Jaagsiekte sheep retrovirus proviral clone JSRV(JS7), derived from the JS7 lung tumor cell line, induces ovine pulmonary carcinoma and is integrated into the surfactant protein A gene

Abstract

Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or Clara cells caused by a type D/B chimeric retrovirus, jaagsiekte sheep retrovirus (JSRV). Here we report the isolation, sequencing, pathogenicity, and integration site of a JSRV provirus isolated from a sheep lung tumor cell line (JS7). The sequence of the virus was 93 to 99% identical to other JSRV isolates and contained all of the expected open reading frames. To produce virions and test its infectivity, the JS7 provirus (JSRV(JS7)) was cloned into a plasmid containing a cytomegalovirus promoter and transfected into 293T cells. After intratracheal inoculation with virions from concentrated supernatant fluid, JSRV-associated OPC lesions were found in one of four lambs, confirming that JSRV(JS7) is pathogenic. In JS7-cell DNA, the viral genome was inserted in the protein-coding region for the surfactant protein A (SP-A) gene, which is highly expressed in ATII cells, in an orientation opposite to the direction of transcription of the SP-A gene. No significant transcription was detected from either the viral or the SP-A gene promoter in the JS7 cell line at passage level 170. The oncogenic significance of the JSRV proviral insertion involving the SP-A locus in the JS7 tumor cell line is unknown.

FIG. 1

Features of the JS7 cell line. (A) Confluent monolayer showing the polygonal, epithelial appearance of JS7 cells and presence of perinuclear granules (arrows) (bar = 20 μm). (B) Transmission electron micrograph showing ultrastructural features of JS7 cells including surface microvilli (mv), desmosomes (arrows), and intracytoplasmic lamellar body-like structures (arrowheads) (bar = 10 μm). (C) Protein immunoblot analysis using goat antiserum to Mason-Pfizer monkey virus p27 and iodinated rabbit anti-sheep F(ab)₂ to detect the 26-kDa JSRV CA (arrowhead) in lung fluid from a natural case of OPC (lane a) and in supernatants from a JS7 cell culture, passage 8 (lane b). Immunoglobulin light chain (faint fuzzy band above the JSRV CA band in lung fluid [lane a]) is absent in preparations from the JS7 culture supernatant.

FIG. 2

Alignment of JSRV_JS7 provirus restriction maps. (A) Results of Southern blot analysis of JS7 genomic DNA digested with restriction endonucleases EcoRI (E), HindIII (H), and SacI (S). The membrane was hybridized with probes specific to the U3 and TM regions of JSRV (1). (B) Full-length proviral lambda clone 2-1. (C) Partial-length proviral lambda clone 5-1.

FIG. 3

CMV-based JSRV_JS7 plasmid constructs. Schematic representation of the full-length JSRV_JS7 provirus clone in which the 5′ U3 region was replaced by the CMV promoter inserted 5′ to the JSRV_JS7 RNA start site. The insert shows the context of the region joining the CMV promoter with the 5′ R region of JSRV_JS7. Also shown are pCMV-J:gag-pol and pCMV-J:env. Both of these constructs were designed to overexpress viral proteins by using the CMV promoter to drive transcription. Standard retrovirus notation is used. These constructs were transfected into 293T cells for production of JSRV virions.

FIG. 4

Production of JSRV in 293T cells. (A) RT activity in supernatants of 293T cells or in lung fluid of an OPC-affected lamb detected by measuring the amount of BrdU incorporation into an immobilized RNA template (Lenti-RT; Cavidi Tech AB). Colorimetric quantitation of BrdU incorporation was achieved by the binding of a BrdU-specific antibody conjugated with alkaline phosphatase. Supernatant was prepared from 293T cells transiently transfected with the JSRV plasmids described in Fig. 3 and pooled. Combined supernatant was then concentrated ∼20-fold by ultrafiltration; the ultrafiltrate was also examined for RT activity. RT activity in lung fluid from an experimental case of OPC (85/13) was used as an indicator for acceptable levels of JSRV_JS7 concentrated virus for lamb inoculations. (B) Western immunoblot analysis of concentrated JSRV_JS7 virus from transfected 293T cells using rabbit antiserum to JSRV CA. Lane a represents 30 μl of the lung fluid (85/13) described above. Lane b represents 30 μl of concentrated supernatant from 293T cells. OD, optical density.

FIG. 5

Experimental induction of OPC by JSRV_JS7. Lung tissue from a JSRV_JS7-inoculated lamb was fixed in neutral formalin, sectioned at 4 to 6 μm, and stained. (A) Microscopic tumor nodule detected in lamb number 1615 (hematoxylin and eosin stain; bar = 40 μm). Note the increased numbers of alveolar macrophages in alveoli surrounding the tumor nodule (arrows). (B) JSRV antigen presence in the same tumor nodule detected by immunohistochemistry using rabbit antiserum to JSRV capsid developed using Vector Red and using Carazzi's hematoxylin as a counterstain (bar = 40 μm).

FIG. 6

SP-A is the site of JSRV_JS7 proviral integration. (A) JSRV_JS7-provirus is integrated in a reverse orientation to the 5′ untranslated region of SP-A exon 1. Both JSRV_JS7 and SP-A sequence fragments are shown to scale. Ovine SP-A exons are shaded and introns are white. US is a 25-nucleotide upstream “enhancer” region which shares high homology to sequences in human and baboon. (B) The boundaries of the integration site are shown in detail. Exon 1 is predicted to begin approximately 25 nucleotides downstream of the TATA box. Integration of JSRV_JS7 interrupts the proposed six-amino-acid exon 1 SP-A translation product. The dotted line represents the JSRV_JS7 provirus. GGCTGT is the retroviral target sequence duplicated by the host upon integration. Amino acids are indicated above some of the codons in the nucleotide sequences as follows: M, Met; G, Gly; C, Cys; V, Val; R, Arg; A, Ala. us, upstream; ds, downstream.