133-1140. DYADKOVA, A. M., and KUZNETSOV, 0. K. (1970). An attempt of hybridization of Rous sarcoma virus and parotitis virus. Neoplasma 17, 59-64. ENDERS, J. F., HOLLOWAY, A., and GROGAN, E. (1967). Replication of poliovirus I in chick embryo and hamster cells exposed to Sendai virus. Proc. Nat. Acad. Sci. USA 57, 637-644. FARMILO, A. J., and STANNERS, C. P. (1972). Mutant of vesicular stomatitis virus which allows deoxyribonucleic acid synthesis and division in cells synthesizing viral ribonucleic acid. J. Viral. 10, 605613. HALPERN, M. S., WADE, E., RUCKER, E., BAXTERGABBARD, K. L., and LEVINE, A. S. (1973). A study of the relationship of reticuloendotheliosis virus to the avian leukosis-sarcoma complex of viruses. Virology 53, 287-299. HANAFUSA, H. (1964). The nature of defectiveness of Rous sarcoma virus. Nat. Cancer Inst. Monogr. 1’7, 543-556. HANAFUSA, H., and HANAFUSA, T. (1971). Noninfectious RSV deficient in DNA polymerase. Virology 43, 313-316. HANAFUSA, H., MIYAMOTO, T., and HANAFUSA, T. (1970). A cell-associated factor essential for formation of an infectious form of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 66, 314-321. HUANG, A. S., BESMER, P., CHU, L., and BALTIMORE, D. (19731. Growth of pseudotypes of vesicular stomatitis virus with N-tropic murine leukemia virus coats in cells resistant to N-tropic viruses. J. Virol. 12, 659-662.

MURPHY

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