Vol. 66, No. 9

JOURNAL OF VIROLOGY, Sept. 1992, p. 5696-5702

0022-538X/92/095696-07$02.00/0 Copyright X) 1992, American Society for Microbiology

Interference Established in Mice by Infection with Friend Murine Leukemia Virust THOMAS MITCHELL* AND REX RISSER McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Madison, Wisconsin 53706 Received 9 April 1992/Accepted 11 June 1992

Retroviral interference is manifested in chronically infected cells as a decrease in susceptibility to superinfection by virions using the same cellular receptor. The pattern of interference reflects the cellular receptor specificity of the chronically infecting retrovirus and is mediated by the viral envelope glycoprotein, which is postulated to bind competitively all cellular receptors available for viral attachment. We established retroviral interference in mice by infecting them with Friend murine leukemia virus and then measured susceptibility to superinfection by challenging the mice with the erythroproliferative spleen focus-forming virus. Infection of approximately 10lo of nucleated splenocytes rendered mice 1% as susceptible to superinfection as untreated controls. The magnitude of this effect was the same in mice incapable of producing neutralizing antibodies or genetically deficient for T cells. The results indicated that retroviral interference in vivo was established rapidly with infection of a fraction of the host cell population and that the decrease in susceptibility to superinfection occurred without a detectable contribution by immunologic factors.

Retroviral interference is associated with the chronic infection of susceptible host cells. It is manifested as a marked decrease in susceptibility to superinfection by retroviruses sharing the same subgroup of cellular receptors (34). Murine leukemia viruses (MuLV) in particular demonstrate interference patterns in vitro which reflect their distinct amphotropic, dualtropic, ecotropic, and xenotropic host ranges (12, 16, 31). Because the envelope glycoprotein (Env) of retroviruses determines host range by directing attachment of the virion to cellular receptors, the receptor binding specificity of this molecule has been postulated to result directly in retroviral interference by occupying all receptor attachment sites (14, 38, 39). This postulate is strongly supported by reports that env expression exclusive of other retroviral genes is sufficient to confer resistance to superinfection (4, 9) and that human immunodeficiency virus Env forms a stable complex (24) with its cellular receptor CD4 (40). Murine Fv-4 (41) and Rmcf (17) and the avian resistance alleles (29) are similarly associated with retroviral interference in vivo. Each of these loci encodes membrane glycoproteins related in sequence to the corresponding envelope glycoprotein of presumptive parental retroviruses (3, 5, 20, 32), which are speculated to have undergone deletion of the gag and pol coding regions concurrent with or subsequent to integration into the host genome (19). The Fv-4r allele protects mice from infection by the ecotropic spleen focusforming virus (SFFV) complex which induces a progressive erythroleukemia in susceptible mice (13). The gene assigned to Fv-4r has been cloned; characterization of its sequence established relatedness to ecotropic MuLV env (19). Cells cultured from BALB/c Fv-4' mice in vitro showed resistance to ecotropic MuLV infection (21), demonstrating that, at least in cultured cells, expression of the Fv-4 membrane glycoprotein was sufficient to establish interference. Moreover, Salter and Crittenden (37) generated a transgenic *

chicken line that resembled those carrying avian ev resistance alleles by introducing a defective avian leukosis virus genome into the germ line of avian leukosis virus-free chickens. After inoculation with replication-competent avian leukosis virus, one of the integrated genomes analyzed had undergone mutation such that env alone was expressed. Chickens harboring this provirus were resistant to further infection by avian leukosis virus in a subgroup-specific manner, which indicated that retroviral interference had been established. Thus, it is likely that the only viral gene product necessary to establish interference in vivo is Env. It is not known whether host immune factors contribute to this effect or whether transfer of a dominantly interfering gene such as env to somatic cells rather than to germ cells as a transgene is sufficient to protect recipients from viral disease. To characterize retroviral superinfection interference in vivo, we used a quantitative assay to measure retroviral interference in mice. The extent of retroviral interference is commonly measured in tissue culture by superinfecting chronically infected cells with a transforming retrovirus. The transforming activity of the challenge virus serves as a measure of the susceptibility or resistance of the target cells to superinfection. Interference can similarly be established and measured in mice susceptible to the spleen focusforming activity of SFFV. SFFV first induces expansion of the erythroid compartment of the blood cell system, which is followed by fully malignant leukemia. During the early stage of this disease, erythroid progenitor cells infected with SFFV rapidly proliferate to form macroscopic foci of transformed cells as early as 9 days after exposure to the virus (2). We used formation of these foci as an indicator of susceptibility to superinfection and analyzed the extent and specificity of interference established by infection with replication-competent MuLV in immunocompetent and genetically

immunodeficient mice. Retroviral interference established in vivo. We characterized retroviral interference in mice by infecting them with replication-competent Friend MuLV (F-MuLV) and then challenging them with infection with a complex of ecotropic

Corresponding author.

t Dedicated to the memory of Rex Risser. 5696

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TABLE 1. Retroviral interference in BALB/c mice Preinfection virus

% Virus-producing

splenocytesa

Challenge virus

0 0.09 ± 0.14 1.3 + 0.3

SFFV(F-MuLV)

Expt 1 None AM-MuLV F-MuLV

SFFV(F-MuLV)

SFFV(F-MuLV)

Relative'

In vivo titer" of SFFV

susceptibility

2.3 x 104 ± 0.6 x 104 1.8 x 104 + 0.6 x 104 2.9 x 102 ± 1.2 x 102

0.8 (P = 0.12)d 0.013 (P = 0.001)

1.0

Expt 2 1.0 2.6 x 102 ± 0.3 x 102 SFFV(AM-MuLV) 2.9 (P > 0.5)d 7.6 x 102 ± 1.6 x 102 SFFV(AM-MuLV) 35 (P = 0.006) -9 X 103 SFFV(AM-MuLV) a Determined by the S+L- (AM-MuLV) or the UV-XC (F-MuLV) infectious center assay. Uninfected cells showed no activity in either assay. NT, not tested. bDilutions of SFFV that produced 20 to 60 foci per spleen were preferentially used to determine the in vivo titer of SFFV(F-MuLV) or SFFV(AM-MuLV).

None AM-MuLV F-MuLV

NT 0.03 ± 0.02 1.6 ± 0.2

c Calculated by dividing the value for the in vivo titer of SFFV observed with preinfected mice by that observed with mice that had not been preinfected. d Statistical significance of the differences in susceptibility relative to control mice was determined by a one-sided Wilcox rank sum test.

or amphotropic MuLV and replication-defective SFFV. Adult male and female BALB/cBy and SWR mice 4 to 6 weeks old were obtained from Charles River Laboratories (Portland, Mich.) or were grown in our mouse colony at the McArdle Laboratory. CD1 nu/nu outbred mice 4 to 8 weeks old were obtained from Charles River Laboratories. Virus pools of F-MuLV clone 57 (28) were harvested as culture supernatants from chronically infected NIH 3T3 fibroblasts, and PFU were determined by the UV-XC plaque assay (33). Pools of amphotropic MuLV clone 4070A (15) were harvested from chronically infected NIH 3T3 cells, and focus-forming units (FFU) were determined by the mink S+L- transformation assay (30). Recombinant AM-MuLV (26) was obtained in proviral form as plasmid pAM (gift from D. Miller), which was transfected into NIH 3T3 cells by the calcium phosphate precipitation technique of Hopkins et al. (18). AM-MuLV pools were harvested after passaging transfected cells for at least 2 weeks, and titers were determined by the mink S+L- assay. Pseudotypes of polycythemic SFFVApL (35) were generated by infecting the SFFV nonproducer cell line Tf-2 (gift from S. Ruscetti) with F-MuLV, 4070A, or AM-MuLV at a multiplicity of infection of 1:10. Rescued SFFV pseudotype pools were harvested from infected cells after they had been passaged for at least 2 weeks. All virus stocks were collected as culture supernatants from cell cultures incubated for 24 h and were frozen at -70°C until use. To measure the extent of viral spread, spleen cells from infected mice were perfused in Dulbecco's modified Eagle medium supplemented with 10% calf serum (HyClone) and exposed to 3,000 rads in a 137CS irradiator. Serial log dilutions of infected cells were prepared in a final volume of 0.2 ml and added to NIH 3T3 cells for analysis in the UV-XC assay (F-MuLV-infected cells) or to mink lung S+L- cells in the S+L- assay (amphotropic MuLV-infected cells). Retroviral interference in BALB/c mice. BALB/c mice are fully permissive for F-MuLV infection (6, 10) and are susceptible to the spleen focus-forming activity of SFFV. Interference established in vivo should therefore be measurable in these mice by using SFFV as an indicator of susceptibility to superinfection. Ten BALB/c mice were preinfected with 2 x 105 XC PFU of F-MuLV and 12 were preinfected with 2 x 105 S+L- FFU of AM-MuLV. AMMuLV consists of ecotropic long terminal repeat, gag, and pol sequences derived from Moloney MuLV and amphotropic env sequences derived from MuLV 4070A. This NBtropic virus replicates in Fv-lb BALB/c mice far more

efficiently than N-tropic 4070A, and the gag-pol sequences are closely related to those of F-MuLV. Because AM-MuLV uses a cellular receptor distinct from that of F-MuLV to infect host cells, however, preinfection with this virus was not expected to establish interference with superinfecting SFFV(F-MuLV). Nine days after infection, one uninfected mouse, two AM-MuLV-infected mice, and four F-MuLVinfected mice were sacrificed to obtain splenocytes for the infectious center assay. On the same day, 6 F-MuLVinfected mice were challenged with an undiluted dose of SFFV(F-MuLV); 10 AM-MuLV-infected and 6 uninfected mice were challenged with serially diluted doses of SFFV(FMuLV), with 2 to 3 mice tested per dose. Nine days after SFFV(MuLV) challenge all mice were sacrificed, and macroscopic spleen foci were scored. The susceptibility of mice preinfected for 9 days with F-MuLV to the spleen focusforming activity of SFFV(F-MuLV) was reduced to approximately 1% of that of mice that had not been preinfected, while those infected with AM-MuLV showed no change (Table 1, experiment 1). The failure of AM-MuLV to protect mice from superinfection was inconclusive with respect to the specificity of interference, because the extent of AM-MuLV viral spread measured by the infectious center assay of the number of virus-producing cells at the time of SFFV challenge was roughly a factor of 10 lower than that of F-MuLV (Table 1). If the determination of the extent of virus spread was accurate, the failure of AM-MuLV to establish interference to SFFV(F-MuLV) could be explained by a relative failure to infect SFFV target cells and not by a specific failure to occupy all cellular receptors for SFFV(F-MuLV) after AMMuLV has infected the target cells. To determine whether the UV-XC and mink S+L- infectious center assays provided equally sensitive measures of the extent of virus spread, we performed the assays with cells chronically infected in vitro with either ecotropic or amphotropic MuLVs. Irradiated NIH 3T3 fibroblasts that produced F-MuLV or SFFV(F-MuLV) plated with an average efficiency of approximately 50% in the UV-XC plaqueforming assay, and AM-MuLV- or 4070A-producing cells plated with approximately 40% efficiency in the mink S+Lassay (data not shown). By this measure, the UV-XC and mink S+L- assays were equally sensitive tests for ecotropic and amphotropic virus-producing cells, respectively. The failure of AM-MuLV to spread in vivo to the extent that F-MuLV did was, therefore, not artifactual. Flow cytometric analysis of F-MuLV-infected splenocytes.

5698

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NOTES

The value for the number of F-MuLV-producing splenocytes shown in Table 1 suggested that only -1% of nucleated splenocytes became infected and expressed gp7O surface protein (SU) at the time of SFFV challenge. Because the susceptibility to superinfection by SFFV(F-MuLV) was decreased by 2 orders of magnitude in these mice rather than by just 1%, we concluded that either F-MuLV preferentially infected the cellular target for SFFV-induced transformation or the plating efficiency of splenocytes in the UV-XC infectious center assay was low. Indirect immunofluorescence quantified by flow cytometry was used to measure directly the number of gp70 SU' splenocytes in F-MuLV-infected mice (Fig. 1). Splenocytes explanted from the mice analyzed for Table 1 (experiment 1) or cultured mouse erythroleukemia (MEL) cells were pelleted, treated with erythrocyte lysis buffer (Sigma Chemical Co.), washed in phosphate-buffered saline (PBS), and incubated in a solution of 0.2% sodium azide to prevent antigen capping. To detect cell surface expression of gp70 SU, 106 cells per sample were stained in a 1:600 dilution of either goat anti-Rauscher-MuLV gp70 SU antiserum (National Cancer Institute) or normal goat serum, rinsed three times in PBS, stained with 12.5 ,ug of rabbit anti-goat immunoglobulin (Ig) antibody conjugated to fluorescein isothiocyanate (FITC) (Kirkegaard & Perry) per ml, and washed four times. Fluorescence intensity was determined with an Epics Profile II Cytometer (Coulter Corp.). For graphic presentation, cytometric datum points were converted to Apple computer text files and plotted by using Cricketgraph version 1.3.1. MEL cells produced a strongly fluorescent signal when incubated with goat anti-gp7O SU but not when incubated with normal goat serum (Fig. 1A). Splenocytes from AMMuLV-infected mice (Fig. 1B) or uninfected mice (data not shown) showed no relative increase in fluorescence intensity, whereas the splenocyte population from F-MuLVinfected mice was weakly positive for cell surface gp7O (Fig. 1C). The cytometric profile of F-MuLV-infected splenocytes showed a small but uniform shift in peak fluorescence, observed with all four F-MuLV-infected samples tested in this experiment and in eight other experiments. In comparison to the profile seen for MEL cells, this result indicated either that all of the cells were weakly positive or that few of the splenocytes were positive for cell surface Env. SRBC rosette formation. To quantify more clearly the subpopulation of F-MuLV-infected splenocytes that expressed gp7O SU, cell samples were also tested for sheep erythrocyte (SRBC) rosette formation. SRBC antibody conjugates were prepared by rinsing SRBC (Colorado Serum Co.) twice in 0.9% NaCl and twice in 0.35 M mannitol-0.01 M NaCl (MSS) before suspension to 0.5 ml at 10% (vol/vol) in MSS. Affinity-purified swine anti-goat Ig (Boehringer Mannheim) antibody, 100 ,ul at 1.25 mg/ml of MSS, was preincubated with 10 ,ul of 1-ethyl-3-(3-diethylaminopropyl)carbodiimide at 20 mg/ml of MSS for 2 min at room temperature and was added to the SRBC for 90 min on ice. The reaction was stopped by rinsing SRBC in PBS twice and suspending them to 1% (vol/vol) PBS. To detect cell surface gp7O SU, cells were prepared as for flow cytometry and incubated with a 1:2,000 dilution of goat anti-gp7O SU antiserum or normal goat serum, rinsed three times, and suspended in 1.0 ml of PBS. SRBC-antibody conjugate was added in a volume of 50 Ru to MEL cells at 2 x 105/0.2 ml or to splenocytes at 6 x 105/0.2 ml, pelleted, incubated on ice for 30 min, resuspended by pipetting, diluted 1:5, centrifuged onto microscope slides, and fixed in methanol. Cells bound to four or more SRBC were scored as positive for rosette

fluorescence FIG. 1. Flow cytometric profile of splenocytes stained for the presence of gp70jfV or surface Ig. Unfixed spleen cell samples (SPL) obtained from the mice tested for Table 1 or MEL cells were stained with goat anti-gp7O SU antiserum or normal goat serum (NGS) and then with rabbit anti-goat Ig-FITC to detect cell surface gp7O SU. (A) Fluorescent profile of MEL cells stained with NGS overlaid with a profile of cells stained with anti-gp7O SU; (B) overlaid profiles of AM-MuLV-infected SPL stained with NGS or anti-gp7O SU; (C) overlaid profiles of F-MuLV-infected SPL stained with NGS or anti-gp7O SU. Cell samples stained in parallel were also tested for cell surface gp7O by the SRBC rosette assay, with boxed values indicating the percentages of cells scored positive.

formation. Background values from negative controls were subtracted to determine the number of cells scored as positive for cell surface expression of gp7O SU. SRBC conjugated with swine anti-goat Ig antibody were incubated with MEL cells or with the cell samples used for Table 1 (experiment 1) after incubation with normal goat

VOL. 66, 1992

serum or goat anti-gp7O SU antiserum, to form rosettes (boxed values, Fig. 1). Ninety-two percent of the MEL cells scored positive for cell surface gp70 by this assay. Ten percent of the F-MuLV-infected splenocytes scored positive for gp70 SU relative to uninfected splenocytes stained with goat anti-gp7O SU. In contrast, only 0.1% of AM-MuLVinfected splenocytes scored positive. In two other experiments, 5 and 16% of splenocytes from F-MuLV-infected mice scored positive for gp70 SU, relative to controls (data not shown). The fidelity of the SRBC rosette assay was tested by enumerating rosette-forming cells bearing surface Ig and comparing values with those obtained by flow cytometric analysis. To detect surface Ig, cells were stained with 1.5 p,g of goat anti-mouse IgG(H+L)-FITC (Boehringer Mannheim) per ml and rinsed four times. The cell samples described previously were stained in parallel with 0.5 ,ug of goat anti-mouse Ig antibody per ml and incubated with SRBC conjugated to swine anti-goat Ig antibody. Two percent of MEL cells scored positive, whereas 34% ± 2.4% of splenocytes from F-MuLV-infected mice scored positive for cell surface Ig by SRBC rosette formation. This value compared favorably to the 31% + 5.9% of the same splenocyte populations which were determined by flow cytometry to express cell surface Ig (data not shown). MEL cells did not register as B cells when similarly stained and analyzed, and uninfected splenocytes showed a profile equivalent to those obtained from F-MuLV-infected mice (data not shown). These experiments showed that the enumerations of splenocytes bearing cell surface molecules by SRBC rosette formation were reliable and therefore that approximately 10% of the nucleated splenocytes present in mice 9 days after inoculation with F-MuLV bore gp7O SU at detectable levels. Time course of viral spread and of retroviral interference. To determine whether the level of retroviral interference reflected the duration of preinfection with F-MuLV, we analyzed the time course of the spread of F-MuLV and of susceptibility to SFFV(F-MuLV) (Fig. 2). BALB/c mice were preinfected with F-MuLV for 1, 4, 9, or 18 days prior to superinfection by SFFV(F-MuLV). Two mice were assayed at each time point for splenic infectious centers, and the remainder were challenged with serially diluted doses of SFFV(F-MuLV). Control mice were preinfected with AMMuLV for 18 days prior to challenge. Figure 2 (left axis, boxed squares) shows that the spread of F-MuLV was rapid: by day 4 of the preinfection period, the number of virusproducing splenocytes, as determined by the infectious center assay, had increased by 3 orders of magnitude. Correspondingly, the in vivo titer of SFFV(F-MuLV) was decreased to 2.4% of that in AM-MuLV-infected mice (Fig. 2, right axis, circled squares). The in vivo titer of SFFV(FMuLV) was decreased to 0.4 and 0.7% after 9 and 18 days of F-MuLV preinfection, respectively. These results were reproduced a total of four times for the 4-day time point and two times for the 1-, 9-, and 18-day time points, with values similar to those shown in Fig. 2. Because significant protection from superinfection was established as soon as viral production was detectable, and because the susceptibility of mice to SFFV(F-MuLV) after preinfection for 18 days was not lower than that of mice preinfected for 9 days, this experiment showed that the level of retroviral interference correlated with the number of F-MuLV-producing splenocytes and not with the duration of the preinfection period. Specificity of retroviral interference in vivo. The extent of AM-MuLV spread in infected mice was consistently lower than that of F-MuLV in the previous experiments. Without a

NOTES

5699

Days post-infection

FIG. 2. Time course of viral spread and of susceptibility to challenge infection by SFFV(F-MuLV). The graph depicts viral spread 1, 4, 9, and 18 days after inoculation with F-MuLV or 18 days with AM-MuLV pools, expressed as infectious centers per 106 nucleated splenocytes (left axis, boxed squares). Shown also is a representation of susceptibility to SFFV(F-MuLV) infection of mice inoculated with F-MuLV 1, 4, 9, or 18 days prior to challenge or with AM-MuLV 18 days prior, expressed as spleen FFU3 pe,r milliliter of SFFV pool (right axis, circled squares).

suitable control for the specificity of the cellular receptors, we could not demonstrate that the observed decrease in susceptibility to SFFV superinfection was specific for the pseudotype of the superinfecting virus, as might be expected for retroviral interference. To demonstrate that the resistance of F-MuLV-infected mice to superinfection by SFFV(F-MuLV) was dependent on the pseudotype of the challenging virus, we prepared amphotropic pseudotypes of SFFV and tested them in ecotropic or amphotropic MuLV-infected mice. Eight BALB/c mice were preinfected with 2 x 105 XC PFU of F-MuLV, 10 were preinfected with 2 x 105 S+L- FFU of AM-MuLV, and 9 were not infected. Seventeen days after infection, two AM-MuLV-infected mice and two F-MuLVinfected mice were sacrificed to obtain splenocytes for the infectious center assay. On the same day, the remaining mice were challenged with serially diluted doses of SFFV (AM-MuLV) in sets of two to three mice per dose. Nine days after challenge, all mice were sacrificed and macroscopic spleen foci were scored. Table 1 (experiment 2) shows that BALB/c mice preinfected with F-MuLV showed no decrease in susceptibility to spleen focus formation induced by SFFV(AM-MuLV). In fact, F-MuLV-infected mice were significantly more susceptible to the spleen focus-forming activity of SFFV(AM-MuLV), which may indicate that F-MuLV preinfection can increase the number of target cells for SFFV-induced focus formation. The failure of F-MuLV preinfection to reduce susceptibility to SFFV(AM-MuLV) indicated that reduced superinfection by SFFV after F-MuLV infection is dependent on the ecotropic pseudotype of SFFV. AM-MuLV preinfection did not reduce susceptibility to SFFV(AM-MuLV) challenge in this experiment. This result may indicate that the extent of AM-MuLV preinfection was insufficient to establish amphotropic retroviral interference

5700

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TABLE 2. Specificity of retroviral interference in SWR mice Preinfection

%

Virus-producing

titers' CalnevrsIn ofvivoSFFV

Relative'

virus

splenocytesa

Expt 1, part 1 None 4070A F-MuLV

NT 0.03 + 0.02 0.56 ± 0.05

SFFV(F-MuLV) SFFV(F-MuLV) SFFV(F-MuLV)

2.4 x 105 1.2 x 105 10

1.0 2.0 (P > 0.5)d 0.00004 (P = 0.005)

Expt 2, part 2 None 4070A F-MuLV

NT 0.03 ± 0.02 0.56 ± 0.05

SFFV(4070A) SFFV(4070A) SFFV(4070A)

1.9 x 104 4.7 x 104 8.5 x 104

1.0 2.5 (P > 0.05) 4.5 (P > 0.05)

Challenge virus

susceptibility

a Determined by the S+L- (4070A) or the UV-XC (F-MuLV) infectious center assay. NT, not tested. b Dilutions that produced 20 to 60 foci per spleen were preferentially used to determine the in vivo titer of SFFV(F-MuLV) or SFFV(4070A). c Calculated by dividing the value for the in vivo titer of SFFV observed with preinfected mice by that observed with mice that had not been preinfected. d Statistical significance of the differences in susceptibility relative to control mice was determined by a one-sided Wilcox rank sum test.

or that amphotropic retroviruses are incapable of establishing interference in vivo (see discussion below). Retroviral interference in SWR mice. SWR mice were analyzed to determine whether this pattern of interference was generalizable to mice other than BALB/c. Because the amphotopic virus 4070A is N-tropic and SWR mice are Fv-1n, this mouse strain was used to determine whether interference with amphotropic pseudotypes of SFFV could be established with the nonrecombinant progenitor of AMMuLV, 4070A. Twenty-eight SWR mice were preinfected with 2 x 105 S+L- FFU 4070A-MuLV, 20 were preinfected with 2 x 105 XC PFU of F-MuLV, and 14 were not infected. Seven days after infection, two 4070A-MuLV-infected mice and two F-MuLV-infected mice were sacrificed to obtain splenocytes for the infectious center assay. On the same day, the remaining mice were challenged with serially diluted doses of either SFFV(F-MuLV) or SFFV(4070A) in sets of two to three mice per dose. Nine days after challenge, all mice were sacrificed and macroscopic spleen foci were scored (Table 2). As before, F-MuLV preinfection signifi-

cantly reduced the spleen focus-forming activity of SFFV(FMuLV) but not of SFFV(4070A), and 4070A-MuLV preinfection did not reduce susceptibility to either SFFV(FMuLV) or SFFV(4070A). Retroviral interference in T-cell-deficient mice. Some strains of mice are resistant to the outgrowth of SFFVinfected cells. This response is mediated primarily by cytotoxic T cells (see discussion below) but could appear as retroviral interference in our system. We therefore tested the ability of F-MuLV to establish interference in outbred CD1 nulnu mice. Eight CD1 nu/nu mice were preinfected with 2 x 105 XC PFU of F-MuLV, eight were preinfected with 2 x 105 S+L- FFU of AM-MuLV, and six were not infected. Eighteen days after infection, two AM-MuLV-infected mice and two F-MuLV-infected mice were sacrificed to obtain splenocytes for the infectious center assay. On the same day, the remaining mice were challenged with serially diluted doses of SFFV(F-MuLV) in sets of two to three mice per dose. Nine days after challenge, all mice were sacrificed and macroscopic spleen foci were scored. CD1 nu/nu mice were 0.9% as susceptible to superinfection by SFFV(FMuLV) when preinfected with F-MuLV than when preinfected with AM-MuLV (data not shown). The degree of this response was similar to that observed with BALB/c mice. Thus, T-cell-mediated immune responses did not contribute detectably to the magnitude of the decrease in susceptibility we observed after establishing retroviral interference in mice by preinfection with F-MuLV.

In summary, we demonstrated that BALB/c mice infected with F-MuLV were approximately 1% as susceptible to superinfection by SFFV(F-MuLV) as uninfected controls, when measured by the spleen focus-forming activity of SFFV. Flow cytometric analysis of F-MuLV-infected splenocytes before superinfection demonstrated that the decrease in susceptibility correlated with weak expression of gp7O SU in nucleated spleen cells. Enumeration of gp70+ cells by SRBC rosette formation showed that 10% of these splenocytes were infected. Superinfection by SFFV(FMuLV) was reduced in BALB/c mice as early as 4 days after infection with F-MuLV and was also observed with T-celldeficient nu/nu mice 18 days after exposure to F-MuLV. Experiments with amphotropic Env pseudotypes of SFFV showed that preinfection by F-MuLV did not prevent superinfection by SFFV(AM-MuLV) in BALB/c mice or by SFFV(4070A) in SWR mice. We did not detect any decrease in susceptibility to superinfection by SFFV(AM-MuLV) or SFFV(4070A) when mice were preinfected with AM-MuLV or 4070A. The genome of SFFV encodes the virally derived oncogene gp55 (1, 36), which has been shown to induce the proliferation of cells expressing the receptor for erythropoietin (23). The target cell for this activity in the spleen is the erythroid progenitor BFU-e (for burst forming unit-erythroid) (22) and has been defined in tissue culture by the ability to form clusters of hemoglobinized cells (25). The resistance of F-MuLV-infected mice to SFFV(F-MuLV), but not to SFFV(AM-MuLV), challenge was consistent with the receptor saturation model for retroviral interference, which predicts that expression of ecotropic gp70flV in BFU-e would saturably occupy cellular ecotropic Env receptors and block attachment of SFFV virions coated with ecotropic Env. SFFV virions pseudotyped with amphotropic Env, however, would bind, enter, and stimulate the proliferation of BFU-e even if they expressed ecotropic Env. Mice inoculated with amphotropic MuLV showed no establishment of resistance to superinfection by SFFV(FMuLV), as expected, but the failure of AM-MuLV or 4070A to spread to the extent that F-MuLV did prevented us from concluding that this pattern was a reflection of retroviral interference. Murine antibodies raised against the transmembrane protein of ecotropic Env can cross-react with amphotropic Env (7), and so infection with amphotropic MuLV provided a useful negative control for possible immune responses which could have reduced the activity of SFFV(FMuLV) in amphotropic MuLV-infected mice. The receptor saturation model for retroviral interference

VOL. 66, 1992

Zlso predicts that spleen cells expressing amphotropic Env should be less susceptible to superinfection by SFFV virions bearing amphotropic Env. We did not detect any reduction in spleen focus-forming activity of SFFV (amphotropic) after preinfection with either AM-MuLV in BALB/c mice or with 4070A-MuLV in SWR mice. Cultured cells chronically infected with amphotropic MuLV exhibit greatly reduced susceptibility to amphotropic MuLV challenge (for example, see reference 31), so it is clear that amphotropic viruses are capable of establishing interference. Because our preparations of both SFFV(AM-MuLV) and SFFV(4070A) induced spleen foci, it was not possible that BFU-e cells simply lack the cellular receptor for amphotropic Env. It was far more likely that amphotropic MuLV, while able to infect some fraction of BFU-e, failed to infect and express amphotropic Env in a number of BFU-e sufficient to establish measurable interference. The reduction in susceptibility to SFFV(F-MuLV) challenge in mice preinfected with F-MuLV could be explained by the production of neutralizing antibodies specific for ecotropic Env rather than by retroviral interference. BALB/c mice and other mice susceptible to Friend virus infection by virtue of harboring the sensitivity allele at the immune response locus Rfr-3, however, are known not to produce neutralizing antibodies to F-MuLV (6, 10), and in fact they remain viremic after F-MuLV infection until natural death. Humoral responses that normally clear virus from infected mice therefore could not have contributed to the prot,ection established in BALB/c mice after infection with F-MIuLV. The generation of neutralizing antibodies in strains of mice that are capable of such a response takes 15 to 20 days (27). Our observation that SFFV superinfection was reduced in BALB/c mice preinfected with F-MuLV for only 4 days further precluded the possibility that a humoral response could contribute to the magnitude of reduction in susceptibility to superinfection that we measured. Some strains of mice are susceptible to the initial proliferative activity of SFFV but eventually recover by suppressing infected cells with cytotoxic T-cell activity (8, 11). Again, BALB/c mice are fully susceptible to the proliferation and outgrowth of erythroid progenitor cells infected by SFFV. We have also shown that spleen focus formation by SFFV(F-MuLV) was reduced in T-cell-deficient nu/nu mice preinfected with F-MuLV to a degree similar to that observed with BALB/c mice. Thus, we measured a decrease in susceptibility to superinfection which was dependent only on retroviral interference. Establishment of interference that reduced susceptibility to superinfection to 1% in BALB/c mice was correlated with production of virus from only 1% of spleen cells at the time of challenge when measured by the infectious center assay. Analysis by SRBC rosette formation, however, showed that gp7O SU was expressed at the surface of approximately 10% of nucleated splenocytes. This discrepancy can best be explained by a low plating efficiency of primary spleen cells in the infectious center assay, although it was also possible that many more cells expressed gp7O SU than productively shed virus. More important, the significant resistance to SFFV(F-MuLV) challenge detected as few as 4 days after infection with F-MuLV must mean that not all spleen cells need to be infected to establish interference. At this time point, susceptibility to superinfection was reduced by a factor of 40, but the number of virus-producing cells increased by a factor of 10 from days 4 to 7 of infection. The true number of virus-producing cells present 4 days after infection could not therefore have exceeded 10% of the

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overall population. Taken together, these experiments showed that expression of Env in a fraction of the overall host tissue cell population resulted in a decrease in susceptibility to superinfection of up to 2 orders of magnitude. Retroviral interference established in cultured cells, in contrast, can reduce superinfection by 5 or more orders of magnitude (for example, see reference 31). It was not possible to determine whether we had achieved 100% infection of target cells such that the susceptibility of a given spleen cell was reduced on average to 1% of that of an untreated control or whether 99% of cells became infected and were resistant to the degree seen in cultured cells while 1% remained uninfected and were fully susceptible to infection by SFFV. In any event, the establishment of interference was strikingly efficient and apparently specific for the hematopoietic compartment targeted for transformation by SFFV, indicating that not all cells bearing receptors for viral attachment in vivo need be infected to protect a given subpopulation of cells from superinfection. Thanks are due to S. Ruscetti for cell line Tf-2; to D. Miller for plasmid pAM; and to B. Chesebro, W. Sugden, and H. Temin for comments on the manuscript. This work was supported by Public Health Service grant CA22443 and predoctoral training grant T32-CA09135 (to T.M.) from the National Institutes of Health.

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