Expression of the jaagsiekte sheep retrovirus envelope glycoprotein is sufficient to induce lung tumors in sheep

Abstract

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses env under the control of its own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses env in trans for JS-RD particles and provides the genomes necessary to deliver JSRV env upon infection. During the optimization of the JS-RD system we determined that both R-U5 (in the viral 5' LTR) and the env region are important for JSRV particle production. Two independent experimental transmission studies were carried out with newborn lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4 and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that JSRV can induce OPA in the absence of viral spread.

FIG. 1.

Schematic representation of the plasmids used in this study. JS-RD particles were derived by transfecting 293T cells with pJS-XE and pGPP-MX. VR1, variable region 1; CMV, cytomegalovirus immediate early promoter; Ψ, packaging signal; MCS, multiple cloning site.

FIG. 2.

Optimization of the JSRV packaging construct. Cell lysates (bottom panel) and virus pellets from supernatants (top panel) of transfected cells were analyzed 48 h posttransfection by SDS-PAGE/Western blotting employing an antiserum against the JSRV p23 (matrix). (A) 293T cells were transfected with the plasmids indicated above each lane. Note that only cells transfected with plasmids containing the R-U5 region of the JSRV-proximal LTR released viral particles into the supernatant. Note that the bands below Gag in the cell lysates are likely the product of partially cleaved Gag (present also in panel C). (B) Quantification of virus release was done by chemifluorescence on Western blots of viral pellets. Shown are the means (± standard errors) obtained in three independent experiments. (C) A positive effect on JSRV particle release was observed in 293T cells cotransfected with pGPP-MX and increasing amounts (from 0.5 to 14 μg) of an expression plasmid for Sam68.

FIG. 3.

The JSRV env region is important for optimal viral particle production. (A) 293T cells were transfected with the plasmids indicated above each lane. Virus pellets from cell supernatants (top panel) and cell lysate (50 μg) were analyzed 48 h posttransfection by SDS-PAGE/Western blotting. Where indicated, Env was supplied in trans by the JS-XE plasmid. Note that the bands below Gag in the cell lysates are likely the product of partially cleaved Gag (present also in panel D). (B) Quantification of virus release was performed by chemifluorescence on Western blots of virus pellets. Shown are the means (± SE) obtained in three independent experiments. (C) 293T cells were transfected with the plasmids indicated. Forty-eight hours posttransfection supernatants were harvested. Virus pellets were analyzed by SDS-PAGE/Western blotting employing an antiserum raised against the JSRV Env. As expected, neither JSRVΔEnv nor JSRVStop1 expresses detectable Env. (D) 293T cells were transfected with the indicated plasmids and p23/Gag was detected as described above. Note that cotransfection of pGPP-MX, pJS-XE, and pCDNA3-HA-Sam68 yields JS-RD virus particles in quantities similar to those of replication-competent JSRV.

FIG. 4.

Macroscopic and microscopic lesions induced by JS-RD. Three of the five JS-RD-inoculated lambs showed numerous isolated lesions creamy in color and of hard consistency (A and B). (C) Histology from a lung section of a JS-RD-inoculated lamb shows the presence of a papillary to acinar expanding nodule that compresses the surrounding normal alveoli. (D and E) Immunohistochemistry in lung sections from a JS-RD-inoculated lamb shows expression of the JSRV Env in tumor cells (characterized by the intracytoplasmic brown color). Micrographs are shown at both low (D) and high (E) magnification. (F and G) Immunohistochemistry of lung sections of JSRV-inoculated lamb no. B689. Note the presence of neoplastic foci of various sizes in close proximity. Micrographs are shown at both low (F) and high (G) magnification. (H and I) Phosphorylated Erk1/2 is detectable both in JSRV- and JS-RD-induced tumors. Detection of phospho-Erk1/2 was achieved by immunohistochemistry both in lung sections from JSRV-inoculated lambs (I) and JS-RD-inoculated lambs (H). A positive reaction is characterized by a cytoplasmic brown color. Bars, 100 μm (panels E, G, H, and I) and 500 μm (panels C, D, and F).

FIG. 5.

Detection of JS-RD in experimentally inoculated lambs. Representative examples of PCRs from lambs inoculated with JS-RD, JSRV (positive control), or packaging construct only (mock). DNA was extracted from seven anatomical regions (indicated with letters A to G and corresponding, respectively, to the cranial part of the left cranial lobe, caudal part of left cranial lobe, left diaphragmatic lobe, right diaphragmatic lobe, right middle lobe, right cranial lobe, and accessory lobe) and tested by PCR specific for either JS-XE (no. 1 and 2) or JSRV (no. 3 and 4) as indicated in Materials and Methods. Specific PCR products for JS-XE are found only in JS-RD-infected lambs (no. 77 and 74). JSRV is amplified only by PCRs no. 3 and 4 in DNA extracted from tumor tissue of lamb no. 685 (lanes indicated with +). PCR for enJSRV DNA was used to control for the quality of the DNA preparations.