Sequence Flexibility in the Polytropic env gp70-Derived Region of the Membrane Glycoprotein (gp55) of Friend Spleen Focus-Forming Virus Affects Its Biological Activity

Abstract

We previously reported (N. Watanabe, M. Nishi, Y. Ikawa, and H. Amanuma, J. Virol. 65:132–137, 1991) that the mutant Friend spleen focus-forming virus (F-SFFV_MS), which encodes a mutant gp55 membrane glycoprotein with an ecotropic env gp70 sequence, was nonpathogenic. Here we injected the F-SFFV_MS–Friend murine leukemia virus (F-MuLV) clone 57 complex into newborn DBA/2 mice. We obtained four groups of pathogenic variant F-SFFV complexes, each showing a different degree of pathogenicity in adult mice and a different gp55 profile. Of these, group 1 variant F-SFFV was particularly interesting, because it was the most frequently obtained and because it produced doublet bands of gp55 (59 and 57 kDa), neither of which reacted with the nonecotropic gp70-specific monoclonal antibody, and because its DNA intermediate did not hybridize with the nonecotropic env-specific probe. Cloning and DNA sequence analysis of the env region of one isolate of the group 1 variant F-SFFV revealed that this virus consisted of two distinct F-SFFV genomes; one (clone 117) differed from the other (clone 118) due to the presence of a 39-bp in-frame deletion. Reconstitution to full-length F-SFFV genomes and a pathogenicity assay showed that each reconstituted F-SFFV was pathogenic, with clone 117 showing a higher degree of pathogenicity than clone 118. Both reconstituted F-SFFVs caused activation of the mouse erythropoietin receptor in the factor-independent cell proliferation assay, although much less efficiently than the wild-type polycythemia-inducing isolate F-SFFVp. Clone 118 produced a gp55 of 59 kDa, while clone 117 produced one of 57 kDa. Clone 118 had a substitution by the F-MuLV clone 57 gp70 sequence, indicating that it was derived from the F-SFFV_MS env gene by a homologous recombination with the F-MuLV clone 57 env gene. The site of the 39-bp deletion in clone 117 corresponded to the portion of the clone 118 sequence which was unique to the ecotropic env genes. These results indicated the importance for the biological activity of gp55 of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.

Friend spleen focus-forming virus (F-SFFV), a replication-defective mouse type C retrovirus contained in the Friend virus complex, causes an acute erythroleukemia in adult mice of susceptible strains in the presence of a helper virus, such as the replication-competent Friend murine leukemia virus (F-MuLV) (reviewed in references 8 and 23). F-SFFV does not encode a viral oncogene, but its defective env gene product, gp55, plays a critical role in inducing the disease. In vitro biochemical evidence demonstrated that gp55, a membrane glycoprotein encoded by the polycythemia-inducing isolate of F-SFFV (F-SFFVp), specifically binds to a mouse erythropoietin receptor (EPO-R) and activates it, causing mitogenic signal transduction in the absence of the natural ligand erythropoietin (EPO) (12). Since the EPO-R is expressed in the erythroid progenitor cells, and the interaction between EPO and EPO-R regulates the level of erythropoiesis (15), it is assumed that the continuous expression of F-SFFV gp55 results in an abnormal proliferation of these cells, leading to leukemic transformation after several additional cytogenetic changes (3).

gp55, although closely related to the Env protein of MuLV, is not incorporated into the retrovirus particles, but stays inside the cells, mainly in the rough endoplasmic reticulum membrane, with a small fraction of the molecules (3 to 5%) processed through the Golgi apparatus to the cytoplasmic membrane (6, 21, 24). Cell surface-localized gp55 is then shed from the cells, probably after proteolytic cleavage from the transmembrane domain (6, 20, 21).

There are three major differences between the primary structures of gp55 of F-SFFVp and the Env protein of ecotropic MuLV (1, 4, 31). These are, from the N terminus, a substitution by the polytropic (dualtropic) env gp70 sequence, a 585-bp deletion which eliminates the proteolytic cleavage site for gp70 and p15E, and a 6-bp duplication and a single base insertion which cause premature termination of translation and loss of a 34-amino-acid peptide at the C terminus. By constructing F-SFFV encoding a mutant gp55, analyzing its pathogenicity in vivo, and also obtaining spontaneous revertant F-SFFVs from the mutant F-SFFV, we demonstrated that each of these three structural differences is essential for the pathogenicity of gp55 (2, 27–29). We previously reported (28) that the constructed F-SFFV (F-SFFV_MS) encoding the mutant gp55, in which the polytropic gp70 sequence had been replaced by the ecotropic F-MuLV clone K-1 gp70 sequence, was nonpathogenic in adult mice. The polytropic gp70-derived sequence in gp55 appears to contain a binding site for EPO-R (32). The purpose of the present study was to obtain spontaneous revertants from mice neonatally injected with F-SFFV_MS and to analyze the structure of their gp55s, with the goal of pinpointing the sequence required for pathogenicity and activation of EPO-R. The results indicated the importance of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.

MATERIALS AND METHODS

Viruses and infection of mice.

Construction of the F-SFFV_MS genome DNA (pULSM) and an analysis of its env gene product as well as its pathogenicity in adult mice were described previously (28). In the present study, the MS-13 NIH 3T3 cell clone was used as nonproducer F-SFFV_MS DNA-transfected cells, instead of the MS-22 clone, which was used previously (28). Superinfection of the MS-13 cells with F-MuLV clone 57, recovery of the F-SFFV_MS–F-MuLV complex, and confirmation of the successful rescue were performed by methods previously described (2). The titer of F-MuLV in the complex was 3.3 × 10⁵ XC PFU/ml.

DBA/2J mice were purchased from Charles River Japan, Inc. Newborn mice were obtained by mating. Aliquots of about 0.1 ml of the F-SFFV_MS–F-MuLV complex were intraperitoneally injected into newborn mice. Six- to eight-week-old male mice were also used for pathogenicity assay. These mice were intravenously injected with 0.1 to 0.2 ml of the virus sample via the tail vein. At the indicated times, splenic enlargement and hematocrit values were determined. Cell-free spleen homogenates and sodium dodecyl sulfate (SDS) lysates of spleen cells were prepared as described previously (2), except that a mixture of protease inhibitors (Complete; Boehringer Mannheim) was used for preparing the spleen cell lysates.

Immunoblotting.

The immunoblotting procedures used to detect Env proteins present in cell lysates were the same as those described previously (2) except for the following elements. The proteins were electrophoretically transferred to a membrane filter with a buffer consisting of 0.25 M Tris and 1.87 M glycine and reacted with either the goat antiserum against Rauscher MuLV gp70 (obtained from the National Cancer Institute, Frederick, Md.) or the 7C10 rat anti-nonecotropic gp70 monoclonal antibody (30) (ascites fluid) (kindly provided by S. Ruscetti, National Cancer Institute). After extensive washing, the filter was treated with either horseradish peroxidase (HRP)-conjugated rabbit anti-goat immunoglobulin G (Bio-Rad) or HRP-conjugated goat anti-rat immunoglobulin G (TAGO, Inc.). The reacting bands were visualized with an enhanced chemiluminescence (ECL) reagent (Amersham) and by exposure to X-ray film.

Preparation of viral DNA intermediates and the Southern hybridization analysis.

A group 1 or group 4 pathogenic variant F-SFFV–F-MuLV complex contained in the spleen homogenate was allowed to expand by infection to NIH 3T3 cells in the presence of Polybrene (4 μg/ml). These viruses were then used to isolate unintegrated viral DNA intermediates. Specifically, NIH 3T3 cells were infected with the virus in the presence of Polybrene, and the Hirt supernatant (7) was prepared 24 h later. DNAs (10 μg each) in the Hirt supernatants were separated by electrophoresis in 0.7% agarose gels, blotted onto nylon membrane filters (Hybond-N⁺; Amersham), and hybridized with 10 ng of either the HRP-labeled FM or the HRP-labeled BE probe per ml at 42°C for 12 h in ECL Gold hybridization buffer (Amersham) containing 5% (wt/vol) blocking agent and 0.5 M NaCl. The filters were then washed twice with 0.5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0) containing 6 M urea and 0.4% SDS at 42°C for 20 min and twice with 2× SSC at room temperature for 5 min. The hybridized bands were visualized by soaking the filters in ECL reagent (Amersham) and by exposure to X-ray film. The FM probe was the 8.8-kb EcoRI fragment of the pFM plasmid that contained the full-length permuted F-MuLV (K-1) DNA (18); the BE probe was prepared by linearization by EcoRI of the pBE plasmid (3.6 kb), which contained the 5′ half of the env gene of the Friend mink cell focus-inducing virus (F-MCFV) (2). These probe DNAs were labeled with HRP with the ECL direct nucleic acid labeling system (Amersham) according to the manufacturer’s instructions.

PCR amplification and cloning of the env gene of F-SFFV_MSR1.

PCR amplification of the env gene of F-SFFV_MSR1 was performed with the Hirt supernatant DNA as a template and the following primers: 5′-TTACGGCCGCTCTCAAAGTAGGATC-3′ (sense primer) and 3′-GGTGGTCGATTTTGGTGATC-5′ (antisense primer). The sense primer included most of the BamHI site (double underline), which corresponded to the 5′ end of the ecotropic env sequence in the F-SFFV_MS genome (28) and an EagI site (single underline), which was added for the purpose of cloning the amplified DNA. This sense primer was chosen so as to not amplify the env gene of the associating F-MuLV DNA. The antisense primer corresponded to the sequence of the F-SFFV_MS genome between the termination codon for gp55 and the start of the 3′ long terminal repeat sequence. The reaction mixture (100 μl) contained 1 μg of template DNA, 50 μM (each) primer, 20 mM (each) deoxynucleoside triphosphate, 1.25 mM MgCl₂, and 2.5 U of Taq DNA polymerase (Promega). Forty-five reaction cycles, consisting of denaturation at 94°C for 90 s, annealing at 55°C for 2 min, and elongation at 72°C for 2 min, were performed. The PCR product was subjected to electrophoresis in a 1.4% agarose gel, and the 1.5-kb fragment was isolated from the gel. This fragment was digested with EagI and BanIII and cloned into the pBluescript SK II(+) vector, which had been treated with the same enzymes.

DNA sequence analysis.

All plasmid DNA sequencing was conducted with the Exo/Mung DNA sequencing system (Toyobo, Osaka, Japan) and the BcaBEST dideoxy sequencing kit (Takara). Electrophoresis was run in 6% polyacrylamide gels containing 7.8 M urea.

Reconstitution and a pathogenicity assay of the cloned env genes of F-SFFV_MSR1.

Full-length F-SFFV genome DNAs were reconstituted from env clones 118 and 117. For this purpose, the sequence encoding the wild-type gp55 (EagI-BanIII fragment) in pBLSF was replaced by the corresponding sequence of the 118 or 117 env clone. The pBLSF plasmid contained the same full-length F-SFFVp (K-1) DNA insert as that in the pLSF4 plasmid (2) in the pBluescript vector. Isolation of transfectant NIH 3T3 clones expressing the reconstituted F-SFFV genomes, F-SFFV_MSR118 and F-SFFV_MSR117, preparation of rescued F-SFFV–F-MuLV complexes, and a pathogenicity assay were conducted by the methods described previously (2).

In vitro EPO-R activation assay.

A cell subline (BER28C) which expresses the mouse EPO-R and requires interleukin-3 (IL-3) or EPO for growth was established from IL-3-dependent mouse pro-B-lymphoid Ba/F3 cells (16) and will be described elsewhere. Ba/F3 or BER28C cells cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and IL-3 (1.2 × 10⁷ cells/0.8 ml) were transfected by electroporation (975 μF at 310 V) with a mixture of two linearized plasmid DNAs, 100 μg of a plasmid harboring F-SFFV DNA and 20 μg of pUCSVBSD encoding the blasticidin S (BS)-resistant gene (9). Two days after transfection, cells were subjected to growth selection in the presence of 15 μg of BS per ml for 10 days. A similar level of expression of gp55 in the BS-resistant cells was confirmed by subjecting the cell lysates to immunoblotting. A factor-independent cell proliferation assay was carried out as follows. Each BS-resistant cell population, with or without adaptation to medium containing EPO instead of IL-3, was plated in 24-well multiwell plates (0.4 ml/well) at cell densities ranging from 2.5 × 10⁵ to 2.5 × 10¹ cells/ml with medium containing neither EPO nor IL-3. Twenty-four wells were used for each density of cells. Five and ten days after the cells were plated, a number of wells containing growing cells was scored by microscopic observation. In addition, 5 days after the cells were plated, cells in 8 of 24 wells were separately harvested, washed to remove 2-mercaptoethanol, and replated. Twenty-four hours later, the relative number of growing cells in each well was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (17). Cells were incubated with MTT for 3 h.

RESULTS

Occurrence of spontaneous pathogenic variants in mice neonatally infected with the mutant F-SFFV_MS–F-MuLV complex.

More than 100 newborn DBA/2 mice were intraperitoneally injected with a preparation of F-SFFV_MS–F-MuLV complex, which had demonstrated no pathogenic effects when injected into adult DBA/2 mice (28). These mice were periodically (from 6 to 13 weeks postinfection) sacrificed and analyzed for splenomegaly and hematocrit. Of the 107 mice analyzed, about 60% (64 mice) showed an enlarged spleen with a wet weight of more than 0.2 g (Fig. 1A). These mice also demonstrated mild polycythemia (Fig. 1B). It appeared that the splenomegaly and polycythemia occurred transiently; peaks were observed at around 8 to 10 weeks postinfection. It should be noted, however, that only those mice having less severe splenomegaly could have survived for more than 10 weeks postinfection. Since the mouse strain used was DBA/2, injection of F-MuLV alone into the newborn mice did not cause splenomegaly or a change of hematocrit (data not shown) (22).

FIG. 1.

Splenomegaly (A) and polycythemia (B) observed in DBA/2J mice neonatally infected with the mutant F-SFFV_MS–F-MuLV complex. Each open circle represents an average value of samples analyzed, the number of which is indicated in the parenthesis. Vertical bars show the standard errors. Uninjected 6- to 9-week-old normal DBA/2J mice had 0.08 to 0.15 g (wet weight) of spleen and a hematocrit value of about 44%.

We considered two possibilities for the identity of viruses which caused the disease in these mice; one is that the mutant F-SFFV_MS might have been transiently pathogenic itself when it was injected into the newborn mice, and the other is that the mutant F-SFFV_MS was nonpathogenic in newborn mice and that pathogenic variant (PV) F-SFFVs occurred in these mice. To determine which was the case, we prepared spleen homogenates of the mice with splenomegaly and intravenously injected them into adult DBA/2 mice. If the virus present in the spleen homogenate was F-SFFV_MS, it would be nonpathogenic in adult mice. Of 30 different spleen homogenates examined, 29 caused splenomegaly and polycythemia in adult mice within 20 days postinfection, suggesting the presence of PV F-SFFVs (Table 1). Since the subsequent analysis confirmed the presence of PV F-SFFVs and showed that these could be divided into four groups (Fig. 2), data are grouped accordingly in this table. Obviously, the degree of pathogenicity was dependent on the group. A group 4 virus was almost as pathogenic as the wild-type F-SFFVp, whereas group 1 viruses, which were most frequently obtained (23 of 29 cases), showed the weakest pathogenicity; group 2 and group 3 viruses were intermediate.

TABLE 1.

Pathogenicity of the variant F-SFFVs obtained after neonatal passage of the mutant F-SFFV_MS–F-MuLV

Virus samplesa	No. of samples	Spleen wt (hematocrit value) for mice sacrificed atb:
		10 Days postinfection	20 Days postinfection
Control
Wild-type F-SFFVp– F-MuLV		1.20 ± 0.1 (49 ± 1)	2.23 ± 0.25 (73 ± 1)
F-SFFVMS–F-MuLV		0.09 (45 ± 1)	0.11 ± 0.02 (46 ± 3)
Variant F-SFFV–F-MuLV
Group 1	23	0.28 ± 0.02 (48 ± 1)	0.84 ± 0.07 (61 ± 1)
Group 2	3	0.39 ± 0.16 (52 ± 2)	1.06 ± 0.18 (65 ± 2)
Group 3	2	0.77 ± 0.19 (53 ± 2)	1.00 ± 0.07 (65 ± 6)
Group 4	1	0.23 ± 0.03 (44)	2.19 ± 0.15 (66 ± 2)

^aEach virus sample (0.2 ml) was intravenously injected into four 6-week-old male DBA/2J mice. 

^bThe spleen weights (expressed in grams) and hematocrit values are presented as averages ± standard errors. 

FIG. 2.

Immunoblotting detection of gp55 in the enlarged spleens of mice which had been injected with different groups of PV F-SFFVs 20 days before. Spleen cell lysates (10 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis (8% acrylamide), transferred to membrane filters, and probed with goat anti-Rauscher MuLV gp70 serum (A) or nonecotropic gp70-specific monoclonal antibody 7C10 (B). Reactive bands were visualized with an HRP-conjugated secondary antibody and ECL reagent and by exposure to X-ray film. As controls, lysates of NIH 3T3 cells (10 μg of protein) expressing either wild-type (wt F-SFFVp) or mutant (F-SFFV_MS) gp55 were used. Molecular mass standards are indicated (in kilodaltons) on the right. This figure depicts only the typical results, and all other spleen cell lysates of each group showed the same gp55 profile as that shown in this figure.

Preliminary molecular characterization of the PV F-SFFVs.

In order to confirm the presence of PV F-SFFVs, we examined the expression of gp55, which is a hallmark of F-SFFV. Spleen cell lysates were prepared from the 29 mice which showed splenomegaly and polycythemia 20 days postinfection (Table 1) and used for immunoblotting analysis to detect gp55. Figure 2A shows the typical results obtained by using goat anti-Rauscher MuLV gp70 serum, which has a broad immunospecificity against the MuLV envelope glycoproteins. Besides the F-MuLV gp70, all of the 29 spleen cell lysates showed either single or multiple gp55 bands, and four distinct groups of gp55 profiles were recognized (groups 1 to 4). No spleen cell lysates showed a single gp55 band with a molecular mass of 59 kDa, excluding the possibility that the mutant F-SFFV_MS itself had caused the disease.

When the same 29 spleen cell lysates were electrophoresed, transferred, and probed with nonecotropic gp70-specific monoclonal antibody 7C10, some of the gp55 bands also reacted with this antibody, indicating the presence of a nonecotropic gp70 sequence in these gp55 molecules, like the wild-type gp55 (Fig. 2B). This antibody did not react with the gp55 band of the mutant F-SFFV_MS, consistent with the fact that the nonecotropic (polytropic) gp70 sequence of the wild-type gp55 had been replaced by the ecotropic gp70 sequence in this mutant (28). Interestingly, the monoclonal antibody did not react with either of the doublet gp55 bands of the group 1 PVs detected by the goat antiserum.

We were interested in the group 1 and 4 PVs, since (i) group 1 PVs were by far the most frequently obtained and their doublet gp55s were unusual in that they did not react with the nonecotropic gp70-specific monoclonal antibody and (ii) the group 4 PV was very similar to the wild-type F-SFFVp, as judged by the degree of pathogenicity and the gp55 properties, such as molecular mass and immunological reactivity.

To know the env gene structures of group 1 and 4 PV F-SFFVs, viral DNA intermediates of the representative PV F-SFFV of either group 1 or 4 were isolated. Specifically, spleen homogenates were prepared from the adult mice which had developed splenomegaly (Table 1) and been examined for the presence of gp55 in enlarged spleen cells by immunoblotting (Fig. 2). Viruses present in these homogenates were allowed to expand by infection to NIH 3T3 cells and were then used to isolate unintegrated viral DNA intermediates in the Hirt supernatants. Viral DNA intermediates were detected by Southern hybridization with two different probes. Figure 3A shows the results obtained with the whole F-MuLV (K-1) DNA (FM) as a hybridization probe. This probe reacted with both the wild-type F-SFFVp and mutant F-SFFV_MS DNAs (data not shown). The viral DNA intermediates of the group 4 virus (lane 1) showed two reactive bands (8.8 and 6.0 kb), probably corresponding to the F-MuLV and the PV F-SFFV (designated F-SFFV_MSR4) linear DNA, respectively. Those of the group 1 virus also gave two bands (8.8 and 5.9 kb) (lane 2), the upper band being the F-MuLV linear DNA and the lower band being the PV F-SFFV (designated F-SFFV_MSR1) linear DNA.

FIG. 3.

Southern hybridization analysis of unintegrated viral DNA intermediates of group 1 and group 4 PV F-SFFVs. Group 1 and group 4 viruses in the spleen homogenates were allowed to expand, and their viral DNA intermediates were isolated in the Hirt supernatants 24 h after infection of NIH 3T3 cells. DNAs (10 μg each) of the Hirt supernatants were separated by 0.7% agarose gels, blotted onto nylon membrane filters, and hybridized with either the HRP-labeled FM (A) or the HRP-labeled BE probe (B). Hybridized bands were detected with the ECL reagent. Lane 1, Hirt supernatant DNA isolated from the group 4 virus-infected cells; lane 2, Hirt supernatant DNA isolated from the group 1 virus-infected cells.

A hybridization analysis was performed with the BE probe to determine whether these PV F-SFFV DNA intermediates contained a nonecotropic env sequence in their env regions (Fig. 3B). This probe, derived from the env gene of F-MCFV, reacted with the wild-type F-SFFVp DNA, but not with the mutant F-SFFV_MS DNA (data not shown). The F-SFFV_MSR4 DNA intermediate reacted with this probe (lane 1), indicating the presence of a nonecotropic env sequence, but that of the F-SFFV_MSR1 did not react with this probe (lane 2), like the F-MuLV DNA intermediate, indicating the absence of a nonecotropic env sequence. These results were consistent with the findings that the gp55 product of F-SFFV_MSR4 reacted with the nonecotropic gp70-specific monoclonal antibody, while those of F-SFFV_MSR1 did not, as described above.

Molecular cloning of the env region of F-SFFV_MSR1 and determining its nucleotide sequence.

To further characterize the F-SFFV_MSR1 genome, its env region was amplified by PCR with the Hirt supernatant DNA as a template. The PCR product, which appeared as a single 1.5-kb band, was cloned after digestion with EagI and BanIII. When recombinant plasmid DNAs were prepared from several clones and analyzed by BamHI digestion, we found that there were two types of clones differing slightly from each other in the size of the 0.9-kb BamHI fragment. Representative clones of both types were selected, clones 118 and 117, and their nucleotide sequences were determined.

Figure 4 shows the nucleotide sequence of clone 118, together with those of the F-SFFV_MS and F-MuLV clone 57 env genes. It is clear from this figure that the nucleotide sequence of clone 118 is very similar to the F-SFFV_MS env sequence. A long open reading frame uses the same translation initiation and termination codons as those used by the F-SFFV_MS env gene. Scattered differences in the nucleotides from the F-SFFV_MS env sequence were observed in the portion of the clone 118 sequence extending from nucleotide 296 to nucleotide 1171. A comparison of the sequence of this portion with that of the corresponding portion of the F-MuLV clone 57 env gene revealed that the sequences were identical. Since F-MuLV clone 57 was used as a helper virus to rescue F-SFFV_MS for in vivo mouse experiments, it is likely that the clone 118 env resulted from a homologous recombination between the env genes of F-SFFV_MS and F-MuLV clone 57. The 5′ recombination point could be between nucleotides 232 and 295, and the 3′ recombination point could be between nucleotides 1172 and 1175.

FIG. 4.

Alignment of nucleotide sequences in the env clone 118 and env genes of F-SFFV_MS and F-MuLV clone 57. The sequence of clone 118, from the EagI site to the BanIII site, is shown at the top. For the sequences of F-SFFV_MS and F-MuLV clone 57, only the nucleotides which differ from those of clone 118 are shown. Dots indicate the nucleotides identical to those of clone 118. The sequences of F-SFFV_MS and F-MuLV clone 57 are from published data (1, 10, 18). We determined the sequence of the F-SFFV_MS env gene in this study and confirmed the published results. 5′ Rec, 5′ recombination point; 3′ Rec, 3′ recombination point; DR, differential region; CR, constant region; ——, 5-bp direct repeats; ∗, 6-bp duplication and a single base insertion. A 585-bp sequence of the F-MuLV clone 57 env gene is omitted where indicated by an upward arrow. Other features of the sequences are indicated in the figure.

When the nucleotide sequence of clone 117 was compared with that of clone 118, it was readily recognizable that the former differs from the latter only by a 39-bp deletion, the location of which is indicated in Fig. 4 (from nucleotide 361 to nucleotide 399). The deletion is in frame and results in a loss of a 13-amino-acid peptide from the protein product encoded by the clone 118 env. Interestingly, 5-bp direct repeats (TCAGG) are found in the clone 118 sequence at sites corresponding to each end of the 39-bp deletion.

Protein product and pathogenicity of the cloned F-SFFV_MSR1 env regions.

Full-length F-SFFV genome DNA was constructed by substituting the env region of the wild-type F-SFFVp DNA with either a clone 118 or a clone 117 env region. The reconstituted F-SFFV DNA, designated F-SFFV_MSR118 and F-SFFV_MSR117, was introduced into NIH 3T3 cells, and the cell clones expressing gp55 were selected. Figure 5 shows the immunoblotting detection of gp55 of clones 118 (lane 5) and 117 (lane 6) with the goat anti-gp70 serum. Clones 118 and 117 produced single bands with molecular masses of 59 and 57 kDa, respectively, each corresponding to one of the doublet gp55 bands detected in the spleen cell lysate of the group 1 virus-infected mouse (lane 4). Neither the 59- nor the 57-kDa band was detectable when the nonecotropic gp70-specific monoclonal antibody was used (data not shown). These results clearly indicated that the group 1 PV F-SFFV was a mixture of two different F-SFFVs, each env region having given rise to clone 118 or 117. The difference in molecular mass between two gp55s (2 kDa) is consistent with the difference in the env nucleotide sequences of the two clones (39 bp).

FIG. 5.

Detection of gp55 of the reconstituted F-SFFVs, F-SFFV_MSR118 and F-SFFV_MSR117, by immunoblotting. Cell lysates (each 10 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis (8% acrylamide), transferred to membrane filters, and probed with the goat anti-Rauscher MuLV gp70 serum. Reactive bands were visualized with the HRP-conjugated secondary antibody and ECL reagent and by exposure to X-ray film. Lane 1, NIH 3T3 cells (negative control); lane 2, NIH 3T3 cells expressing the wild-type gp55; lane 3, NIH 3T3 cells (MS-13) expressing the mutant gp55 of F-SFFV_MS; lane 4, enlarged spleen cells of the mouse infected with the group 1 virus; lane 5, NIH 3T3 cells expressing gp55 of F-SFFV_MSR118; lane 6, NIH 3T3 cells expressing gp55 of F-SFFV_MSR117; lanes 7, 8, and 9, enlarged spleen cells of the mouse infected with the F-SFFV_MSR118–F-MuLV complex 10, 20, or 30 days before, respectively; lanes 10, 11, and 12, enlarged spleen cells of the mouse infected with the F-SFFV_MSR117–F-MuLV complex 10, 20, or 30 days before, respectively; lane 13, NIH 3T3 cells infected with the wild-type F-SFFVp–F-MuLV complex; lane 14, NIH 3T3 cells infected with the F-SFFV_MS–F-MuLV complex; lanes 15 and 16, NIH 3T3 cells infected with the F-SFFV_MSR118–F-MuLV complex which was obtained 9 or 16 days after the start of the rescue, respectively; lane 17, NIH 3T3 cells infected with the F-SFFV_MSR117–F-MuLV complex. Molecular mass standards are indicated (in kilodaltons) on the right. The F-SFFV_MSR118–F-MuLV complex obtained 9 days after the start of the rescue was used in the pathogenicity assay.

The reconstituted F-SFFVs were rescued by infection of NIH 3T3 nonproducer cells with a helper F-MuLV clone 57. Successful rescue was confirmed by analysis of the NIH 3T3 cells infected with a rescued virus complex (Fig. 5, lanes 15 through 17). The virus complex was then intravenously injected into adult DBA/2 mice, and splenomegaly and hematocrit value changes were monitored, as shown in Fig. 6. The F-SFFV_MSR117–F-MuLV complex was almost as pathogenic as the wild-type F-SFFVp–F-MuLV, although the splenomegaly caused by the former seemed transient and the polycythemia caused by the former was not as severe as that caused by the latter. The F-SFFV_MSR118–F-MuLV was also pathogenic, but at a lower level. We confirmed that the reconstituted F-SFFV itself caused the disease, since we could detect the same gp55 in the enlarged spleen cell lysate (Fig. 5, lanes 7 through 12) as that encoded by the F-SFFV which was injected into mice.

FIG. 6.

Pathogenicity of the F-SFFV_MSR118– and F-SFFV_MSR117–F-MuLV complexes in adult DBA/2J mice. Mice were intravenously injected with 0.2 ml of the virus samples (day 0) and periodically sacrificed, and spleen weights (A) and hematocrit values (B) were monitored. Virus samples used are as follows: ▴, wild-type F-SFFVp–F-MuLV; ▪, F-MuLV alone; □, F-SFFV_MS–F-MuLV; ○, F-SFFV_MSR118–F-MuLV; •, F-SFFV_MSR117–F-MuLV. Each point represents an average value of 2 to 5 mice, and a vertical bar shows the standard error.

EPO-R activation by gp55s of the clones 118 and 117.

Since the activation of EPO-R is an intrinsic property of the wild-type gp55, we examined whether the gp55s encoded by F-SFFV_MSR118 and F-SFFV_MSR117 show this activity. BER28C cells expressing gp55 were examined for growth in the absence of EPO. Results are shown in Table 2. The wild-type gp55 caused EPO-independent growth of BER28C cells, whereas the gp55 of F-SFFV_MS did not cause any. Both gp55s of the clones 118 and 117 also caused EPO-independent growth, although with much less efficiency than the wild-type gp55. Ba/F3 cells were not converted to being factor independent by the introduction of any F-SFFV genome DNA (data not shown). Measurement of the relative number of growing cells by the MTT method indicated that in the case of the BER28C cells expressing gp55 of the clone 118, the number of cells growing in the absence of EPO was only about 6% of that obtained after the BER28C cells expressing the wild-type gp55 were cultured in the absence of EPO (Table 2). In addition, gp55 of the clone 117 showed a weaker activity than gp55 of the clone 118 in this assay, contrasting with the results of the pathogenicity assay (Fig. 6). Possible reasons for the discrepancy between in vivo and in vitro results will be discussed below.

TABLE 2.

Factor-independent growth of BER28C cells expressing a different F-SFFV genome

F-SFFV genome	No. of wells in a 24-well plate with growing cells 5 (10) days after inoculation of the indicated no. of cellsa
	105	104b	103	102	101
None	0	0 [0.01]	0	0	0
F-SFFVMS	0	0 [0.01]	0	0	0
Wild-type F-SFFVp	24	24 [2.04]	24	24	24
F-SFFVMSR118	24 (24)	24 [0.13] (16)c	23 (23)	17 (14)	10 (8)
F-SFFVMSR117	6 (2)d	1 (1)d	0 (0)	0 (0)	0 (0)

^aData at 5 days postinoculation are shown, and those at 10 days postinoculation are in parentheses. 

^bCells in eight wells were separately measured for growth by the MTT method 5 days after inoculation. Average values of MTT reduction in optical density units are shown in brackets. 

^cA total of 16 wells were examined for growing cells. 

^dExpression of gp55 of the clone 117 was confirmed for these cells. 

Comparison of the deduced amino acid sequences of gp55 among F-SFFV_MS, env clones 118 and 117, and the wild-type F-SFFVp.

Since both the reconstituted env clones 118 and 117 were biologically active in vivo and in vitro, it is interesting to compare the amino acid sequences of gp55s of these clones with those of F-SFFV_MS and the wild-type F-SFFVp. Figure 7 shows alignment of the sequences, yielding the following conclusions. (i) The amino acid identity between the F-SFFV_MS gp55 and the wild-type gp55 is 31.1% in the differential region with gaps introduced. (ii) Twelve amino acids are different between gp55s of F-SFFV_MS and clone 118 in the differential region; four of these cause an increase in the identity score from 31.1% (F-SFFV_MS versus wild-type F-SFFVp) to 32.0% (clone 118 versus wild-type F-SFFVp). (iii) The 13-amino-acid deletion in clone 117 is located in the region unique to ecotropic env sequences, thus increasing the identity score to 36.2% (clone 117 versus F-SFFVp). This region is included in structural element I defined by Linder et al. (14).

FIG. 7.

Alignment of the deduced amino acid sequences of gp55 from F-SFFV_MS (1, 18), env clones 118 and 117, and the wild-type F-SFFVp (1). Amino acids are shown from the N terminus of the mature protein to the residue just N terminal to the site of the 195-amino-acid deletion. Single-letter amino acid symbols are used. Sequence alignment, including gaps, was according to Koch et al. (11). Positions of amino acid identity in all four sequences are indicated by shading. Structural elements I through III are from Linder et al. (14). ∗, amino acids identical with those of the F-SFFV_MS gp55; —, gaps; ↓, positions where the amino acids of the clone 118 gp55 are different from those of the F-SFFV_MS gp55 and the same as those of the wild-type gp55; DR, differential region; CR, constant region.

DISCUSSION

This study provided evidence that the polytropic MuLV env gp70-derived sequence is not essential for the biological activity of gp55 of F-SFFVp. Our previous studies (28) demonstrated that the mutant F-SFFVp (F-SFFV_MS), which encodes the ecotropic env-containing gp55, was nonpathogenic in adult mice. In the present study, we obtained a weakly pathogenic variant gp55 (env clone 118) which also has a substitution of an ecotropic env sequence. The former ecotropic env sequence was derived from the F-MuLV clone K-1 (18), while the latter was derived from the F-MuLV clone 57 (19). There are 12 amino acid differences between gp55s of F-SFFV_MS and the clone 118 in the differential region; these differences could account for the difference in biological activity. It should be noted that the 3 amino acid differences out of the 12, where the amino acids of the clone 118 gp55 are the same as those of the wild-type gp55, are clustered close (between amino acid residues 159 and 175 of the clone 118 sequence [Fig. 7]). This region is located between structural elements II and III. F-MuLV clone 57 is known to be highly erythroleukemogenic in newborn mice (19, 25), while clone K-1 is not (data not shown). This difference in pathogenicity of F-MuLV may correlate with the difference in pathogenicity of gp55 of F-SFFV. As shown in Fig. 5, lanes 7 through 9, a 59-kDa band was detected in the spleen cell lysates of mice showing mild splenomegaly 10, 20, and 30 days after injection of the F-SFFV_MSR118–F-MuLV complex, indicating that F-SFFV_MSR118 itself caused splenomegaly. In addition to the 59-kDa band, a very faint band with a molecular mass of about 57 kDa was observed (Fig. 5, lanes 7 through 9), raising the possibility that the splenomegaly and polycythemia were due to the presence of F-SFFV, which encoded this band. This 57-kDa band, however, is too faint to account for the degree of pathogenicity observed and, furthermore, the intensity of this band does not seem to increase at 20 (lane 8) and 30 (lane 9) days postinfection over that at 10 days postinfection (lane 7), a result which does not parallel the development of the disease (Fig. 6). We have not yet examined the gp55 profile after further serial passages of this virus complex through mice.

The more severe pathogenicity of the clone 117 env relative to that of the clone 118 env must be due to the presence of the 13-amino-acid deletion. The site of this deletion is in the region of the clone 118 sequence, which is unique to the ecotropic env genes. It is possible that the sequence of gp55 responsible for pathogenicity, and consequently for activation of EPO-R, resides in the regions of the MuLV env sequence, which are included in the differential region and contained in both polytropic and ecotropic env genes. For example, there is a 9-amino-acid sequence which is contained among gp55s of the clones 118 (amino acid residues 153 to 161) and 117 and the wild-type F-SFFVp (Fig. 7). F-SFFV_MS gp55 has one amino acid difference in this sequence: proline instead of serine at residue 159. It is worth noting that the 13-amino-acid deletion is likely to disrupt structural element I, which is thought to be important for receptor choice by the ecotropic envelope glycoprotein (14). Consequently, the clone 117 gp55 may not cause interference with the ecotropic MuLV receptor, while the clone 118 gp55 may do so. In fact, lanes 15 through 17 of Fig. 5 show more efficient rescue of F-SFFV_MSR117 from the cultured nonproducer NIH 3T3 cells by the ecotropic helper virus than F-SFFV_MSR118. More efficient rescue in vivo will cause a faster spread of the defective virus through spleen cells, contributing to the faster kinetics of the disease. At the same time, the 13-amino-acid deletion could be the cause of a weaker activity in the in vitro EPO-R activation assay. Due to the deletion, cellular processing of the clone 117 gp55 to the cell surface may be worse than that of the clone 118 gp55. Only those gp55 molecules processed to the cell surface are considered competent for activation of the EPO-R (5, 13, 26). To sum up, the 13-amino-acid deletion in the clone 117 gp55 likely plays dual roles, causing a decrease in the intrinsic biological activity of gp55 while favoring an increase in the titer of F-SFFV_MSR117 in vivo. Still, another factor may contribute to the pathogenicity of the clone 117 gp55. Coexpression of the F-MuLV clone 57 Env in the same cells may facilitate the processing of the clone 117 gp55 to the cell surface, thus complementing the defect in cellular processing and stimulating the erythroblastosis.

We isolated 23 independent group 1 PV F-SFFVs. All of them exhibited the same gp55 profile, indicating that they probably contain the same env genes as those represented by clones 118 and 117. Based on the nucleotide sequences, derivation of the clone 118 and 117 env genes may be as follows: the clone 118 env gene occurred first by a homologous recombination between the env genes of F-SFFV_MS and the helper F-MuLV clone 57, and then the clone 117 env gene appeared after the 39-bp deletion. Whether these modifications of the F-SFFV_MS env gene took place in the individual mouse or whether they occurred during the rescue from the nonproducer NIH 3T3 cells (MS-13 cells) by F-MuLV clone 57 is not known. Apparently, the F-SFFV_MS–F-MuLV complex used for injection into newborn mice did not contain the clone 117 env sequence, as evidenced by the absence of a detectable level of a 57-kDa band in the NIH 3T3 cells infected with this virus complex (Fig. 5, lane 14).

In general, a nonpathogenic retrovirus variant will be overwhelmed by pathogenic variants in tissue as a result of selective proliferation of the pathogenic variants due to the increased growth potential of cells infected with them. Consistent with this phenomenon, known as pathogenic selection, both of the surviving group 1 F-SFFV variants were found to be pathogenic. We found that an almost constant ratio of the intensities of the doublet gp55 bands was maintained (59 kDa:57 kDa ≅ 2:1) among the spleen cell lysates of mice that were infected with each of the 23 independent group 1 F-SFFV complexes. The ratio of band intensities was unchanged after a secondary passage of one of the group 1 F-SFFV complexes through adult mice (data not shown). Intensity of the band likely reflects the titer of the virus in the tissue. Whether the apparent stability of the group 1 PV F-SFFV complex can be implicated is presently not clear.

We cloned the env gene of F-SFFV_MSR4 by the same method as that used for cloning the F-SFFV_MSR1 env gene. As expected from the results of Southern hybridization (Fig. 3), nucleotide sequencing revealed that it had regained a whole differential region of a polytropic env gene, which was slightly different from that contained in the wild-type F-SFFVp (K-1) env, indicating that it is a true revertant (data not shown). Judging from the gp55 profiles, the group 2 and group 3 PVs probably contain several F-SFFVs. Further analysis of these groups of PVs has not yet been carried out.