The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain

Thomas E. Allen1, Kate J. Sherrill1, Sara M. Crispell1, Matthew R. Perrott1, Jonathan O. Carlson2 and James C. DeMartini1

Departments of Pathology1 and Microbiology2, Colorado State University, Fort Collins, CO 80523, USA

Author for correspondence: Thomas Allen. Fax +1 970 491 0603. e-mail allent{at}colostate.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Ovine pulmonary adenocarcinoma, caused by jaagsiekte sheep retrovirus (JSRV), is a naturally occurring retrovirus-induced pulmonary neoplasm of sheep. We report here that expression of the JSRV env gene is sufficient to transform an avian embryo fibroblast cell line, DF-1. DF-1 cells transfected with an avian sarcoma–leukaemia retroviral expression vector containing the JSRV env gene [pRCASBP(A)-J:env] exhibited changes consistent with transformation, including contraction and rounding of cells with formation of dense foci. Transfection with a reporter construct expressing the green fluorescent protein did not induce morphological changes in DF-1 cells, eliminating the possibility that the vector, the transfection protocol or culturing techniques were responsible for the transformed phenotype. When pRCASBP(A)-J:env-transfected cells were inoculated into nude mice, tumours formed, verifying that the DF-1 cells were tumorigenic. Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring retrovirus-induced pulmonary neoplasm of sheep, which shares certain characteristics with human bronchioloalveolar carcinoma (BAC) (DeMartini & York, 1997 ; Palmarini et al., 1997 ; Palmarini & Fan, 2001 ). Both OPA and BAC are well-differentiated tumours of alveolar type II (ATII) or non-ciliated bronchiolar (Clara) cells exhibiting multifocal growth and a peak occurrence in adults (Bonne, 1939 ; Liebow, 1960 ; Perk & Hod, 1982 ; Palmarini & Fan, 2001 ; Platt et al., 2002 ). The incidence of BAC, which is only weakly associated with smoking, has dramatically increased since 1955 and now represents 24% of lung cancers, or as many as 40000 deaths annually (Wu et al., 1988 ; Barsky et al., 1994 ; Ginzburg et al., 1996 ). While the aetiology of BAC is unknown, OPA is one of only a few epithelial cancers associated with a retrovirus aetiology (Morris & Cardiff, 1987 ). Thus, OPA is an excellent model with potential to contribute significantly to our understanding of retroviral oncogenesis as well as pulmonary biology.

Classified as a betaretrovirus (type B/D chimeric retrovirus), jaagsiekte sheep retrovirus (JSRV) clone JSRVJS7 has been shown to cause OPA (York et al., 1992 ; Palmarini et al., 1999 ; DeMartini et al., 2001 ). JSRV is considered a simple retrovirus and contains no obvious oncogene within its genome although an alternative open reading frame of unknown function within the pol gene has been identified and is referred to as orfX (Bai et al., 1999 ; Palmarini et al., 1999 ). Although JSRV does not contain an obvious oncogene, OPA pathogenesis is reminiscent of an acute transforming retrovirus in that OPA lesions can be seen in experimentally inoculated lambs within weeks to months after inoculation (Sharp et al., 1983 ; DeMartini et al., 1987 ; Rosadio et al., 1988 ). Recently, it has been reported that the JSRV env gene is capable of transforming murine NIH-3T3 cells as well as a rat fibroblast cell line, F208 (Maeda et al., 2001 ; Rai et al., 2001 ). The purpose of this study was to test the hypothesis that the JSRVJS7 env gene was sufficient to transform an avian embryo fibroblast cell line, DF-1, and that the transformed cells would form tumours in mice. In addition, we examined the possible role of a conserved SH2 binding domain in the cytoplasmic tail within the transmembrane domain (TM-CD) of the envelope protein.

To examine JSRV genomic components for oncogenic effects, we used a series of avian sarcoma–leukaemia retroviral (ASLV)-derived vectors [pRCASBP(A)] to express JSRV subgenomic components in the cell line DF-1 (Federspiel & Hughes, 1997 ; Schaefer-Klein et al., 1998 ; Fisher et al., 1999 ). The DF-1 cell line is a non-transformed, immortalized avian cell line that efficiently supports replication of ASLV-derived vectors and has been used for the study of oncogenic transformation (Himly et al., 1998 ). In addition, the DF-1 cell line was derived from an EV-0 embryo, which was free of endogenous retroviral sequences with high homology to RAV-0, thus eliminating the possibility of recombination with the introduced recombinant virus (Schaefer-Klein et al., 1998 ). The pRCASBP(A) vectors were constructed by removing the src gene from the virus genome and replacing it with a multiple cloning region (Federspiel & Hughes, 1997 ). JSRVJS7 env, orfX and the surface glycoprotein (SU) component of the env gene were each cloned separately into pRCASBP(A) vectors and their effect on DF-1 cells was monitored.

We report here that only the env gene of JSRVJS7 is necessary for transformation of DF-1 cells. Furthermore, in contrast to recent data suggesting that a conserved SH2 binding domain in the TM-CD of the envelope protein is necessary for transformation in murine NIH-3T3 cells (Palmarini et al., 2001 ), the elimination of this binding site did not abrogate JSRV env-induced transformation of DF-1 cells. These findings suggest that more than one mechanism may be involved in JSRV env-induced transformation in vitro and in neoplastic events in OPA.


   Methods
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Introduction
Methods
Results
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References
 
{blacksquare} Cell culture.
DF-1 cells (ATCC CRL-1203) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum (FBS; Sigma), 100 U/ml penicillin and 100 µg/ml streptomycin (DMEM) at 39 °C in 10% CO2.

{blacksquare} Plasmid construction and nomenclature.
All plasmid propagation, ligations and PCR amplifications were performed as described in Sambrook et al. (1989) . The vector pGreen-Lantern (Gibco BRL) was used as a template for amplification of the gene for green fluorescent protein (GFP). The vector pRCASBP(A) was generously supplied by Galen H. Fisher (NIH) and has been described (Federspiel & Hughes, 1997 ; Fisher et al., 1999 ). Primers used to create the pRCASBP(A)-based vectors are listed in Table 1. Primers were designed to create a 5' NotI site and a 3' ClaI site for cloning into the pRCASBP(A) expression vector. Primers used for PCR were: TA-64F/65R for pRCASBP(A)-J:orfX, TA-66F/67R for pRCASBP(A)-J:env, TA-56F/57R for pRCASBP(A)-J:SU, TA-66F/105R for pRCASBP(A)-J:(-99) and TA-48F/49R for pRCASBP(A)-J:gfp. PCR amplification cycles for each primer set consisted of: 94 °C for 2 min (1 cycle); 94 °C for 15 s, 55  °C for 30 s, 72 °C for 45 s (30 cycles); and 72 °C for 10 min (1 cycle).


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Table 1. List of primer sequences

 
{blacksquare} Transfection of the DF-1 cell line.
All transfections were performed using LipofectAmine, as described by the manufacturer (Life Technologies). Briefly, DF-1 cells were plated on 60 mm tissue culture plates followed by incubation for ~15 h or until the cells were ~70% confluent. Two µg of plasmid was then mixed with 18 µl LipofectAmine in 400 µl serum-free DMEM (SFM) and allowed to incubate for 40 min at room temperature followed by the addition of 1·6 ml of SFM. DF-1 cells were washed twice with SFM and the transfection mixture was added directly on to cells. Cell were incubated for 5 h at 39  °C in a 10% CO2 incubator followed by the addition of 2 ml DMEM containing 20 % FBS. Cells were incubated for ~15 h followed by removal of the transfection mixture and replacement with DMEM.

{blacksquare} Infection of DF-1 cells.
Tissue culture supernatants from pRCASBP(A)-J:env-transfected, JSRV env mutant-transfected and pRCASBP(A)-J:gfp-transfected DF-1 cells were harvested and tested for reverse transcriptase (RT) activity. DF-1 cells were plated on 60 mm tissue culture plates and allowed to grow overnight so that they would be ~70% confluent at the start of the experiment. Virus-containing supernatants were then added to the DF-1 cells and virus was allowed to adsorb to the cells for ~5 h at 39 °C. Cells were then washed twice in DMEM and maintained for the duration of the experiment in DMEM at 39  °C in 10% CO2.

{blacksquare} RT activity.
RT activity was monitored by measuring bromodeoxyuridine incorporation into an immobilized template (Lenti-RT; Cavidi Tech) as described by the manufacturer.

{blacksquare} RNA isolation and Northern analysis.
RNA was isolated from DF-1 cells and from mouse tissue using the RNeasy RNA isolation kit (Qiagen). Northern blots were prepared as described (Sambrook et al., 1989 ). Probes used for Northern blot analysis were created using digoxigen-11-uridine 5'-triphosphate as described by the manufacturer (Roche). Primers used to produce probes were: TA-48F/49R for gfp, TA-64F/65R for orfX, TA-56F/57R for SU and TA-60F/61R for env (Table 1).

{blacksquare} RT–PCR.
RT–PCR was performed using a one-step RT–PCR method as described by the manufacturer (Life Technologies). Conditions for cDNA production involved incubation at 50  °C for 30 min. Subsequent PCR amplification cycles for each primer set consisted of: 94 °C for 2 min (1 cycle); 94 °C for 15 s, 55 °C for 30 s, 72 °C for 45 s (35 cycles); and 72 °C for 10 min (1 cycle). Primers (Table 1) for production of cDNA and PCR amplification were the same as those used for creation of the pRCASBP(A) plasmids listed above with the addition of TA-88F/91R for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

{blacksquare} DNA isolation and PCR.
DNA from DF-1 cells and mouse tissue was isolated using a DNeasy DNA purification kit (Qiagen) as described by the manufacturer. DNA PCR amplification cycles and primer sets were the same as described above for RT–PCR except that 30 cycles were performed instead of 35.

{blacksquare} Limiting dilution analysis.
Tissue culture supernatant from cells in culture 20 days after transfection with pRCASBP(A)-J:env was added to DF-1 cells plated 24 h earlier on 60 mm tissue culture plates (in triplicate). At the time of inoculation, cells were ~70% confluent. A series of tenfold dilutions of the virus-containing supernatant stock in a final volume of 2 ml DMEM was added to cells and the virus was allowed to adsorb for ~5 h at 39 °C. Cells were then washed in DMEM and cultured at 39 °C with 10% CO2 in DMEM. Cells were trypsinized and diluted (1:5) on days 3, 5 and 7 post-transfection. Cell monolayers were monitored daily for changes in morphology and phenotypic behaviour. Supernatant collected on day 9 was tested for RT activity as described above. Cell cultures were monitored for a minimum of 13 days to ensure sufficient time to observe JSRV env-related results.

Experiments designed to test the relative efficiency of transformation of the JSRV Env mutants produced using site-directed mutagenesis (see below) were performed essentially as described above with the following modifications. Virus stocks used for the limiting dilution assay were collected from transfected cells on day 15. RT assays (see above) on the various viral stocks were performed to ensure that equal titres of virus were used in each experiment. A series of twofold dilutions were used (in triplicate) to determine the end-point dilution at which no phenotypic changes consistent with transformation were observed. Supernatants collected from infected cells were tested for RT activity on both day 9 and day 13. Finally, cell cultures were monitored for 17 days to ensure sufficient time to observe JSRV env-related results. The Kärber method was used to estimate the 50% endpoint for transformation (Payment & Trudel, 1993 ).

{blacksquare} Tumor induction in nude mice and histology.
To determine the capacity of transfected DF-1 cells to form tumours in vivo, three female athymic nude mice (Harlan) were inoculated subcutaneously at separate sites with 105, 106 and 107 pRCASBP(A)-J:env-transfected DF-1 cells and two mice were inoculated with the same concentrations of DF-1 cells transfected with pRCASBP(A)-gfp. For inoculation, transfected cells were harvested 20 days after transfection, washed in PBS and resuspended in PBS. Cell suspensions (200 µl) containing the indicated cell concentration were inoculated subcutaneously into nude mice and the sites of implantation were observed for 40 days after which the mice were sacrificed. Tissue masses were processed by fixation in 4% paraformaldehyde for 24 h followed by paraffin embedding. Following sectioning (5 µm), slides were stained with eosin and haematoxylin. Additional sections of tumour tissue were stained by histochemistry using the Alcian blue technique for acid mucopolysaccharides and Masson's trichrome technique for collagen. Statistical relevance of tumour formation in mice was analysed using Fisher's Exact Test.

{blacksquare} Site-directed mutagenesis.
Mutation of the JSRVJS7 env gene was performed by site-directed mutagenesis using a PCR-based, commercially available kit (Quikchange XL; Stratagene) as described by the manufacturer. Primers used for introducing mutations were: TA-118F/119R for pRCASBP(A)-J:env(Y{Delta}A), TA-120F/121R for pRCASBP(A)-J:env(Y{Delta}S), TA-122F/123R for pRCASBP(A)-J:env(M{Delta}V), TA-124F/125R for pRCASBP(A)-J:env(M{Delta}L). Temperature cycling involved 94 °C for 1 min (1 cycle); 94 °C for 50 s, 60 °C for 50 s, 68 °C for 12 min (18 cycles); and 68 °C for 7 min (1 cycle).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Transfection of DF-1 cells
To examine whether expression of JSRV subgenomic components is capable of inducing cellular changes, we developed a series of pRCASBP(A) plasmid-based viral vectors to express JSRVJS7 subgenomic components individually in the avian embryo fibroblast cell line, DF-1 (Federspiel & Hughes, 1997 ; Schaefer-Klein et al., 1998 ). The pRCASBP(A) vector was created by removing the src gene of the virus and replacing it with a multiple cloning region flanked by a 5' NotI site and a 3' ClaI site (Federspiel & Hughes, 1997 ). Vectors for the expression of JSRVJS7 orfX, env and the SU portion of env were created by PCR amplification as described in Methods (Table 1). In addition, a vector containing the gene for GFP was created to act as a control for JSRV non-specific events. Plasmid-based viral constructs were transfected into DF-1 cells and transfection efficiencies determined (data not shown). Although initial transfection efficiencies were low, the robust production of virus and subsequent infection of surrounding cells compensated for the inefficiencies. For each pRCASBP(A) construct, production of recombinant virus was confirmed by testing the cell supernatant for RT activity (data not shown). To monitor virus spread and expression of GFP, cells transfected with pRCASBP(A)-gfp were monitored by fluorescence (data not shown).

JSRVJS7 envelope gene induces changes in DF-1 cells consistent with transformation
Cells transfected with the pRCASBP(A) vectors were monitored for changes in growth characteristics. Whereas no changes were seen with pRCASBP(A)-J:orfX, gfp and the SU component of env, dramatic changes were observed with pRCASBP(A)-J:env (Table 2 and Fig. 1). Between day 5 and day 6 post-transfection, scattered cells throughout the culture appeared to be raised from the monolayer and densely packed. Between days 7 and 8, indications of rounding, contraction and multi-layering of cells were observed. By day 10, all pRCASBP(A)-J:env-transfected cultures displayed microscopic dense cellular foci and a striking decrease in the pH of the cell culture medium compared with that of control cells, suggesting an increase in metabolic activity (data not shown). By 12 days post-transfection, macroscopic foci were observed in all pRCASBP(A)-J:env-transfected cell cultures (Fig. 1).


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Table 2. Morphological changes observed with RCASBP(A)-J:env and pRCASBP(A)-J-env

 


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Fig. 1. JSRVJS7 env-induced morphological changes in DF-1 cells. (A) (magnification 20x) and (B) (40x) represent DF-1 cells at day 12 post-transfection with pRCASBP(A)-gfp. No changes in morphological or growth characteristics were seen in these cells. (C) (20x) and (D) (40x) represent DF-1 cells at day 12 post-transfection with pRCASBP(A)-J:env. Indications of altered growth characteristics and morphological changes can be seen. Cells appear rounded and have contracted into obvious foci containing multi-layered cells. Arrows highlight typical cellular foci.

 
To confirm that RNA transcripts for RCASBP(A)-J:orfX, su, env and RCASBP(A)-gfp were present in transfected cells, Northern blot hybridization analysis was performed. In each case, a band of the correct size was detected (Fig. 2). No corresponding bands were observed with non-transfected DF-1 cells (Fig. 2). To confirm the production of recombinant virus and to test for the ability of the recombinant virus to transform DF-1 cells, tissue culture supernatant from transfected cells was used to infect freshly plated DF-1 cells. Morphological changes in DF-1 cells infected with RCASBP(A)-J:env virus were qualitatively equivalent to cells transfected with the pRCASBP(A)-J:env plasmid vector (Table 2). However, changes were seen at earlier time points, presumably due to rapid widespread infection of the virus carrying the JSRVJS7 env gene. As in the transfection experiments, no morphological changes were observed with RCASBP(A)-gfp virus.



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Fig. 2. Northern blot analysis of DF-1 cells transfected with (A) pRCASBP(A)-J:orfX; (B) pRCASBP(A)-gfp; (C) pRCASBP(A)-J:env; and (D) pRCASBP(A)-J:su. Approximate sizes of transcripts are denoted. The symbol (*) identifies the full-length virus genome containing the indicated gene. The Northern blot analysis demonstrates that the transfected cells contain similar levels of transcripts of the introduced gene.

 
To quantitate the transforming effect of RCASBP(A)-J:env, a limiting dilution strategy was employed. Cultures of DF-1 cells were exposed to virus-containing supernatant that was serially diluted to achieve an endpoint dilution where no JSRV env-induced events were observed. Transformation occurred in all cultures at dilutions of 10-5 in each of three independent experiments. However, at dilutions of 10-6, no transformation was detected in any culture. To confirm the presence of RCASBP(A)-J:env virus in cultures exhibiting the transformed phenotype, RT activity in infected cultures was assayed. Only cultures that displayed a transformed phenotype contained RT activity in tissue culture supernatant (data not shown). These results demonstrate a direct and consistent correlation between the presence of the RCASBP(A)-J:env virus and the transformation of DF-1 cells.

Tumorigenicity of the JSRVJS7 envelope gene
To test for tumorigenicity of DF-1 cells transfected with the pRCASBP(A)-J:env vector, three mice were inoculated subcutaneously with cells from DF-1 cultures at 20 days post-transfection. As a control, two mice received the same number of cells from pRCASBP(A)-J:gfp-transfected DF-1 cells, also collected at 20 days post-transfection. After a few days, the initial swelling observed in all mice resolved. Subsequent development of subcutaneous nodules was then observed in all mice inoculated with 107 cells transfected with the pRCASBP(A)-J:env vector. In two of the three mice, nodules 3–4 mm in diameter were seen at the site of cell implantation at 19 days post-implantation. These grew progressively to 6–7 mm in diameter by 40 days post-implantation, when the experiment was terminated. At the time of termination, the third mouse inoculated with pRCASBP(A)-J:env-transfected cells had developed a 2 mm nodule at the site of cell implantation. In addition, one of the mice developed a lesion at a site inoculated with 106 cells that also progressively grew over 40 days to 6–7 mm in diameter. No growth was observed at any site inoculated with cells transfected with pRCASBP(A)-J:gfp. Using the Fisher Exact Test, tumour formation induced by cells transfected with pRCASBP(A)-J:env was found to be statistically significant (P<0·04) when compared with controls.

At necropsy, white–grey tissue masses were found in the subcutis below the panniculus carnosus muscle at the site of the gross lesions in mice inoculated with pRCASBP(A)-J:env-transfected cells. Histologically, the masses were generally similar, consisting of well-circumscribed cellular masses exhibiting dense cellularity in the peripheral areas and a loose arrangement in the central areas (Fig. 3). The cells were spindle-shaped to polygonal with moderate amounts of cytoplasm and anisokaryotic nuclei, often containing prominent nucleoli. A few multinucleate or binucleate cells were seen. The mitotic index averaged 1–3 per high power field (40x objective). In the central areas, the stellate cells were separated by a myxomatous matrix, revealed by histochemistry to contain acid mucopolysaccharides, and some areas of eosinophillic fibrillar material revealed by histochemistry to contain collagen (data not shown).



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Fig. 3. Histopathology of a subcutaneous nodule from a nude mouse. Two areas of one mass are shown. (A) Densely cellular area consisting of spindle cells with oval nuclei and prominent nucleoli, and occasional mitotic figures (arrowhead, inset). (B) Loosely arranged central area consisting of spindle cells separated by mucopolysacharide matrix (M) and collagen (C), and larger, anaplastic cells with two mitotic figures (arrowheads, inset). Bar, 50 µm.

 
To confirm the presence of the JSRVJS7 env gene in the tumour tissue, DNA PCR was performed using primers specific for the TM component (Table 1). In the two mice tested, PCR amplification demonstrated the presence of the env gene (Fig. 4). The presence of JSRV env RNA also was confirmed by RT–PCR analysis for the JSRVJS7 env transcript (Fig. 4).



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Fig. 4. Confirmation of transcription and integration of the JSRVJS7 env gene in cells isolated from mouse tumours. DNA and RNA was isolated from tumour masses from two mice inoculated with transformed DF-1 cells (M-env). Liver tissue was used to isolate DNA and RNA from a mouse inoculated with pRCASBP(A)-gfp-transfected DF-1 cells (M-gfp). Primers for the TM component (714 bp) of the JSRVJS7 env gene were used for each analysis. RT, lanes depicting RT–PCR analysis; + and -, RT–PCR experiments with or without RT; DNA, lanes containing samples from DNA PCR analysis. Only the mice that received transformed DF-1 cells were shown to contain both integrated JSRVJS7 env and the transcript of the gene. The lane marked G represents a control DNA PCR for GAPDH (208 bp) to confirm the quality of DNA from the control mouse.

 
Deletion mutant analysis
Comparison of the predicted envelope protein sequence from four JSRV and three endogenous sheep retroviruses related to JSRV (ESRV) revealed that the greatest dissimilarity occurred in the C terminus of the TM domain of the envelope protein (Fig. 5A) (Palmarini et al., 2000 ). This region also contains the cytoplasmic tail of the envelope protein, a site of potential cellular protein interactions. To determine if this variable region plays a role in mediating JSRV env transformation, a series of three deletion mutants were constructed that removed sequential, non-overlapping coding sequences starting from the 3' end of the env gene, each corresponding to a deletion of 33 amino acids per mutant construct (Fig. 5A).



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Fig. 5. Partial alignment of the predicted JSRV and ESRV envelope proteins. (A) Four JSRV and three ESRV sequences are shown. The thick arrow indicates the predicted start of the cytoplasmic tail of the TM domain (TM-CD). The narrow arrow indicates the last amino acid (valine) present in the construct pRCASBP(A)-J:(-99). All amino acids downstream of this site were removed. (B) Graphical representation of the region surrounding the Y-X-X-M motif in JSRV. This region is compared with the same region in ESRV. Arrows denote amino acid residues that make up the motif. These two amino acids were mutated as described in the text.

 
In three independent experiments, DF1 cells were transfected individually with each of the plasmid constructs. In each case, no transformation occurred. Thus, analysis focused on construct pRCASBP(A)-J:(-99), which contained the full-length env gene with the last 99 nucleotides of the 3' end of the gene removed. To ensure that the loss of the transformed phenotype was not due to reduction in transcription efficiency, Northern blot hybridization analysis was performed. Transcription levels of pRCASBP(A)-J:(-99) were comparable with pRCASBP(A)-J:env (Fig. 6A).



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Fig. 6. Northern analysis of JSRV mutants. (A) RNA transcription levels of the deletion mutant, pRCASBP(A)-J:(-99), in DF-1 cells 9 days after transfection with the plasmid construct. No band is seen in untransfected controls (DF-1). (B) RNA transcription analysis of JSRV mutants created by site-directed mutagenesis of the Y-X-X-M sequence, a known binding site of SH2 domains in the p85 subunit of PI3-K. Transcription levels for each mutant are comparable with cells transfected with the non-mutated counterpart, pRCASBP(A)-J:env. No signal is seen in untransfected controls. The symbol (*) identifies the full-length virus genome containing the indicated gene.

 
Site-directed mutagenesis
Further analysis of the predicted TM-CD of the JSRV envelope protein contained within the pRCASBP(A)-J:(-99) construct identified a stretch of three amino acids containing methionine (595), lysine (596) and tyrosine (597) residues, which are not present in isolates of ESRV (Fig. 5B). The juxtaposition of the additional tyrosine (597) residue three amino acids upstream from a conserved methionine (600) residue creates the known SH2 binding domain, Y-X-X-M (Fig. 5B) (Palmarini et al., 2001 ). To test whether this domain may play a role in JSRV env transformation of DF1 cells, site-directed mutagenesis was performed to disrupt the SH2 binding motif.

When the methionine (600) residue in this motif was mutated to valine or leucine [pRCASBP(A)-J:env(M{Delta}V) and pRCASBP(A)-J:env(M{Delta}L)], transformation of DF-1 cells occurred. Furthermore, when the tyrosine residue (597) was mutated to alanine or serine [pRCASBP(A)-J:env(Y{Delta}A) and pRCASBP(A)-J:env(Y{Delta}S)], the mutated JSRV env was still competent to induce transformation of DF1 cells. In each case, mRNA levels were found to be comparable with cells transfected with the non-mutated JSRV env construct, pRCASBP(A)-J:env (Fig. 6B).

To compare the relative transformation efficiencies of the mutants described above with the wild-type Env construct, a limiting dilution assay was performed to determine the 50% endpoint for transformation (50% EP). For the Env wild-type construct (pRCASBP(A)-J:env), the 50% EP was estimated to be 10-3·66/ml while this value for the mutants was 10-3·46/ml for pRCASBP(A)-J:env(Y{Delta}S) and 10-3·56/ml for pRCASBP(A)-J:env(M{Delta}V). Since the 50% EP values are within a twofold dilution of each other, these data support the conclusion that the relative transformation potential of the mutant constructs are similar to that of the wild-type Env protein and that the mechanism by which they induce DF-1 transformation is most likely identical. Furthermore, only dilutions that induced transformation were found to contain virus, as determined by measuring RT activity in the tissue culture supernatants (data not shown). These results demonstrate a direct and consistent correlation between the presence of the RCASBP(A) viral construct expressing the JSRV env gene and the transformation of DF-1 cells.


   Discussion
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Methods
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Discussion
References
 
The production of pRCASBP(A)-based viral vectors permitted experiments to test the cytopathic effects of subgenomic components of JSRVJS7 in DF-1 cells. In these experiments, only the JSRVJS7 env gene induced observable changes consistent with transformation (Fig. 1). In addition, the ability of DF-1 cells transfected with the env gene to induce tumours when transplanted into mice confirmed that the cells were tumorigenic (Shin et al., 1975 ). It is interesting to note that the SU component of the env gene did not by itself cause transformation. This result suggests that the TM domain is either directly responsible for transformation or is required for functional presentation of the SU protein, possibly by influencing protein folding, modification and/or trafficking. Experiments designed to determine directly a role for the TM protein in transformation using our system were inconclusive since little TM mRNA was observed in DF-1 cells transfected with constructs designed to overexpress this protein.

Clinical and pathological features of JSRV-induced OPA are consistent with both an acute and non-acute transforming virus. In experimentally induced disease, OPA lesions are seen weeks to months after inoculation (DeMartini et al., 1987 ; Rosadio et al., 1988 ). The rapid onset and multifocal nature of the lesions suggest that JSRV may act by directly inducing cell proliferation, thus resembling an acute transforming virus. However, in naturally occurring cases, OPA is seen almost exclusively in adults, and lesions are typically a single lung tumour mass (DeMartini & York, 1997 ). In this way, JSRV may resemble a non-acute transforming virus. Since it can be assumed that younger sheep are exposed to the virus, the more frequent occurrence of OPA in adult sheep suggests a long latent period during which virus replication and cellular proliferation may be followed by JSRV-induced insertional mutagenesis. Genetic changes in the target cells may then lead to expansion of a monoclonal or oligoclonal cell population and tumour formation. The recent description by our laboratory of an integration event into the SP-A surfactant gene of an ATII cell line isolated from a natural occurring tumour is of interest, especially since a hallmark feature of OPA is the abundant production of lung fluid that is rich in SP-A (Sharp et al., 1983 ; DeMartini et al., 1987 ). Work is in progress to determine whether a common integration site can be found in OPA tumours. The diverse manifestations of OPA suggest that JSRV-induced OPA may be an example of multistep carcinogenesis (Morris & Cardiff, 1987 ; Fan, 1997 ). However, the evidence in this report supports the hypothesis that the JSRV env gene product may be sufficient to cause neoplasia.

With the observation that the env gene causes transformation and tumour production, attention turns to the possible mechanism of action. There are few precedents of a retroviral structural protein directly causing neoplasia. Most acute transforming retroviruses contain within their genome an oncogene that is responsible for rapid development of tumours (Kung et al., 1991 ; Kung & Liu, 1997 ). These viruses are usually replication defective and require a non-defective ‘helper virus' to replicate and spread (Kung et al., 1991 ; Kung & Liu, 1997 ). For example, the spleen focus-forming virus directly causes the initial proliferative stage of erythroleukaemia in infected mice (Fang et al., 1998 ). The interaction of the gp55 component of the env gene with the erythropoietin cell surface receptor triggers massive erythroid proliferation (Fang et al., 1998 ). In contrast, the avian hemangioma retrovirus is a replication-competent virus whose env gene has been recently shown to cause proliferation of NIH-3T3 cells directly, although the mechanism of action is unknown (Alian et al., 2000 ).

The presence of a conserved SH2 binding motif in the predicted cytoplasmic tail of the JSRV envelope suggests a role for phosphatidylinositol 3-kinase (PI3-K) in the transforming properties of this protein. This domain is present in all isolates of JSRV but not in the related endogenous proviruses, ESRV (Palmarini et al., 2000 ). Recent work in murine NIH-3T3 cells has implicated a role for PI3-K and AKT kinase in mediating transformation (Palmarini et al., 2001 ). In that report, mutational analysis of the Y-X-X-M motif demonstrated that both the tyrosine and methionine residues were necessary to induce transformation. In addition, AKT kinase was activated in transformed cells. Proteins of the PI3-K/AKT kinase cascade have been implicated as determinants in many human cancers (Moore et al., 1998 ; Datta et al., 1999 ; Kobayashi et al., 1999 ; Shayesteh et al., 1999 ). For example, AKT genes are overexpressed or amplified in breast, pancreatic, ovarian and prostate cancers (Datta et al., 1999 ). Relevant to retroviruses, the avian sarcoma virus 16, which induces hemangiosarcomas in chickens, has been shown to contain an oncogene that is derived from the cellular catalytic subunit of PI3-K (Chang et al., 1997 ). This gene has been shown to be a potent transforming factor in chicken embryo fibroblast cells and to activate AKT kinase (Chang et al., 1997 ). However, the results presented in this report do not support a direct role for this motif in DF-1 transformation since elimination of either the tyrosine (597) or the methionine (600) residues contained within this motif did not abrogate transformation. Nevertheless, the elimination of the last 33 amino acids of the TM-CD of the Env protein does eliminate transformation, suggesting a role for the cytoplasmic domain in transformation, although this role may be as trivial as influencing the correct folding and/or trafficking of the protein.

The discrepancy in observed results regarding the SH2 binding domain between the DF-1 system and the NIH-3T3 system is intriguing. A possible explanation for the observed differences is that PI3-K is activated in both DF-1 and NIH-3T3 cells, but PI3-K does not directly interact with the JSRV Env protein. Alternatively, it is possible that at least two distinct mechanisms are involved in transformation. Recent efforts in our laboratory have focused on the development of JSRV Env-specific antibodies, which should prove invaluable to the further study of Env-induced transformation. Of particular interest is the examination of the phosphorylation state of the tyrosine in the Y-X-X-M motif, a prerequisite for direct interaction with PI3-K.

The DF-1 cell system using pRCASBP(A) vectors should prove to be a useful model for dissecting the molecular mechanism involved in JSRV tumorigenesis. It should be noted, however, that the events observed in this report involve an avian fibroblast cell line, whereas the predominant cell type transformed in OPA is an epithelial cell of the lung, the ATII cell. While it seems unlikely that the transformation event seen in this report is specific to DF-1 cells, confirmation of these results will need to be made in the ovine system. Recently, our laboratory has demonstrated the ability to isolate and culture ovine primary ATII cells (T. Allen, unpublished results), which will allow for a direct examination of JSRV env-induced events in the predominant neoplastic cell type found in OPA. By analysing and comparing the mechanism of JSRV env-induced DF-1 cell transformation with env-induced effects on ATII cells, a more thorough examination of the role that JSRV env plays in OPA will be possible.


   Acknowledgments
 
This work was supported by grant CA 59116 from the National Cancer Institute and by a grant from the College Research Council of the College of Veterinary Medicine and Biomedical Sciences, Colorado State University. We thank Dr Galen Fisher for his gift of the pRCASBP(A) vector and Dr A. D. Miller for the gift of the PJ/LAPSN cell line. Also a special thanks to J. A. Platt for critical reading of this manuscript. Dr DeMartini is a member of the University of Colorado Cancer Center.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 1 March 2002; accepted 5 July 2002.