VIROLOGY

‘77,

797-810 (1977)

Murine Xenotropic II. Phenotypic

Mixing

Type C Viruses

with Mouse and Rat Ecotropic

Type C Viruses

JAY A. LEVY Cancer

Research

Institute

and

the Department San Francisco, Accepted

of Medicine, California November

University 94143

of California

Medical

Center,

28,1976

Phenotypic mixing between the murine xenotropic (X-tropic) and ecotropic type C viruses occurs readily and may account for heterogeneity of viral RNA in preparations of mouse type C viruses. Similarly there is no interference between mouse and rat endogenous type C viruses, and they can undergo phenotypic alterations. Various phenotypic and, perhaps, genotypic changes can happen as these viruses pass through heterologous hosts. Occurring perhaps as early as 1 hr after infection, phenotypic mixing enhances the potential for spread of oncogenic genomes among all animal species. It enables Xtropic viruses to infect and replicate in mouse cells, anobservation suggesting the major block to X-tropic virus infection in mouse cells is at the surface or penetration level. These studies also demonstrate the selective intracellular regulations of type C virus replication in various host cells. X-tropic virus replicates leas efficiently than ecotropic virus in mouse cells. Ecotropic virus replication is diminished in heterologous cells compared to that of X-tropic virus. An intracellular block in rat cells for the production of endogenous rat type C virus is also present. The nature of these intracellular regulating systems remains to be determined. INTRODUCTION

viruses (Levy, 1974; Levy et al., 1975b). The product of this infection is progeny Mouse type C viruses (MuLV), which composed of not only parental but also are xenotropic (X-tropic), productively in- mixed types, i.e., ecotropic viruses with fect only cells from animals which are for- xenotropic envelope antigens and xenoeign to the host species (Levy, 1973). Their tropic viruses with the type-specific deterhost range for exogenous infection in- minant of ecotropic type C viruses. Such cludes cells from rat, human, mink, and phenotypic mixing has been observed with duck but not mouse cells (Levy, 1973, the avian type C viruses (Vogt, 1967; 1975a). They constitute a class of endoge- Weiss et al., 1973). It can also occur benous viruses which differ from ecotropic tween unrelated viruses (Huang et al., type C viruses which preferentially infect 1974; Choppin and Compans, 1970) and, in and grow in cells from their own host spe- the case of vesicular stomatitis virus and cies (Levy, 1974). These ecotropic viruses RNA tumor viruses has been used to anaexogenously infect mouse and rat cells. lyze host range determinants of the type C Only ecotropic viruses induce XC plaque viruses (Zavada, 1972; Huang et al., 1973; formation in tissue culture (Levy, 1973, Krontiris et al., 1973; Weiss et al., 1974; 1974). Roettiger et al., 1975). This report defines Endogenous ecotropic viruses interfere the interference patterns for xenotropic with other members of this class. Cells and ecotropic viruses and describes the infected by one type of ecotropic virus are phenotypic mixing that results from coinresistant to superinfection by another fection of mouse or rat cells with these two (Sarma et al., 1967). We have shown that different murine type C virus classes. Ohcells infected with xenotropic type C vi- servations on similar interactions with the ruses permit superinfection by ecotropic endogenous rat type C virus are included. 797 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0042-6822

798

JAY MATERIALS

AND

A.

METHODS

Cells. Primary NIH Swiss mouse embryo cells (NIH-ME) and Fischer rat embryo (RE) cells were obtained from Microbiological Associates, Bethesda. Cultures of human foreskin cells (HuF) were provided by Ms. Miriam Debby, San Francisco, or were established in our laboratory from fresh specimens obtained from the nursery of the University of California, San Francisco. They were used up to passages lo-12 when their sensitivity to Xtropic viruses decreased. Normal rat kidney (NRK) cells (Due-Nguyen et al., 1966) were originally provided by Dr. R. Ting, Bethesda; a clone of these cells which was very sensitive to X-tropic and ecotropic viruses was employed. The SC-l line, established from a clone derived from embryo cells from a feral mouse (Hartley and Rowe, 1975), was given to us by Dr. J. W. Hartley, Bethesda. Although sensitive to infection by xenotropic MuLV, this cell line is at least 1 log more sensitive for detection of ecotropic virus (Hartley and Rowe, 1975; Levy, unpublished observations). Mink lung fibroblasts (American Type Culture Collection CCL64) were given to us by Dr. P. Peebles, Bethesda. Primary duck embryo fibroblast cells (DEF) were established from fertilized eggs obtained from Kimball Farms, Petaluma. Cells from other animal species were provided by Dr. W. Nelson-Rees, Oakland. From an established line of NZB embryo cells (the NZB-Q line), provided by Ms. M. Lander, Bethesda, two clones were derived which varied in production of X-tropic NZB type C viruses (NZB-MuLV) (Levy et al., 1975b). Clone 4 produces about 100 infectious particles/ml of NZB-MuLV. Clone 35 produces over 10,000 infectious NZBMuLV particles/ml. A continuous line of human (RD) cells producing the C57 Leaden X-tropic virus (C57L-MuLV) (Arnstein et al., 1974) and the D17 canine sarcoma cell line producing C57L-MuLV (the C57L-D line) were supplied by Dr. P. Arnstein, Berkeley. They produced lo4 and lo6 infectious viruses/ml, respectively. The quantity of X-tropic virus produced by these cell lines was measured by end-titration of the culture supernatants in human

LEVY

foreskin cells with subsequent cocultivation assays (Levy et al., 1975b). The NRKHarvey line is a non-virus-producing Harvey murine sarcoma virus-transformed NRK cell line derived in this laboratory (Levy, 1971). BALB S+L- cells were provided by Dr. A. Hackett, Oakland. This line was established from BALB 3T3 cells infected by murine sarcoma virus (Bassin et al., 1971). It does not release infectious virus. Media. Cell lines were maintained in minimal essential medium Eagle’s (EMEM) supplemented with 10% fetal calf serum and 1% glutamine and antibiotics (250 U/ml of penicillin and 250 Fg/ml of streptomycin). For focus formation assays, EMEM with 5% heated (56, 30 min) calf serum and 1% glutamine and antibiotics were employed. Viruses. The AKR, Gross, and Rauscher murine leukemia viruses (AKR-MuLV, GMuLV, R-MuLV) and the Harvey strain of murine sarcoma virus (H-MSV) were provided by Dr. Hartley and passaged in NIHME in this laboratory. Preparations of pseudotype sarcoma viruses were made of each of the above ecotropic MuLV and of the NZB and C57L X-tropic viruses by cocultivation of appropriate cell lines with the NRK-Harvey cell line as already described (Levy et al., 1975b). The rat type C virus pseudotype of MSV was obtained by inducing NRK-Harvey cells with iododeoxyuridine (IUDR) (100 pglml) by standard techniques (Lowy et al., 1971). Seru. Rat antiserum specific for AKRMuLV was,provided by Dr. Hartley. Rabbit anti-rat type C virus serum was supplied by Dr. J. Gruber, National Cancer Institute. A rabbit anti-X-tropic virus serum was prepared in this laboratory by the procedure previously described (Levy et al., 197513). Virus assay. The quantity of X-tropic MuLV was determined by end-titration in human foreskin cells with subsequent cocultivation assays (Levy et al., 197513). Focus formation for MSV detection and XC plaque assay for quantitation of ecotropic MuLV in mouse as well as other cells were performed as described (Hartley and Rowe, 1966; Rowe et al., 1970; Levy et al., 197513). All cells were pretreated with di-

XENOTROPIC

AND

ECOTROPIC

ethylaminoethyldextran to increase their sensitivity to virus infection (Duc-Nguyen, 1968). Neutralization tests. Neutralization tests were conducted by standard techniques (Levy, 1973, 1974; Levy et al., 1975b). In brief, for X-tropic viruses, 0.1 ml of the NZB pseudotype of MSV was incubated at room temperature with an equal amount of serum with effective neutralizing activity (2-4 u). After 30 min, the mixture was diluted to give approximately 100 focus forming particles/O.4 ml and inoculated onto NRK cells. Control cultures received virus incubated with EMEM instead of serum. Similar procedures were followed for ecotropic viruses which were measured by the XC plaque assay (Rowe et al., 1970). Neutralization was considered effective when 67% of foci or plaques formed was suppressed as compared to control cultures. RESULTS

Virus Interference

To illustrate the lack of interference between the X-tropic and ecotropic viruses, experiments were conducted using NRK cells chronically infected with either ecotropic or xenotropic MuLV. As shown by Table 1, interference was class specific. It occurred among all subgroups of the ecoTABLE INTERFERENCE XENOTROPIC

PATTERNS MURINE

Pseudotype sarcoma virusb

Focus NRK

R-MuLV AKR-MuLV NZB-MuLV c51 L-MULV

1 WITH TYPE

400 220 100 90

ECOTROPIC AND C VIRUSES”

formation

NRK-R 0 0 120 NT’

NRK-A 0 0 110 NT

in NRK-X 400 200 0 0

a NRK cells were infected with Rauscher CR), AKR (A), or NZB (X) murine type C viruses (MuLV). After seven passages in culture, the cells were inoculated with 0.4 ml of various pseudotypes of the Harvey murine sarcoma virus. The results are given as the average number of foci in duplicate cultures of the cell lines listed. * Harvey strain of murine sarcoma virus with the type-specific envelope antigens indicated. c NT, not tested.

PSEUDOTYPES

799

tropic viruses as has been reported (Sarma virus isolates. NRK cells infected with either the FMR (Rauscher) or AKR-Gross subgroups of ecotropic MuLV inhibited focus formation by all ecotropic pseudotypes of MSV. The xenotropic NZB-MuLV pseudotype of H-MSV, however, could form foci in the NRK cells infected with ecotropic MuLV. Similarly, NRK cells chronically infected with NZB-MuLV, blocked focus formation by the xenotropic virus pseudotypes of MSV, H-MSV(NZB-MuLV), and H-MSV(C57L-MuLV) but not the ecotropic pseudotypes of H-MSV. Progeny production mirrored the above results. NRK cells chronically infected with ecotropic MuLV.only produced progeny sarcoma virus when superinfected with the xenotropic virus pseudotypes of MSV, not ecotropic pseudotypes of MSV. The progeny produced after superinfection with the X-tropic virus was a mixture of xenotropic and ecotropic pseudotypes of MSV as demonstrated by the induction of foci in mouse, rat, duck, and human cells (data not shown).

et al., 1967) and among the xenotropic

Phenotypic Mixing of Helper Viruses Involved in MSV Replication

These latter experiments and ones reported previously (Levy et al., 1975b) have illustrated that the host range and typespecific antigens of ecotropic or X-tropic pseudotypes of MSV can change after passage of the virus through cells producing the other class of MuLV. Phenotypic mixing results from this superinfection and the progeny sarcoma viruses are both the parental and mixed types. MuLV also undergoes phenotypic alteration which can be illustrated by using the MSV preparations since MSV are genetically defective for the synthesis of certain virion proteins. The host range and replication of the sarcoma genome depends on the accompanying MuLV (Hartley and Rowe, 1966; Huebner et al., 1966; Parkman et aZ., 1970; Aaronson et aZ., 1970; Levy, 1971; Levy and Rowe, 1971). Focus formation and progeny MSV production, therefore, directly reflect the infectivity and replicative ability of the helper MuLV. Rat

JAY

800

A.

cells are sensitive to exogenous infection by both ecotropic and X-tropic MuLV, so that coinfection of these cells is possible (Levy et al., 1975b; Levy, 1975a). NRK cells, chronically infected with G-MuLV, were superinfected with a virus preparation containing the xenotropic virus pseudotype of MSV, MSV(NZB-MuLV), and its helper virus, NZB-MuLV. The rat cells became completely transformed, and the virus progeny produced foci in rat (NRK), human (HuF), DEF, and NIH-ME cells (Fig. 1). The host range indicated that both ecotropic and X-tropic pseudotypes of MSV were produced. The undiluted supernatant from the mouse culture was subsequently inoculated onto NRK, NIH-ME, HuF, and DEF cells. Focus formation was noted in all cells including the duck. Thus, NZBMuLV was replicated in the mouse cells since direct infection of mouse embryo cells by X-tropic virus is blocked (Levy, 1973, 1975a; Fischinger et al., 1975) and ecotropic MSV infection of NIH-ME has never activated the endogenous X-tropic NIH-MuLV (Fischinger and Nomura, 1975; Levy, unpublished observations).

LEVY

The NZB-MuLV must have infected the mouse cells while in an ecotropic virus coat. Confirmation that the mouse cells were producing X-tropic MSV was provided by neutralization tests. Supernatant from a mouse cell culture (see Fig. 1 (a)) was incubated with antisera against NZBMuLV and AKR-MuLV. Only the antiNZB-MuLV serum prevented the focus formation in DEF (Table 2). When supernatants from the DEF culTABLE NEUTRALIZATION

OF THE

PRODUCED

Virus

2 PSEIJD~TYPE

BY MOUSE

MSV

CELLS”

sample

Foci

Supernatant from virus-infected NIHME cell@ 1. Incubated with medium 2. Incubated with anti-NZBMuLV serum 3. Incubated with anti-AKR-MuLV serum

80 0 60

a Neutralization tests were conducted as described in text. Values represent the average number of foci formed in duplicate plates of duck embryo cells. b See virus passage (a), Fig. 1.

Phmofypfc mixing ofxenotroplc NRK chron,colly infected wth G-MuLV and supermfected wth K? FFU of MSV (NZB-MuLVl I MiF

DiF

G-L-I MEF (%I

f

(b)

tt

NgRgK“Z.5 “,“4’ 16.31

DEF 5.6

NFfK 6.6

r-T-L-2;) NRK 5.5

N&F 49

MEF

4.4 (6.3)

HUF

3.4

M&F 1.9 (4.4)

65

5.1

M&F 09 63 I331

5.3 t-h DEF MEF NRK 4.4 1.4 5.7 (3.4)

FIG. 1. Passage of the NZB xenotropic pseudotype of Harvey murine sarcoma virus, H-MSV(NZBMuLV) with NZB-MuLV through NRK cells chronically infected with the Gross strain of murine leukemia virus (GMuLV). Filtered undiluted supernatants (0.4 ml) from the cell cultures tested were passed as indicated by arrows. The letters (a) and (b) refer to virus preparations used for experiments described in the text. The numbers represent the titer of focus forming virus (log FFU per milliliter) of the inoculation as determined by the average number of foci detected in duplicate plates of the various cell lines listed. Figure in parentheses under mouse cultures represent the titer of MuLV (log PFU per milliliter) as determined by the XC plaque assay (Rowe et al., 1970). NIH Swiss mouse embryo cells (MEF) register ecotropic viruses. Human foreskin (HuF) and duck embryo fibroblasts (DEF) register the xenotropic viruses. NRK cells register both ecotropic and xenotropic pseudotypes of MSV.

XENOTROPIC

AND

ECOTROPIC

tures were inoculated on NIH-ME, foci and XC plaques were observed (Fig. 1). These results indicated that G-MuLV was present. Since DEF are resistant to infection by G-MuLV (Levy, 1975a) the virus must have gained entry via an NZB virus envelope. Cells in which the pseudotype MSVs were passed influenced the type of progeny virus produced. Human or duck cells yielded low titers of ecotropic viruses as measured in NIH-ME but high titers of xenotropic virus. Mouse and rat cells favored production of ecotropic virus pseudotypes of MSV. These results correlated with the relative ability of the helper MuLV to replicate in the host cells (Levy, 1975a). A similar result has been observed with MuLV pseudotypes with Rous sarcoma virus (RSV) (Levy, 1977). A low initial input of virus may have been responsible for some of the results although the ecotropic pseudotype of MSV and its helper MuLV replicated efficiently in mouse cells even when inoculated at a low input multiplicity (see Fig. 1 (b)). The influence of virus input on phenotypic mixing was studied by superinfecting NRK cells chronically producing G-MuLV (the NRK-G line) with MSV(NZB-MuLV) at various titers. Supernatants from the NRK-G cultures receiving 106, 104, and lo2 FFU/ml of the NZB pseudotype of MSV were used to infect NRK, HuF, DEF, and NIH-ME cells. As illustrated by Fig. 2, the Effect

NRK chrmcally

lo6 FFU

DE;

Replication Cells

wth

f-L NRK 54

Virus

in Mouse

nvxmg

G-MuLV and

MSV(NZB-MuLV)

at the

titers

10’ FFU

MS&F

of X-Tropic

NIH-ME infected with X-tropic virus in an ecotropic virus envelope produced substantial amounts of the xenotropic pseudotype of MSV as indicated by focus formation in human and duck cells (Fig. 1). Passage of this virus mixture through MEF did not reduce its titer. The efficiency of this X-tropic virus production by mouse cells was studied by comparing its replication in NIH-ME to that in rat cells (NRK and Fischer RE). MSV(NZB-MuLV) was inoculated onto NRK cells producing GMuLV. This coinfection resulted in phenotypic mixing; the NZB-MuLV and the replication-defective MSV emerged in ecotropic coats derived from G-MuLV. These phenotypically changed viruses could now infect mouse as well as rat cells. The virus preparation was first incubated with rabbit anti-NZB-MuLV serum to eliminate all MSV(NZB-MuLV) and NZB-MuLV which could have infected the rat but not the mouse cells. The effectiveness of this neutralization was confirmed by complete

Infected wth

followng

r-47 N&K

extent of phenotypic mixing correlated with virus input. DEF were the most sensitive to the effects of input levels of MSV. The results support the observation that replication of ecotropic virus is favored over X-tropic virus in rat cells. In human and duck cells, xenotropic virus replication is preferred (Fig. 1).

of virus mputon extent of phenotypic

superlnfected

801

PSEUDOTYPES

DEF MEF 17 44

I 10’ FFU

F-l-7 NRK 34

MF 14

MEF 34

FIG. 2. An NZB xenotropic pseudotype of Harvey mouse sarcoma virus, H-MSV(NZB-MuLV), was inoculated at various titers onto NRK cells chronically infected with the Gross strain of murine leukemia virus (GMuLV). Nine-day supernatants from these cultures were inoculated onto rat (NRK), duck (DEF), and NIH Swiss mouse embryo (MEF) cells. The numbers represent the titer of transforming virus (log FFU per milliliter) as determined by the average number of foci detected in duplicate plates of the cell lines listed.

802

JAY

A.

LEVY

virus replication exists in mouse cells but it is not complete. The nature of this intracellular block to X-tropic virus replication was explored further in cocultivation experiments. We placed BALB/c S+L- cells in culture with dog thymus cells chronically infected with the C57L xenotropic virus (the C57L-D line). After 6 days of cocultivation, the supernatant was removed, filtered and assayed in rat and human cells. Whereas the S+L- cells alone produced no infectious MSV, the cocultivated cultures produced up to 200 FFU/ml (Table 4) of MSV which were susceptible to neutralization by antiNZB-MuLV serum (data not shown). Since the MSV rescue requires cell to cell contact (Svoboda and Dourmashkin, 19691, it appears that the X-tropic MuLV produced by the dog thymus cells continued to replicate after cell fusion and provided the envelope for MSV. These results support the con-

elimination of all focus formation in HuF cells. The virus preparation was then inoculated onto NIH-ME and rat cells. Virus replication was quantitated by infectivity assays. Progeny ecotropic and xenotropic viruses were both measured in NRK cells. Selective evaluation of X-tropic virus replication was made in human cells. Assays on SC-l and NIH-ME demonstrated the amount of ecotropic virus present. The results of one of four experiments are given in Table 3. As expected, ecotropic virus replication was most efficient in mouse cells which produced titers of virus 2 logs higher than rat cells. In all of these host cells, but particularly in the was mouse, ecotropic virus replication more efficient than X-tropic virus replication. We have noted that X-tropic virus production occurs best in heterologous cells, particularly human cells, where titers as high as 10’ imectious particles/ml are obtained (Levy, 1975a). Nevertheless, in these experiments, xenotropic virus production was substantial in mouse cells (up to 4 logs), equalled that obtained in NRK cells, and was only slightly less than the yield from Fischer RE cells. The highest titer of endogenous X-tropic MuLV produced spontaneously by mouse cells has also ranged between lo3 and lo4 infectious particles/ml (Levy et al., 1975b). Therefore, the quantity of X-tropic viruses produced by mouse cells, both spontaneously and after exogenous infection via an ecotropic virus coat, (e.g., 4 logs) is lower than that of ecotropic MuLV in these same cells (-6 logs). These observations suggest that some intracellular block to X-tropic

TABLE Cell S+LS+LS+LS+LC57-D

NIH-ME

OF X-TROPIC

progeny

NRK

HuF

SC-l

NIH-ME

NRK

HuF

6.5

4.1

6.9

7.3

5.2

4.2

n An NZB xenotropic virus pseudotype of Harvey through NRK cells chronically infected with Gross culture was incubated for 30 min at room temperature was then inoculated onto NIH Swiss mouse embryo Seven-day supernatants from these cultures were (HuF), SC-l, and NIH-ME cells. Values for NRK, (log) per milliliter detected. Values for NIH-ME milliliter as measured by the XC cell assay.

4 BALBlc

S+L-

cultures

CELJ.S~ Foci

cells (P41) alone (P41) + C57L-D cells cells (P18) alone (P18) + C57-D cells cells alone

0 150 0 200 0

3

MuLV NRK

FROM

a S+Lcells (1 x lo51 at passages 18 and 41 were. mixed with 2 x IO5 canine sarcoma cells chronically infected with the C57 Leaden xenotropic virus (the C57L-D line). Six-day supernatants were removed, filtered, and assayed on NRK monolayer cells. Values represent the average number of foci on duplicate plates inoculated with 1 ml of the cocultivation culture supernatant.

TABLE REPLICATION

OF MSV

RESCUE

IN MOUSE

AND

RAT

progeny

CELLS” Fischer

RE progeny

SC-l

NIH-ME

NRK

HuF

SC-l

NIH-ME

5.2

5.4

5.2

4.5

5.1

4.7

mouse sarcoma virus [H-MSVCNZB-MuLV)l was passed murine leukemia virus. The 7-day supernatant from this in the presence of rabbit anti-NZB-MuLV serum. It (ME), rat (NRK), and Fischer rat embryo (RE cells. titered on duplicate plates of NRK, human foreskin HuF, and SC-l represent the titer of focus forming virus represent the titer of plaque forming units (log) per

XENOTROPIC

AND

ECOTROPIC

cept that the block to X-tropic virus replication in mouse cells is not complete. The data contrast somewhat with those of others who found that mouse-human cell hybrids did not replicate X-tropic virus (Gazdar et al., 1974; Scolnick and Parks, 1974). Host cell restriction of virus penetration was probably the reason for the lack of Xtropic virus production in these previous studies. Kinetics

of Phenotypic

Mixing

To determine the time interval from initial infection to release of phenotypically mixed viruses, quadruplicate plates of the clone 35 line of NZB embryo cells were inoculated with H-MSV (m.o.i. = 1). After 1 hr of virus adsorption, the cells were fed with fresh maintenance medium. Supernatants were removed from alternate cultures hourly for 13 hr and then daily for 7 days. These fluids were filtered and assayed on HuF cells. Results representative of five experiments are presented in Fig. 3. One to four infectious xenotropic virus pseudotypes of MSV were detected as early as 1 hr after virus adsorption (or 2 hr after virus inoculation) and in every hourly sample up to 11 hr. At 13 hr, 42 infectious X-tropic pseudotypes of MSV particles were detected. The X-tropic MSV yield reached its peak by 48 hr with a titer of 104.6 infectious particles/ml. Ecotropic virus was also present in the supernatant throughout the time period. As expected, its quantity dropped soon after virus inoculation when the input virus was absorbed or inactivated. Ecotropic progeny virus production reached its highest level at 4872 hr (Fig. 3) when 106.3 infectious particles/ml were detected. Similar results on X-tropic virus production were noted in experiments in which the cell cultures were washed twice before refeeding them with fresh maintenance medium. Moreover, in one experiment in which the cells were trypsinized, washed, and replated 1 hr after virus inoculation, a xenotropic pseudotype of MSV was detected at 3 hr. H-MSV alone did not induce focus formation in human cells, and mixing NZBMuLV with H-MSV did not lead to infection of HuF with MSV.

803

PSEUDOTYPES

Alteration MuLV

in Host

Range

of Ecotropic

Phenotypic mixing of MuLV was further demonstrated by passing AKR-MuLV in NZB-ME cells. Mouse cells are up to 3 logs more sensitive to AKR-MuLV infection than rat cells (Table 5). After inoculating AKR-MuLV in NZB-ME cells, the 7-day supernatant from the culture was assayed on NRK and NIH-ME cells. The titer of recovered virus, measured by the XC plaque assay, was now threefold higher in rat cells than in mouse cells (Table 5). This result suggested that parental and phenotypically mixed MuLV were present in the supernatant. Since NZB-MuLV cannot directly infect mouse cells, the AKR-MuLV particles in the NZB-MuLV envelope, AKR-MuLV(NZB-MuLV), were blocked from infecting these cells. Nevertheless, those same AKR-MuLV(NZB-MuLV) particles could infect rat cells efficiently and gave rise to detectable progeny AKR-

-SC-I d-4 -.-a

2

4

6 6 IO 12 14 HOURS

TIME

AFTER

VIRUS

NRK HuF

1234567 DAYS

INOCULATION

FIG. 3. Kinetics of phenotypic mixing. The ecotropic Harvey strain of murine sarcoma virus (MSV) was inoculated at a multiplicity of 1 into quadruplicate plates of NZB-ME cells producing approximately 10’ infectious NZB-MuLV/ml. Supernatants were collected at the times specified and assay for infectious virus by focus formation in tissue culture. The titer in human foreskin cells (HuF) represents xenotropic MSV, that in SC-1 represents primarily ecotropic MSV, and that in NRK represents both xenotropic and ecotropic MSV. Titers are expressed as focus forming units (FFU) per milliliter.

a04

JAY A. LEVY TABLE

5

INFECTIVITY OF ECOTROPIC MURINE VIRUS A~-~ER PASSAGE THROUGH

Virus

LEUKEMIA NZB CELLS=

Mouse 10=

Rat

AKR-MuLV 10= 10s.’ 105.’ AKR-MuLV(NZB-MuLV)* u AKR murine leukemia virus (AKR-MuLV) with a titer of W5 plaque forming units (PFU)/ml in mouse and W5 PFUlml in rat cells was inoculated at a 1:lO dilution onto NZB cells. The quantity of AKR-MuLV in a ‘I-day supernatant from that culture was measured by XC plaque formation in NIH Swiss mouse and NRK rat cells. The titer per milliliter is given. b AKR-MuLV passed once through NZB mouse embryo cells.

MuLV which induced the XC plaque formation. The presence of these phenotypitally mixed ecotropic viruses was confirmed by neutralization tests. Rabbit anti-NZB-MuLV serum reduced plaque formation in rat cells considerably, and only anti-AKR-MuLV serum significantly suppressed XC plaque formation in NIHME. Effect of Phenotypic Assays

Mixing

on XC Plaque

Pincus et al., (1971) demonstrated that NZB cells are of the N type but are less sensitive for detection of the N-tropic subgroup of ecotropic MuLV by the XC plaque assay than other N-type cells. NZB cells make X-tropic MuLV which does not induce XC plaque formation in tissue culture (Levy and Pincus, 1970; Levy, 1973, 1974). We wondered if the reduced sensitivity of NZB cells in XC plaque assays results from the phenotypic mixing of the viruses since ecotropic virus in a xenotropic coat would not be able to induce the syncytial formation. To examine this possibility, we titered ecotropic MuLV on NZB cell lines derived from two clones of the NZB-Q line: clone 4 and clone 35. These NZB clonal cell lines differ in the yeild of NZB-MuLV (Levy et al., 1975b). Clone 4 produces approximately 100 infectious NZB-MuLV/ml, whereas clone 35 produces over 10,000 infectious particles/ ml. If phenotypic mixing reduced XC plaque formation, we would expect clone

35 to be less sensitive to XC plaque formation than clone 4. When ecotropic MuLV was titered on clone 4, no significant difference in titer from control cultures was detected, but a reduction in its titer was noted in clone 35 (Table 6). The kinetics of virus infection were also different for the two clones. Clone 4 showed one-hit kinetics, whereas clone 35 had variable patterns. Moreover, some XC plaques were smaller in clone 35 cells than in clone 4 cells. Both clones were free of mycoplasma infection, and did not make detectable levels of interferon (experiments performed by Ms. M. Debby, San Francisco). In addition, both clones produced an equal quantity of the type C virus inhibitor associated with NZB cells (Levy, 1975b). These experiments then are consistent with a role for phenotypic mixing in reduced XC plaque efficiency in NZB cells. Infection of Heterologous Cells with XTropic Virus Pseudotypes ofEcotropic MuL V

The experiments illustrated in Figs. 1 and 2 indicate that ecotropic MuLV and the ecotropic pseudotype of MSV in xenotropic virus envelopes can infect cells previously resistant to these viruses. Further evidence of this fact was obtained by inoculating supernatants from NZB embryo TABLE TITER

6 NIH SWISS AND CELLS” Clone 35 Clone 4

OF ECOTROPIC VIRUSES IN NZB MOUSE EMBRYO

Viru;i;rculaR-MuLV AKR-MuLV

NIH-ME

6.3 5.7 6.3 6.0 4.9 5.8 D Secondary NIH Swiss mouse embryo (ME) cells and NZB mouse cells were infected with varying dilutions of standard ecotropic viruses. The viruses were detected by the XC plaque assay (Rowe et al., 19701. The numbers given represent an average of the virus titers (log PFU per milliliter) at several dilutions of each virus preparation. Clone 4 produces approximately 100 infectious NZB-MuLV particles/ ml. Clone 35 produces 10,000 ‘NZB-MuLV particles/ ml. The NIH Swiss mouse embryo cell line had no detectable MuLV. b R-MuLV, AKR-MuLV: Rauscher and AKR murine type C viruses, respectively.

XENOTROPIC

AND

ECOTROPIC

cells infected with AKR-MuLV onto cells from different animal species. Successful infection of these cells by the ecotropic virus was demonstrated by the presence of AKR-MuLV in the supernatants of the infected cells as detected by XC plaque formation in NIH-ME. As illustrated in Table 7, duck, gazelle, human, marmoset, mink, and mongoose cells were resistant to infection by AKR-MuLV. However, these same host cells, inoculated with the AKR-MuLV(NZB-MuLV) virus preparation, produced progeny AKR-MuLV which was detected by induction of XC plaques in NIH-ME. Moreover, direct XC assays on these heterologous cell lines showed some syncytial cells (see Table 8). Preparations of AKR-MuLV (NZB-MuLV) contained lo3 to lo4 infectious phenotypically mixed particles as measured by end-dilution on heterologous cells. This number correlates with the titer of X-tropic MuLV (103-lo4 infectious particles/ml) spontaneously produced by these NZB mouse embryo cells (Levy et al., 197513). Neutralization tests provided further evidence that this production of AKRMuLV by heterologous cells resulted from infection with an NZB virus pseudotype of MuLV. When the ARK-MuLV (NZBMuLV) preparation alone or after incubation with anti-AKR-MuLV serum was added to gazelle cells, production of AKRMuLV was noted. Gazelle cells receiving the virus incubated with anti-X-tropic virus serum, however, yielded no progeny AKR-MuLV (Table 8). Infection of NIHME cells, as expected, was blocked by the anti-AKR-MuLV serum (Table 8). We have used this assay on heterologous cells to examine culture fluids containing

large quantities of ecotropic MuLV for the presence of low titers of X-tropic MuLV. Supernatants from the cultures are inoculated first onto mink cells. After 7 days, the fluids from these mink cultures are assayed for ecotropic MuLV on NIH-ME by the XC plaque technique. Since ecotropic virus can only infect mink cells in a xenotropic virus coat, the presence of any ecotropic MuLV in the mink culture supernatants would indicate that xenotropic MuLV is present in the original culture and participates in phenotypic mixing with the ecotropic MuLV. This procedure has been faster and more sensitive for TABLE

Virus

AKR-MuLV AKR-MuLVfNZB-MuLV)*

OF AKR-MuLV

AFTER

Virus

AKR-MuLV(NZB-MuLV) 1. Incubated with medium 2. Incubated with antiNZB-MuLV serum 3. Incubated with antiAKR-MuLV serum

>300

cells

NIHME’

Progeny virus

2

600

150

0

0

140

2

400

0

7 OF VARIOUS Progeny

0 25

Vmus

’ The AKR murine leukemia virus passed once through NZB cells [AKR-MuLV(NZB-MuLV)] was incubated with the antisera listed and inoculated on gazelle cells. Seven-day supematants of the gazelle cultures were assayed for AKR-MuLV on NIH-ME cells. Values indicate the average number of plaque forming units (PFU) per milliliter as measured on duplicate plates by the XC plaque assay. b Direct XC cell assay on gazelle cells. ’ The AKR-MuLV(NZB-MuLV) preparation assayed directly on NIH mouse embryo cells. Values are PFU per milliliter.

inoculated Gazelle

Gazelle XV

INFECTION

Duck

8

NEUTRALIZATION OF THE XENOTROPIC PSEUDOTYPE OF AKR-MuLV”

TABLE DETECTION

805

PSEUDOTYPES

0

ANIMAL

CELL

LINES

production”

Human

Marmoset

Mink

0 85

0 92

>500

a Seven-day supernatants from cell lines infected with each virus preparation were formation on NIH-ME cells. The numbers given represent the quantity of infectious 0.4 ml as determined in duplicate cultures. b AK&MuLV passed once through NZB embryo cells.

Mongoose 0

0 102

assayed for XC plaque AKR-MuLV particles/

806

JAY

A.

detection of X-tropic virus than inoculating the fluids on heterologous cells and testing for the virus by cocultivation procedures (Levy et al., 1975b). Numerous attempts to infect heterologous cells (e.g., gazelle, HuF, and DEF) simultaneously with high titers of X-tropic and ecotropic pseudotypes of MuLV have failed to yield progeny ecotropic virus. Likewise, simultaneous infection of mouse embryo cells with ecotropic MuLV and the xenotropic virus pseudotype of MSV has not resulted in infection with the MSV. These observations indicate that phenotypically mixed virus and not simultaneous infection by the two classes of MuLV is responsible for the above results. Phenotypic Mixing with Mouse and Rat Type C Viruses

When NRK-Harvey& cells were cultured in the presence of IUDR (100 pglml), a rat C-type virus pseudotype of MSV was produced which induced foci in NRK as well as in embryo cells derived from Fischer and Osborne-Mendel rats but not in NIHME, DEF, HuF, or mink cells. Replication in rat cells of this pseudotype of MSV however, was not readily detected although the cells were transformed. Progeny virus production only occurred after several transfers of the infected cells. A similar observation has been made by Klement et al. (1973). When the rat pseudotype sarcoma virus was inoculated onto NRK cells chronically infected with murine X-tropic or ecotropic MuLV, production of progeny sarcoma virus was markedly enhanced. The focus forming virus, emerging from the NRK cells producing AKR-MuLV, had the coat antigen of this mouse ecotropic virus and it readily infected mouse cells. DISCUSSION

Data presented in this paper illustrate the lack of interference between the xenotropic and ecotropic murine ‘type C viruses. Interference does exist, however, among members of the same class. This kind of specific interaction has been described for subgroups of avian and feline type C viruses (Rubin, 1960; Sarma and Log, 1971).

LEVY

The absence of virus interference permits the replication of X-tropic and ecotropic MuLV in the same cell. This event results in phenotypic alterations involving the antigenic coat and host range of the progeny viruses. Ecotropic virus may emerge with xenotropic virus characteristics and vice versa. Some particles may exchange other protein components of the virion or exist as mosaics. A genetic recombination between the MuLVs might also occur (Stephenson et al., 1974). Further studies are in progress to examine these possibilities. Recent data suggest that infection with ecotropic MuLV may activate endogenous X-tropic virus from some cells (Fischinger and Nomura, 1975) or enhance the endogenous production of the X-tropic virus (Levy et al., 1975b). These interactions would induce or increase phenotypic mixing of the progeny viruses. Because of phenotypic mixing, MuLV preparations may be antigenically homogenous but have different viral RNAs. In this situation, the inability to detect virus by serologic or host range studies is not conclusive. A phenotypically changed virus particle may be present which will only become apparent after infection of appropriate cells with the virus preparation and assay of the culture supernatant for virus progeny. This fact may explain the heterogeneity of the RNA content of virions observed in preparations of the Moloney murine leukemia-sarcoma complex (Manning et al., 1972; Bondurant et al., 1973). It militates for careful screening of a virus preparation before assuming it represents a homogeneous virus type. Phenotypic mixing probably accounts for the variability in focus formation with H-MSV in NZB-ME cells (Levy et al., 197513)since any NZB pseudotype of MSV produced could not subsequently spread in the NZB-ME to induce focus formation. Likewise, XC plaque formation in NZBME may be affected if a sufficient quantity of the progeny MuLV is phenotypically changed and carries the NZB virus envelope. Since NZB cells themselves vary in the quantity of NZB-MuLV produced, the degree of this phenotypic mixing after su-

XENOTFtOPIC

AND

ECOTROPIC

perinfection with ecotropic MuLV would depend on the extent of NZB-MuLV expression by the particular NZB cells used. This phenomenon was illustrated by the variance in sensitivity for AKR-MuLV detection in clones of NZB cells producing different levels of NZB-MuLV (Table 6). It is probably responsible for the low titers of ecotropic MuLV detected by Pincus et al. (1971) in their XC assays in NZB-ME. It may play a role in reducing the sensitivity of other mouse cells for ecotropic virus detection by the XC plaque assay if sufficient X-tropic viruses are being made by the indicator cells. Phenotypic mixing enables ecotropic MuLV to infect cells from a variety of different animals (Table 7) and could permit the spread of known oncogenic type C virus genomes to other species including human. It may be that these events play a role in evolution possibly by inducing somatic cell changes in addition to transformation. Likewise, ecotropic virus pseudotypes of X-tropic MuLV would permit infection of mouse cells by X-tropic virus. A critical question in these situations is whether phenotypic mixing permits viral RNA to enter previously resistant cells in such a way that viral genes are integrated into the cellular genome even without virus replication (Levy, 19761. These studies of phenotypic mixing have demonstrated the presence of selective intracellular regulating systems for type C viruses. Xenotropic viruses entering mouse cells either by cell fusion or via an ecotropic coat replicate up to 3 logs less efficiently than ecotropic virus. Ecotropic virus production in human and duck cells is 3 logs less than xenotropic virus production. These observations occurred even when host cells were coinfected by both classes of MuLV. Moreover, rat cells appear to have an intracellular block to replication of their own endogenous type C virus. They produce, however, both X-tropic and ecotropic MuLV in substantial quantities although the titer of MuLV rarely reaches 106 infectious particles/ml. The ability of X-tropic MuLV to replicate in mouse cells suggests that the predominant block in mouse cells to X-tropic viruses is

PSEUDOTYPES

807

at the surface or penetration level. Nevertheless, since spontaneous X-tropic MuLV production by mouse cells can be enhanced l-2 logs if the cells are superinfected by ecotropic MSV preparations (Levy et al., 1975b), the coinfection of mouse cells with G-MuLV in these studies may have contributed to the X-tropic virus replication in NIH-ME. The cell fusion results described in this paper with the BALB S+L- cells differ from our previous observations when NZB-ME cells were cocultivated with nonvirus-producing (NP) MSV-transformed BALB/3T3 cells (Levy et al., 1975b). The quantity of X-tropic MuLV. produced by the NZB cells used in these earlier experiments was 3 logs less than that made by the C57L-D cells. Moreover, the extent of transcription of the MSV in the NP BALB 3T3 cell line may be different than that in the S+L- cells. The present results support the concept that the intracellular block to X-tropic virus replication is not complete and can be overcome by a sufficient quantity of incoming virus. Natural cell fusion in mice may, in fact, be a mechanism by which X-tropic viruses can spread from cell to cell. The lack of spontaneous production of endogenous X-tropic virus by mouse cells also suggests that incoming and integrated viral genes respond to different kinds of host cell control. These intracellular regulations are reminiscent of the factors determining the N and B type of mouse cells (Hartley et al., 1970; Lilly and Pincus, 1973; Tennant et al., 1974) where a 2- to 3-log difference in sensitivity to infection by N- and B-tropic MuLV has been noted. Moreover, this Fv1 genetic control mechanism can be overcome by large quantities of incoming virus (Hartley et al., 1970; Pincus et al., 1975). The lack of interference between either class of MuLV and the rat type C virus illustrates the ease with which phenotypic mixing (and perhaps genotypic changes) may take place among type C viruses of other species. One can imagine the variety of viruses that could occur if a rat cell producing its endogenous type C virus were superinfected by both classes of MuLV. Our results with this ecotropic rat virus indicated that it was infectious for its

808

JAY

A.

host species as demonstrated by efficient focus formation in rat cells, an intracellular block prevented detectable progeny Similar results have virus production. been reported by Klement et al. (19’73). These observations suggest that presumed noninfectious viruses may be infectious for the host cell if virus penetration and not just replication is measured. Addition of MuLV to rat cells infected by the rat virus pseudotype of MSV enhanced the replication of MSV. This finding illustrates how phenotypic mixing among rodent type C viruses may increase their spread. Moreover, the efficient production of progeny MSV by rat cells chronically infected with MuLV suggests that type C viruses from heterologous species can serve as helpers for sarcoma genomes when replication of the original helper virus is blocked or reduced. Similar observations have been made with MuLV pseudotypes of RSV (Levy, 1977). Such a mechanism could explain the sudden appearance of MSV after passage of MuLV through rats (Harvey, 1964; Kirsten and Mayer, 1967). Finally, the results obtained by measuring the kinetics of phenotypic mixing using H-MSV have suggested that type C viruses can exchange their envelope prop erties as early as 1 hr after infection. Previous data have indicated that the time interval from MuLV infection to viral DNA and subsequent RNA production is 5-6 hr and that infectious virus is not recovered before 12-14 hr (Salzberg et al., 1973; Manning, J. S., unpublished observations). The possibility that the early infectious viral particles we observed represent incoming viral genes and not replicated ones warrants further studies. ACKNOWLEDGMENTS The author would like to thank Drs. Jo Ann Leong and Adeleine Hackett for helpful discussions on the manuscript. This research was supported by U.S. Public Health Service (NCI) Research Grant CA 13086. JAL is the recipient of Research Career Development Award No. 5 K04 CA 70990 from the National Cancer Institute.

AARONSON, J. (1970).

S. A., Murine

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