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Journal of Virology, August 2006, p. 8069-8080, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00013-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of the Cytoplasmic Tail of Jaagsiekte Sheep Retrovirus Envelope Protein

Stacey Hull1,2 and Hung Fan1,2*

Cancer Research Institute,1 Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, California2

Received 3 January 2006/ Accepted 31 May 2006


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ABSTRACT
 
Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a transmissible lung cancer in sheep, ovine pulmonary adenocarcinoma. JSRV is unique in that the envelope protein functions as an oncogene, since it can morphologically transform fibroblast and epithelial cells in culture and can induce lung tumors in mice. Previous studies indicated that the transmembrane (TM) protein is essential for transformation, and particular attention has focused on a YXXM motif in the cytoplasmic tail. In this study, we carried out systematic mutagenesis of the cytoplasmic tail of JSRV Env. Alanine scanning mutagenesis revealed four classes of mutants: mutants in which transformation was abrogated, those in which transformation was not affected, those with reduced transformation, and those with increased transformation (supertransformers). In general, the alanine mutations did not affect Env protein production or its localization to the plasma membrane. Three functional domains of the cytoplasmic tail were identified: an amphipathic helix at the N-terminal (juxtamembrane) side, a nonessential C-terminal region, and an internal region (including the YXXM motif) where mutations resulted in abrogation, decreases, or increases in transformation. Alanine mutations in the amphipathic helix in both the hydrophobic and hydrophilic faces generally abolished transformation. The mutation R591A showed partial transformation that was consistent with loss of signaling through the Akt-mTOR pathway and signaling predominantly through the Ras-Raf-MEK1/2-extracellular signal-regulated kinase 1/2 pathway. The supertransforming mutants generally showed increased signaling through Akt and reduced activation of p38 MAPK that is inhibitory for transformation. These mutants provide further insight into the role of the TM cytoplasmic tail in JSRV transformation.


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INTRODUCTION
 
Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of ovine pulmonary adenocarcinoma, a contagious lung cancer of sheep (14). In vivo, secretory epithelial cells of the distal airways, type II pneumocytes and Clara cells, are the targets of transformation (21). Ovine pulmonary adenocarcinoma shares morphological similarities with a human lung cancer that is weakly associated with cigarette smoke, bronchioloalveolar carcinoma. Therefore, elucidating the mechanism(s) of oncogenesis by JSRV may provide insight into the development of a human epithelial cell lung cancer.

One of the unique features of JSRV is that the envelope (Env) protein is the viral oncogene. The Env protein alone can induce transformation of rodent and chicken fibroblasts and rodent epithelial cells in culture (2, 13, 17). JSRV and the Env protein can also induce rapid tumor formation in experimental lamb and mouse models, respectively (16, 26). However, the mechanism(s) by which the Env protein transforms cells is still not completely understood. The env gene is expressed as a polyprotein that is cleaved at the cell surface by a cellular protease to generate two proteins, surface (SU) and transmembrane (TM). While SU is responsible for receptor binding, TM spans the lipid bilayer of the viral membrane or the cellular plasma membrane and it anchors SU to the virion or cell via disulfide linkages. The TM protein is composed of three distinct regions: extracellular, membrane spanning, and cytoplasmic tail. We and others have previously reported that the cytoplasmic tail of JSRV TM is necessary to induce transformation of rodent fibroblasts (15).

The cytoplasmic tail of JSRV is only 46 amino acids and does not contain any evident enzymatic domains, such as that of a protein kinase. The cytoplasmic tail does have one tyrosine residue that is in the sequence YRNM (amino acids 590 to 593) (15). If the tyrosine were phosphorylated, this motif would match docking sites for either the p85 regulatory component of phosphatidylinositol 3-kinase (PI3K; YXXM) or growth factor receptor binding protein 2 (YXN) (24). Mutation of the tyrosine amino acid at position 590 (Y590) and methionine at position 593 (M593) abrogated transformation in NIH 3T3 fibroblasts, while mutation of the asparagine at position 592 (N592) did not, which suggested a role for the PI3K signaling pathway in Env-induced transformation (15). Consistent with the mutational analysis, JSRV-transformed fibroblast cells display constitutive phosphorylation (activation) of Akt/protein kinase B, a downstream target of PI3K, and Akt phosphorylation was sensitive to treatment with PI3K inhibitors (1). We subsequently observed that PI3K is not absolutely required for JSRV transformation since JSRV could transform cells in which PI3K function was blocked (12). Nevertheless, in these cells, constitutive phosphorylation of Akt was still observed. This suggests that JSRV Env can also induce PI3K-independent phosphorylation of Akt. Other groups have concluded that the tyrosine in the YXXM motif is also not essential for transformation, although transformation was greatly reduced upon mutation of Y590 (2, 10). This has raised the possibility that the cytoplasmic tail indirectly activates the PI3K/Akt signaling pathway. In addition, in primary and established chicken fibroblasts, both the YXXM motif and Akt phosphorylation are dispensable for transformation (27). We have suggested that multiple mechanisms and pathways may be involved in JSRV Env transformation and that the relative importance of particular pathways may depend on the species and cell type used in the assay (7).

Recently, we identified several signaling pathways that play key roles in transformation (11). Experiments performed with both rodent fibroblast and epithelial cells revealed that signaling through the H/N-Ras-MEK-MAPK and Akt-mTOR pathways was important for JSRV Env-induced transformation, with the H/N-Ras-MEK-MAPK pathway appearing to play a more critical role. In addition, the p38 MAPK pathway had a negative regulatory effect on transformation via regulation of MEK1/2 and extracellular signal-regulated kinase 1/2 (ERK1/2) activity.

Although the requirement for the cytoplasmic tail in JSRV Env-induced transformation is well established, there have been minimal mutational analyses beyond the YXXM motif to determine what regions or amino acids in the cytoplasmic tail are important for transformation. In this report, we performed a comprehensive mutational analysis to examine the role of each amino acid of the cytoplasmic tail in transformation of rodent fibroblast and epithelial cells. The results led to the identification of three functional subdomains with regard to transformation. Analysis of individual mutants provided insight into the transformation process.


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MATERIALS AND METHODS
 
Cells and transfection. Mouse NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). Human 293T, rat 208F, and rat RK3E cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin.

Transformation assays were performed as described previously (11, 13). Briefly, 2 x 106 NIH 3T3 or 208F cells were seeded in 10-cm dishes or 7.5 x 105 RK3E cells were seeded in 6-cm dishes. After overnight incubation, cells were transfected with 28 µg of plasmid DNA for 10-cm dishes (13) or 2.5 µg of DNA for 6-cm dishes (11) as described previously using the CalPhos mammalian transfection kit (Clontech) or FuGENE 6 transfection reagent (Roche). Cells were cultured for up to 4 weeks posttransfection with medium changes every 3 days. Cells were examined under phase-contrast microscopy at 3 to 4 weeks and scored for foci. Cell lines transformed by wild-type JSRV Env (pCMV3JS21{Delta}GP) or a mutant Env were derived from a single focus of RK3E cells transformed by the respective plasmid. To examine the effects of signaling inhibitors on focus formation, RK3E or 208F cells were subjected to FTI-277, U0126, SB202190, rapamycin, or PP2 (Calbiochem) with daily medium changes at the concentrations indicated.

Plasmid constructs. All Env mutation and truncation plasmids were constructed in the context of the wild-type JSRV Env expression plasmid pCMV3JS21{Delta}GP (13) or its Flag-tagged version (11) (Fig. 1). To construct the alanine scanning mutants, site-directed mutagenesis was used to convert each of the 46 amino acids (aa) of the cytoplasmic tail (aa 570 to 615) to alanine. All other amino acid substitutions were also constructed by site-directed mutagenesis methods. To create the cytoplasmic tail truncation plasmids, PCR products were generated that deleted 3, 6, 9, or 12 amino acids from the C terminus.


Figure 1
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  		FIG. 1. Expression plasmids used. Diagrams of the expression plasmids used in these experiments are shown. JSRV Env (pCMV3JS21{Delta}GP) expresses wild-type JSRV Env from a cytomegalovirus (CMV) promoter (13), and the Flag-tagged version is also shown. Wild-type JSRV Env was derived from a full-length JSRV provirus driven by the CMV promoter in place of the upstream viral U3 enhancer/promoter sequences; an internal deletion removed the coding sequences for all genes except env. SU and TM indicate domains of the Env gene; the JSRV long terminal repeat (LTR) is at the 3' end of these constructs, and the R and U5 regions of the LTR are also present at the 5' end. The organization of GFP expression plasmids is also shown. pEGFP contains the enhanced GFP protein driven by the CMV promoter. The 18-aa amphipathic helix from JSRV (aa 570 to 587) was cloned in frame and upstream of EGFP in amphipathic helix-GFP; Gray-shaded boxes, hydrophobic residues; diagonal striped boxes, hydrophilic residues. Variants of these plasmids, in which the hydrophobic residues were mutated (hydrophilic-GFP) and in which the hydrophilic residues were mutated (hydrophobic-GFP), are also shown.

To create the amphipathic helix-GFP reporter plasmids (Fig. 1), sense and antisense oligomers encoding the first 18 amino acids of the cytoplasmic tail were synthesized, annealed, and inserted upstream and in frame with the enhanced green fluorescent protein (EGFP) gene in pEGFP-N1 (Clontech) at BamHI and HindIII sites. To disrupt the amphipathic helix, oligomers were synthesized that mutated either hydrophobic residues to aspartate (hydrophilic-GFP) or hydrophilic residues to proline (hydrophobic-GFP).

All plasmids were confirmed by DNA sequencing.

Immunofluorescence assays. To analyze the localization of Env protein in transfected 208F cells, indirect immunofluorescence assays were performed. Cells were seeded at 7.5 x 104 in four-well chamber slides. After overnight incubation, cells were transfected with 1 µg of plasmid DNA by using Lipofectamine 2000 (Invitrogen). At 48 h posttransfection, cells were washed with phosphate-buffered saline (PBS), dried, and fixed with acetone. Fixed cells were incubated with polyclonal antibody to Flag tag (5 µg/ml; Sigma) for 30 min at 37°C. After being washed with PBS, cells were incubated with goat anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate (Molecular Probes) for 30 min at 37°C. After being washed with PBS, cells were covered with VectaShield (Vector Laboratories, Burlingame, CA) plus DAPI (4',6'-diamidino-2-phenylindole) and observed using a fluorescent microscope.

GFP localization and mitochondrial staining. To analyze the localization of GFP fusion protein in transfected 208F cells, cells were seeded at 7.5 x 104 in four-well chamber slides. After overnight incubation, cells were transfected with 1 µg of plasmid DNA by using Lipofectamine 2000 (Invitrogen). At 48 h posttransfection, live cells were stained with MitoTracker Red 580 (200 nM; Molecular Probes) in growth medium for 45 min at 37°C. After being washed with fresh growth medium, cells were fixed with growth medium containing 3.7% formaldehyde for 15 min at 37°C. After being washed with PBS, cells were permeabilized with acetone to improve signal retention (as per the manufacturer's protocol). Cells were again washed with PBS and subsequently covered with VectaShield plus DAPI and observed using a fluorescent microscope.

Immunoblotting. Cell were lysed with radioimmunoprecipitation assay buffer (20 mM Tris [pH 8.0], 137 mM NaCl, 10% glycerol, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 2 mM EDTA, and complete protease inhibitor cocktail [Roche]) for 15 min. Protein samples (5 to 10 µg per sample) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis. An anti-Flag polyclonal antibody (Sigma) was used as the primary antibody to detect the Flag epitope. Antibodies against Akt, phospho-Akt (Ser473), p38, and phospho-p38 (Thr180/Tyr182) (Cell Signaling) were also used as primary antibodies. A goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Pierce) was used as a secondary antibody. Blots were visualized by the SuperSignal West Pico chemiluminescent substrate (Pierce).


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RESULTS
 
Transformation by mutant Env proteins. To further investigate the role of the cytoplasmic tail in JSRV Env-induced transformation, we performed alanine scanning mutagenesis on the complete 46-amino-acid cytoplasmic tail. Each mutant was subjected to triplicate transformation assays with three different cell lines: mouse NIH 3T3 fibroblasts, rat 208F fibroblasts, and rat RK3E kidney epithelial cells. Transformed foci were counted at 3 to 4 weeks posttransfection. All mutants exhibited similar transformation activities in all three cell lines tested. In Fig. 2, the results from RK3E cells are displayed for all mutants as the percentage of transformation compared to that of wild-type Env (JSRV Env). Of the 46 mutants, 17 had transformation activity that was abrogated, 13 had transformation activity less than that of the wild type, 12 had transformation activity similar to that of the wild type, and 4 had transformation activity greater than that of the wild type (supertransformers). The majority of mutants with abrogated transformation activity localized to the N-terminal half of the cytoplasmic tail, and mutants that displayed transformation activity similar to that of the wild type clustered at the C terminus. Mutants with less or more transformation activity than the wild type were scattered throughout the cytoplasmic tail. The supertransforming mutants were particularly interesting; in fact, at lower DNA inputs, their relative transformation efficiencies compared to that of wild-type Env were greater than the two- to fourfold observed under standard conditions. For instance, in 208F cells, the supertransforming mutant N592A showed three to four times more foci than wild-type Env at 15 days when 28 µg of DNA was used. In contrast, approximately 10-fold more foci were observed with the mutant than with the wild type by using 7 µg of DNA (data not shown). These results demonstrate that amino acids in the cytoplasmic tail outside of the YRNM motif can modulate Env-induced transformation of rodent fibroblast and epithelial cells.


Figure 2
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  		FIG. 2. Transformation efficiencies of cytoplasmic tail mutants. The transformation efficiencies in RK3E cells of alanine mutations at each of the residues in the cytoplasmic tail (CT) are shown relative to that of wild-type JSRV Env (JSRV Env; pCMV3JS21{Delta}GP). Standard assay conditions were used, as described in Materials and Methods. Each mutant was tested three times relative to wild-type Env, and the averages ± standard deviations for each mutant compared to the wild type are shown. Similar assays with NIH 3T3 and 208F fibroblasts gave comparable relative efficiencies for all of the mutants. Typical numbers of foci for wild-type JSRV Env ranged from 40 to 100 foci per plate (see Tables 1 to 5).

To determine whether differences in transformation activity were due to differences in protein production or localization of mutant Env proteins, several mutants from each transformation group were epitope tagged (Flag) to facilitate detection of the Env protein. Western blot analysis of transiently transfected 293T cells indicated that all of the mutant envelopes tested had similar levels of protein production compared to wild-type Env (Fig. 3). Indirect immunofluorescence assays revealed that mutant envelopes from each transformation group localized to the plasma membrane, similar to wild-type Env (Fig. 4). We also tested transformation activity of the Flag-tagged Env mutants. The results demonstrated that although the transforming ability of the Flag-tagged Env proteins was reduced compared to that of the nontagged proteins (ca. 50% activity), the relative efficiencies of transformation compared to that of tagged wild-type Env were not altered by the Flag tag in all three cell lines tested (data not shown). Thus, the different transformation activities for the mutant Env proteins were generally not due to variations in protein production or localization.


Figure 3
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  		FIG. 3. Expression of JSRV Env mutants. Selected Flag-tagged alanine mutation expression plasmids were transfected into human 293T cells, and cell lysates were prepared 48 h after transfection. Cell lysates were subjected to SDS-PAGE, followed by Western blotting with an anti-Flag polyclonal antibody as described in Materials and Methods. The region of the Western blot corresponding to the JSRV Env TM protein (37 kDa) is shown. The more slowly migrating bands correspond to uncleaved or partially cleaved Env polyproteins. The blots were stripped and reprobed with an antibody to ß-actin, as shown at the bottom of the figure, to monitor equal loading of cell extracts.


Figure 4
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  		FIG. 4. Membrane localization of Env mutants. Expression constructs for Flag-tagged mutant Env proteins were transfected into 208F cells, and 48 h later, the cells were fixed and stained with a primary anti-Flag and a fluorescein isothiocyanate (FITC)-tagged secondary antibody as described in Materials and Methods. Immunofluorescent microscopy of the cells is shown.

Role of mTOR and H/N-Ras in mutant Env transformation. In recent studies, we identified signaling through two pathways, H/N-Ras-Raf-MEK-mitogen-activated protein kinase (MAPK) and Akt-mTOR, to be important for JSRV Env-induced transformation, and we also found that p38 MAPK had a negative regulatory effect on transformation (Fig. 5) (11). Thus, inhibitors to all three pathways were used to determine whether selected Env mutants have altered signaling through these pathways, which might account for the differences observed in transformation activity.


Figure 5
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  		FIG. 5. Signal transduction in JSRV-transformed cells. Signal transduction pathways activated in JSRV-transformed cells are shown, as reported by Maeda et al. (11). Depending on the cell line, the relative importances of signaling through Akt-mTOR and through H/N-Ras are different, although in all cases, signaling through MEK1/2 is essential for transformation. In contrast, p38 negatively affects transformation. This figure is adapted from that of Maeda et al. (11).

We hypothesized that mutants with decreased transformation activity potentially might have reduced signaling through one of the two pathways important for Env-induced transformation, either H/N-Ras-Raf-MEK-MAPK or Akt-mTOR. Therefore, we determined the effects of rapamycin (inhibitor of mTOR) and FTI-277 (inhibitor of H/N Ras) on transformation in RK3E cells by two mutants that transform less efficiently than wild-type Env, R591A and H587A (Tables 1 and 2). As expected (11), transformation by wild-type Env was partially reduced by rapamycin and FTI-277. The R591A mutant displayed a slight decrease in transformation activity in response to rapamycin and a much larger decrease in response to FTI-277. Thus, transformation by R591A was highly dependent on the H/N-Ras-Raf-MEK-MAPK pathway and only slightly dependent on the Akt-mTOR pathway. R591 is part of the YRNM (YXXM) motif in the cytoplasmic tail, which has been suggested to be a potential docking site for the p85 regulatory subunit of PI3K. Thus, it is possible that R591A might disrupt the YXXM motif, which could reduce PI3K binding and signaling through the Akt-mTOR pathway.


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  		TABLE 1. Effect of rapamycin on transformation by JSRV Env mutants in RK3E cellsa


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  		TABLE 2. Effect of the H/N-Ras inhibitor FTI-277 on transformation by JSRV Env mutants in RK3E cellsa

In contrast, the H587A mutant showed minimal response to rapamycin or FTI-277 treatment. Thus, the ability of H587A to transform RK3E cells did not appear to be dependent on either the H/N-Ras-Raf-MEK-MAPK or Akt-mTOR pathway. This suggested that this mutant signals through another pathway(s) to facilitate transformation. To investigate the apparent lack of dependence on the Akt-mTOR pathway for the H587A mutant, we analyzed AKT phosphorylation (activation) in H587A-transformed cells. Western blot analysis of protein lysates from serum-starved RK3E cells transformed by H587A is shown in Fig. 6B. The blot was probed with either a phospho-Akt (Ser 473) or total Akt antibody, which was used as a loading control. As demonstrated previously (15), the levels of phosphorylated Akt in cells transformed by wild-type Env were increased compared to those in nontransformed RK3E cells. In contrast, the levels of Akt phosphorylation for H587A-transformed cells were similar to those for nontransformed cells. This result was consistent with H587A transformation not being dependent on the Akt-mTOR pathway.


Figure 6
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  		FIG. 6. p38 and AKT phosphorylation in transformed cells. Transformed foci from RK3E cells transformed by wild-type and mutant JSRV Env proteins were picked and purified. For comparison, RK3E cells stably transfected with the pcDNA3.1 plasmid were also isolated. Cells were grown in low-serum media, cell extracts were prepared and analyzed by SDS-PAGE, and Western blotting was performed with antibodies for either phospho-p38 (Thr180/Tyr182) and total p38 (A) or phospho-Akt (Ser 473) and total Akt (B). (A) Three independent wild-type JSRV Env transformants were studies (clones 1 to 3) in addition to two supertransformers, Q597A and H598A. (B) Two independent wild-type JSRV Env transformants were studied (clones 1 and 2). The H587A mutant has reduced transforming potential, while the other three mutants shown are supertransformers.

Role of p38 in mutant Env transformation. We also analyzed the effect of a p38 inhibitor, SB202190, on transformation by Env mutants with transformation efficiencies greater than that of wild-type Env (supertransformers; N592A, Q597A, H598A, and E607A). We hypothesized that these mutants may have increased transformation potential because they do not activate the p38 MAPK pathway as efficiently as wild-type Env; decreased activation of p38 could lead to increased signaling through the H/N-Ras-Raf-MEK-MAPK pathway and increased transformation. As shown in Table 3, transformation by the v-mos oncogene was unaffected by the p38 inhibitor SB202190, and transformation by wild-type JSRV Env increased 11- to 14-fold, as expected (11). In contrast, SB202190 increased transformation three- to sixfold for the four supertransformers. This twofold reduction in p38 inhibitor stimulation was consistent with these mutant envelopes activating the p38 MAPK pathway less efficiently than wild-type Env.


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  		TABLE 3. Effect of the p38 inhibitor SB202190 on transformation by JSRV Env mutants in RK3E cellsa

To confirm the results of inhibitor studies, we analyzed p38 phosphorylation in two supertransformed RK3E cell lines, Q597A and H598A. Protein lysates from serum-starved RK3E cells were probed with either a phospho-p38 antibody or a total p38 antibody that was used as a loading control. As expected, we observed an increase in phosphorylated p38 levels in cells transformed by wild-type Env compared to those in nontransformed RK3E cells (Fig. 6A). In contrast, phosphorylated p38 levels were reduced in the supertransformed cell lines. These results suggest that one mechanism by which these mutants are capable of supertransformation is by reduced activation of p38.

Role of MEK1/2 in mutant Env transformation. We previously reported that while inhibition of H/N-Ras results in a partial reduction of transformation activity for wild-type JSRV Env in RK3E cells, blocking the downstream effector MEK1/2 results in complete abrogation of transformation. These results suggested that the Env protein signals to MEK1/2 by H/N-Ras-dependent and -independent mechanisms and that activation of MEK1/2 is essential for Env-mediated transformation. It seemed possible that the supertransformers might additionally be utilizing a MEK1/2-independent pathway for transformation. To test this, we performed transformation assays in the presence or absence of the MEK1/2 inhibitor U0126. All supertransforming Env mutants were dependent on MEK1/2 for transformation (Table 4). Thus, transformation by the supertransforming JSRV Env mutants did not involve activation of MEK1/2-independent mechanisms.


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  		TABLE 4. Effect of the MEK1/2 inhibitor U0126 on transformation by JSRV Env mutants in RK3E cellsa

Increased activation of Akt by supertransformers. As described above, cells transformed by wild-type JSRV Env display constitutive phosphorylation (activation) of Akt (15), although whether this activation is PI3K dependent is unclear (12). To test whether the supertransformers displayed increased phosphorylation of Akt, protein lysates from serum-starved transformed cell lines were subjected to Western blot analysis with antibodies against phospho-Akt and total Akt (Fig. 6B). Cells transformed by Q597A, H598A, and E607A displayed increased activation of Akt compared to cells transformed by wild-type Env. These results were confirmed with a second set of cell lines that were cloned from a separate subculture (data not shown). This suggests that another mechanism by which the supertransformers facilitate increased transformation activity is by increased activation of Akt.

Transformation activity can be dependent on amino acid substitution. To determine whether the charge of the amino acid selected for the mutational analysis would alter transformation activity, we substituted aspartic acid (Asp; negative), lysine (Lys; positive), serine (Ser; uncharged polar), or isoleucine (Ile; polar) instead of alanine at four residues. One amino acid residue was selected from each transformation activity group identified in the alanine mutation: M574 (abrogated), M593 (decreased), Q597 (supertransformer), and N605 (similar). Results from triplicate transformation assays with NIH 3T3, 208F, and RK3E cells with both non-Flag- and Flag-tagged versions of mutant envelopes were comparable, and results of Western blot analysis with the Flag-tagged plasmids confirmed similar protein production levels between the wild-type and mutant envelopes (data not shown). The results of transformation assays with 208F cells are shown in Fig. 7. The transformation activity was not dependent on the amino acid substitution for positions 574 and 605. Substitution of Asp, Lys, or Ser for methionine at position 574 resulted in abrogated transformation activity similar to that of M574A, while substitution of Ile resulted in transformation activity that was greatly reduced (8% ± 1% [average ± standard deviation] of that of the wild type). At position 605, substitution of Asp, Lys, Ser, or Ile for asparagine resulted in transformation activity similar to that of wild-type Env. In contrast, transformation activity was dependent on the amino acid substitution at positions 597 and 593. For instance, substitution of Asp, Lys, or Ser for glutamine at position 597 resulted in transformation activity less than that of the wild type and substitution of Ile resulted in transformation activity similar to that of the wild type. Interestingly, substitution of the nonpolar Ile at position 597 resulted in the most efficient transformation activity among the four mutants, which correlated with the supertransforming phenotype of Q597A. Transformation activity was also dependent on amino acid substitution at position 593. Substitution of Asp, Lys, or Ser for the methionine residue at position 593 resulted in abrogated or greatly decreased transformation activity. In contrast, substitution of Ile resulted in a supertransforming phenotype.


Figure 7
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  		FIG. 7. Extended mutational analysis. Four residues in the JSRV TM cytoplasmic tail (CT) were subjected to additional mutations, M574, M593, Q597, and N605. Each residue was mutated to aspartate, lysine, serine, and isoleucine. The transformation efficiency of each mutant relative to wild-type JSRV Env in 208F cells is shown, as described for Fig. 2; the efficiencies of the alanine mutations at these residues are also shown.

The supertransformation by M593I was interesting because substitution of Ile at position 593 created a YXXI motif in the cytoplasmic tail. If the tyrosine in YXXI is phosphorylated, this would be a canonical binding site for the SH2 domain of c-Src (24). To determine whether c-Src played a role in transformation by M593I, we performed transformation assays in the presence or absence of the Src inhibitor PP2 in 208F cells, since PP2 showed toxicity in RK3E cells (Table 5). As expected, transformation was not reduced by PP2 for the M593A mutant, which does not contain a Src binding site. Also, as hypothesized, transformation by M593I showed partial (twofold) inhibition by PP2, consistent with the supertransforming activity involving increased signaling through c-Src. Interestingly, PP2 also reduced transformation activity by wild-type Env, although to a somewhat lesser degree than for M593I. This suggests that wild-type Env transformation might also involve activation of c-Src. Indeed, the YXXM motif in wild-type Env matches a low-affinity binding site for c-Src (24).


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  		TABLE 5. Effect of the Src inhibitor PP2 on transformation by JSRV Env mutants in 208F cellsa

Cytoplasmic tail contains an amphipathic helix. Analysis of the cytoplasmic tail amino acid sequence revealed that the first 18 amino acids (aa 570 to 587) have the potential to form an amphipathic helix, with a hydrophobic patch of amino acids on one face of the helix and hydrophilic amino acids on the opposite face (Fig. 8). We hypothesized that the amphipathic helix may position the cytoplasmic tail so that the hydrophobic patch of amino acids is embedded in the plasma membrane. To test whether the predicted amphipathic helix could associate with a cellular membrane, amino acids 570 to 587 were fused to the N terminus of the EGFP (Fig. 1). Two plasmids that disrupted the nature of the amphipathic helix were also analyzed. The first plasmid had the hydrophobic amino acids from 570 to 587 mutated to aspartate (hydrophilic-GFP), and the second had the hydrophilic amino acids mutated to proline (hydrophobic-GFP). All plasmids were transiently transfected into 208F cells and analyzed for subcellular localization by fluorescent microscopy (Fig. 9). When GFP was expressed alone, fluorescence was detected throughout the nucleus and cytoplasm. In contrast, amino acids 570 to 587 of the cytoplasmic tail altered the localization of GFP, resulting in a punctate pattern of fluorescence throughout the cytoplasm and exclusion from the nucleus. Because mitochondrial targeting signals are typically N-terminal amphipathic helices (6), we utilized a MitoTracker Red dye that labels mitochondria to determine whether amino acids 570 to 587 were targeting GFP to mitochondria. Upon overlay of GFP and MitoTracker Red images, we observed colocalization of GFP fluorescence with the mitochondria. This was consistent with amino acids 570 to 587 of the cytoplasmic tail forming an amphipathic helix in vivo. Moreover, disruption of the predicted amphipathic helix (plasmids hydrophilic-GFP and hydrophobic-GFP) restored the localization of GFP to that observed with GFP alone.


Figure 8
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  		FIG. 8. Putative amphipathic helix in the cytoplasmic tail. The first 18 amino acids of the JSRV TM cytoplasmic tail are plotted on a helical wheel (25). The results show a strong potential amphipathic helix, with hydrophobic residues on one side (filled circles) and hydrophilic residues on the other (open circles).


Figure 9
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  		FIG. 9. Functionality of the putative amphipathic helix. 208F cells were transiently transfected with expression plasmids for EGFP, EGFP containing N-terminal fusions of the putative amphipathic helix of JSRV TM (residues 570 to 587), or fusions with mutant amphipathic helices (hydrophobic-GFP and hydrophilic-GFP). Fixed cells were stained with MitoTracker Red, and the same fields were imaged by fluorescent microscopy with filters specific for EGFP (left) or MitoTracker Red (middle). Merges of the red and green images are shown in the right panels. The bottom row shows images for hydrophobic-GFP; similar images were seen for hydrophilic-GFP. Note that in all three cases, not all of the cells in the field were transfected with the GFP expression plasmids; only the transfected cells show fluorescence in the left panels.

The results of transformation assays demonstrated that substitution of alanine at 16 of the 18 amino acids residues that constitute the predicted amphipathic helix either abrogated or reduced transformation activity. It seemed possible that residues on the hydrophilic face might be interacting with cytoplasmic proteins and that substitution of alanine could have disrupted those interactions. In this case, maintaining the charge of the amino acids might be less deleterious. Therefore, we selected two hydrophilic amino acids in the predicted amphipathic helix, R572 and D577, and constructed mutants that did not change the charge at the amino acid position, R572K and D577E. All mutants were subjected to triplicate transformation assays with NIH 3T3, 208F and RK3E cells. The results were similar between the cell lines, and the results from RK3E cells are shown in Fig. 10. As expected, the R572A mutant demonstrated reduced transformation activity compared to wild-type Env. Interestingly, the R572K mutant that conserved the amino acid charge rescued transformation activity to approximately 50% of that of the wild type, as hypothesized. This result suggests that at position 572, amino acid charge is important for transformation activity by the Env protein. In contrast, transformation activity was abrogated for both the D577A and D577E mutants. All Env mutants were also Flag tagged and tested in transformation assays. The results demonstrated that similar levels of protein were produced for wild-type and mutant envelopes and that the Flag tag did not alter transformation efficiency compared to non-Flag-tagged constructs (data not shown).


Figure 10
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  		FIG. 10. Mutation of hydrophilic residues in the amphipathic helix. JSRV Env mutants with charge-conserving residues in the hydrophilic face of the amphipathic helix were generated (R572K and D557E). The relative transformation efficiencies of these mutants in RK3E cells are shown as in Fig. 2; the efficiencies of the alanine mutations at these residues are also shown.

Cytoplasmic tail C terminus is dispensable for transformation. The results of transformation assays demonstrated that substitutions of alanine at any of the residues from aa 607 to the C terminus (the last nine amino acids) of the cytoplasmic tail had little effect on transformation activity. To determine whether the C terminus of the cytoplasmic tail was dispensable for transformation, Env plasmids were constructed that truncated 3 (Env-612), 6 (Env-609), 9 (Env-606), or 12 (Env-603) amino acids from the C terminus. Truncation mutants were subjected to transformation assays with NIH 3T3, 208F, and RK3E cells. The results demonstrated that transformation activity for the truncation mutants was less than that observed for the full-length Env (Env - 615) but that the nine C-terminal amino acids were not absolutely necessary for transformation (Table 6). Deletion of 12 amino acids from the C terminus abrogated transformation. All truncated envelopes were also Flag tagged, and results of a Western blot analysis demonstrated that similar levels of protein were produced for all truncation mutants and wild-type Env. Transformation assays demonstrated that the Flag tag did not alter transformation efficiency compared to non-Flag-tagged constructs (data not shown). These results correlated with the results of transformation assays with alanine substitution mutants and indicated that the last nine amino acids of the cytoplasmic tail do not play an essential role in Env-induced transformation.


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  		TABLE 6. Transformation by C-terminal truncation mutants of JSRV Enva


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DISCUSSION
 
In this study, we carried out systematic mutagenesis of the cytoplasmic tail of JSRV envelope TM protein. Our goal was to gain insights into the mechanism of transformation, since this short 46-amino-acid domain is essential for this process. The alanine scanning mutagenesis revealed four categories of mutants: those that abrogated transformation, those that showed wild-type levels, those that showed decreased transformation, and those that showed increased transformation (supertransformers). Additional mutations and biochemical studies provided additional information as to how these mutations might be affecting the transformation process.

Mutations in JSRV Env protein could affect transformation in several ways. Possible mechanisms include altered protein stability, altered intracellular trafficking, altered interactions with viral proteins (homologous or heterologous), and altered interactions with cellular proteins. It is also important to consider that the JSRV Env protein carries out functions for viral replication. Thus, the cytoplasmic tail mutations could affect transformation and viral replication by independent or related mechanisms, or both. Moreover, if transformation involves interactions of distinct cellular proteins with the TM cytoplasmic tail, it is possible that interactions important for transformation could compete with those involved in replication. In terms of replication, the JSRV TM protein is likely to participate in several processes, by analogy to other retroviruses and, in particular, to the related betaretrovirus Mason-Pfizer monkey virus (M-PMV) that has been well studied (23). The cytoplasmic tail of M-PMV TM has been shown to be important for interaction with viral Gag protein (the MA domain) during viral particle morphogenesis (20), and tyrosine-containing motifs are important for intracellular trafficking (3). Indeed, a YXXL motif in M-PMV is in an analogous position to the JSRV YRNM (aa 590 to 593), and mutation of the tyrosine inhibits virion budding and release (22). The ectodomain of TM also carries a multimerization domain that results in formation of Env trimers on the surface of the virion (8). With regard to these experiments on the role of TM in transformation, the cytoplasmic tail mutations generally did not lead to loss of protein stability or trafficking to the plasma membrane, so these two processes were not likely responsible for the changes in transformation. Thus, the most likely causes for the altered transformation by the mutants would appear to be alterations in interactions with cellular or viral proteins, either by direct effects on binding of critical residues or by induced conformational changes. These transformation assays were performed with plasmids that expressed JSRV Env in the absence of other viral proteins, so potential competing interactions with Gag were not a consideration. Also, in rodent cells, the JSRV SU does not efficiently interact with the rodent homolog of the JSRV receptor (Hyal-2) (5), so Env-receptor interactions also did not likely affect the results.

Taken together, these studies allowed division of the TM cytoplasmic tail into three subdomains with regard to transformation as diagrammed in Fig. 11. The amino-terminal (juxtamembrane) 18 amino acids can be modeled as an amphipathic helix, and experiments with EGFP fusion proteins supported this conclusion. The carboxy-terminal nine amino acids were not essential for transformation, since alanine mutations in any of these residues did not affect transformation. Indeed, truncation of these amino acids also did not abolish transformation. The remaining 19 internal amino acids thus comprise the third subdomain, and they encompass the YXXM motif. Mutations in this subdomain had various effects, including the generation of supertransformers. It seems likely that interactions with cellular proteins necessary for transformation would involve residues in the internal subdomain and/or hydrophilic residues in the amphipathic helix.


Figure 11
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  		FIG. 11. Domains of the JSRV Env cytoplasmic tail. Domains of the cytoplasmic tail are diagrammed based on these studies. The amphipathic helix stretches from residues 570 to 587. C-terminal residues are not essential for transformation (residues 607 to 615). The intermediate region contains the YXXM motif, and mutations in this region can both positively and negatively affect transformation potential. MSR, membrane-spanning region of the TM protein.

The supertransforming mutants are particularly interesting, and they may give new insights into the mechanism of JSRV transformation. In general, cells transformed by the supertransformers showed increased levels of phosphorylated Akt, which supports a role for the Akt-mTOR pathway in JSRV transformation. Also, they showed blunted stimulation of transformation by a p38 MAPK inhibitor, which was consistent with a lower degree of p38 activation (inhibitory for transformation) by the supertransformers. Nevertheless, the supertransformers were still dependent on signaling through MEK1/2, since a MEK1/2 inhibitor abrogated transformation.

Mutations at M593 in the YXXM motif were also informative. We previously reported that M593T abrogates transformation in NIH 3T3 cells (15), while reduced transformation is observed in rat 208F fibroblasts (7). Similarly, the M593A mutation showed partial transformation in rat RK3E cells (and also in NIH 3T3 and 208F cells) (data not shown). It was most interesting that M593I resulted in a supertransforming phenotype, and this mutation also generated a canonical c-Src SH2 domain binding motif. Moreover, transformation by M593I was partially inhibited by treatment with the c-Src inhibitor PP2. This suggests that the supertransforming phenotype of M593I may be due to increased signaling through c-Src. It was also interesting that PP2 partially inhibited transformation by wild-type JSRV Env and that YXXM is in fact a low-affinity c-Src binding site as well. This raises the possibility that wild-type JSRV may also signal through c-Src. It has also been reported that c-Src can activate Akt in a PI3K-independent fashion (4, 9), so this might explain the constitutive phosphorylation of Akt in JSRV-transformed cells with disabled PI3K (12). However, it should be noted that in order for the YXXM motif to function as a docking site for any SH2-containing protein, the tyrosine must be phosphorylated. We and others have failed to detect tyrosine phosphorylation of the TM protein in JSRV-transformed cells (7, 10), although it remains possible that this was due to technical reasons. In particular, low levels of tyrosine phosphorylation could have been sufficient for transformation. Moreover, the only method currently available to detect the Env protein is the addition of a C-terminal epitope tag; pull-downs with anti-epitope antibody could have interfered with simultaneous binding to the cytoplasmic tail (total length, 46 amino acids) of a cellular protein.

Studies of two alanine mutants with reduced transformation were also interesting. For these mutants, we hypothesized that the mutations might have eliminated signaling through one of the two pathways already known to be involved in JSRV transformation (H/N-Ras-MEK1/2-ERK1/2 and Akt-mTOR). The R591A mutant supported this hypothesis in that its reduced transformation potential was not affected by rapamycin but was more sensitive than wild-type JSRV Env to the H/N-Ras inhibitor FTI-277. Thus, R591A appears to signal through H/N-Ras-MEK1/2-ERK1/2 but not through AKT-mTOR. On the other hand, H587A was intriguing because its reduced transformation was not affected by either rapamycin or FTI-277 (although it was sensitive to the MEK1/2 inhibitor). Thus, H587A transformation may involve signaling through novel pathways or through alternates of the known pathways that are resistant to the inhibitors used. For instance, signaling from K-Ras to MEK1/2 and ERK1/2 is not affected by FTI-277, and some downstream signaling from mTOR is resistant to rapamycin (18, 19). Future studies will address these possibilities.

Many of the alanine mutations in the amphipathic helix also abrogated transformation. As mentioned above, some (but not all) of the residues in the hydrophilic face might interact with cytoplasmic proteins during transformation. Indeed, while the R572A mutation severely reduced transformation, the charge-conserving R572K mutation showed substantial transformation (ca. 50% of that of the wild type). This was consistent with R572 being involved in binding of a cellular protein for transformation. However, an alternate explanation could be that charge conservation could have a more general role. Basic residues such as R572 often surround the hydrophobic patch of amino acids in an amphipathic helix. While the hydrophobic resides can embed into membrane lipids, the surrounding positively charged amino acids can enhance membrane binding via interactions with acidic phospholipids.

It was also noteworthy that alanine substitutions in the hydrophobic face of the JSRV amphipathic helix universally abrogated transformation. This was somewhat unexpected since alanines would not be expected to disrupt the hydrophobicity of this face. In fact, others have also reported that alanine substitutions in the hydrophobic faces of other amphipathic helices can change their properties (25). Another possibility is that the hydrophobic face of the JSRV amphipathic helix could interact with hydrophobic regions of the same or other proteins within the lipid bilayer. Alanine substitutions in the hydrophobic face could alter the overall topology of the TM protein, affect multimerization, or change the interactions with other proteins. One possible candidate for interaction with the hydrophobic face of the amphipathic helix could be the membrane-spanning region of the TM protein. We recently made a chimeric JSRV Env in which the membrane-spanning portion of TM was exchanged for the analogous region of an endogenous JSRV-related Env protein (J. Lim, S. Hull, and H. Fan, unpublished data). The resulting Env protein showed changes in transforming potential that suggested alterations in the relative importance of downstream signaling pathways. Additional studies are in progress.

The cytoplasmic tail mutants generated in these studies will also be interesting to study in the context of JSRV replication. As mentioned above, the cytoplasmic tail of TM has been shown to be important for interactions with Gag protein and for efficient release of virus particles for M-PMV. Thus, it will be interesting to introduce these mutations into a full-length genomic clone of JSRV and to study the effects on virus production and replication. These experiments are also in progress.


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ACKNOWLEDGMENTS
 
This work was supported by NIH grant CA94188 to H.F. S.H. was supported by postdoctoral fellowships from the NIH (CA110619) and the American Cancer Society (PF-05-074-01-MBC).

We thank Naoyoshi Maeda and Larry Dearth for advice and Sohail Jahid for technical assistance. Support of the Cancer Research Institute and the DNA Sequencing Shared Resource of the Chao Family Comprehensive Cancer Center is acknowledged.


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FOOTNOTES
 
* Corresponding author. Mailing address: Cancer Research Institute, Sprague Hall, University of California Irvine, Irvine, CA 92697-3900. Phone: (949) 824-5554. Fax: (949) 824-4023. E-mail: hyfan{at}uci.edu. Back


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Journal of Virology, August 2006, p. 8069-8080, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00013-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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