Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA

Abstract

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2-3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21DeltaGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential.

Figure 1

Plasmids used in this study. They are described in Materials and Methods. The restriction sites used for cloning are indicated. The two stop codons in the orf-x reading frame of pCMV2JS21Δorfx are shown as vertical bars underlined by asterisks. The broken lines in pCMV3JS21ΔGP and pCMV3JS21ΔGPΔStuI indicate deletions.

Figure 2

Transformation of NIH 3T3 cells. NIH 3T3 cells were transfected with 28 μg of pCDNA3.1(−) (A) or pCMV2JS21 (B) and maintained in culture for 28 days. A typical focus of transformed cells is shown in B. In parallel experiments, transfected cells were passaged every 3–4 days. After 6 weeks, the cells transfected with pCDNA3.1(−) (C) had the typical appearance of NIH 3T3 cells, whereas the cells transfected with pCMV2JS21 (D) were morphologically altered, with loss of contact inhibition. pCDNA3.1(−)-transfected NIH 3T3 cells did not form large colonies in soft agar (E) whereas NIH 3T3 cells transformed by pCMV2JS21 showed anchorage independent colonies (F). (Magnification: ×40.)

Figure 3

JSRV env transcripts. (A) Northern blotting of total RNA collected from 293T cells transfected with pCMV2JS21, pCMV3JS21ΔGP, and mock-transfected 293T cells. Filters were hybridized with the env-2 probe as described in Materials and Methods. 293T cells transfected with pCMV3JS21ΔGP lack the 7.5-kb band (corresponding to the full-length JSRV genome) because the gag and pol sequences have been deleted. (B) Northern blotting of total RNA from NIH 3T3 cells transformed by pCMV2JS21, parental NIH 3T3 cells, lung from a healthy sheep, and lung from tumor lesions of two sheep affected by OPC. Both the 7.5- and 2.4-kb transcripts are readily visible whereas the 1.2-kb transcript is present at a low level.

Figure 4

Identification of JSRV env transcripts. (A) Northern blotting on 293T cells transfected with pCMV2JS21 vs. mock-transfected cells was carried out to identify the structure of the 1.2-kb env transcript. Blot hybridization was carried out in parallel with a U3 probe and with the env-2 and env-upstream probes. The 1.2-kb transcript is apparent only with the env-2 probe. (B) RT-PCR cloning mapped the 1.2-kb transcript (tr-env) as a prematurely polyadenylated mRNA terminating at position 6301. (C) Transfection of plasmid pCMV3JS21ΔGPΔStuI that expresses only the 1.2-kb tr-env mRNA in 293T cells and Northern blotting.