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
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Abstract |
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Introduction |
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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 sarcomaleukaemia 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.
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Methods |
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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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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.
RT activity.
RT activity was monitored by measuring bromodeoxyuridine incorporation into an immobilized template (Lenti-RT; Cavidi Tech) as described by the manufacturer.
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
).
RTPCR.
RTPCR was performed using a one-step RTPCR 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).
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 RTPCR except that 30 cycles were performed instead of 35.
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 ).
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.
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(YA), TA-120F/121R for pRCASBP(A)-J:env(Y
S), TA-122F/123R for pRCASBP(A)-J:env(M
V), TA-124F/125R for pRCASBP(A)-J:env(M
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).
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Results |
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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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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 34 mm in diameter were seen at the site of cell implantation at 19 days post-implantation. These grew progressively to 67 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 67 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, whitegrey 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 13 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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When the methionine (600) residue in this motif was mutated to valine or leucine [pRCASBP(A)-J:env(MV) and pRCASBP(A)-J:env(M
L)], transformation of DF-1 cells occurred. Furthermore, when the tyrosine residue (597) was mutated to alanine or serine [pRCASBP(A)-J:env(Y
A) and pRCASBP(A)-J:env(Y
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(YS) and 10-3·56/ml for pRCASBP(A)-J:env(M
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.
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Discussion |
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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.
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Acknowledgments |
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Received 1 March 2002;
accepted 5 July 2002.