(1/48) Complete sequence of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep.
The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses. (+info)
(2/48) Jaagsiekte retrovirus is widely distributed both in T and B lymphocytes and in mononuclear phagocytes of sheep with naturally and experimentally acquired pulmonary adenomatosis.
Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus specifically associated with a contagious lung tumor of sheep, sheep pulmonary adenomatosis (SPA). JSRV replicates actively in the transformed epithelial cells of the lung, and JSRV DNA and RNA have been detected in lymphoid tissues of naturally affected animals. To determine the lymphoid target cells of JSRV, CD4(+) T cells, CD8(+) T cells, B lymphocytes, and adherent cell (macrophage/monocyte) populations were isolated from the mediastinal lymph nodes of naturally affected sheep and lambs inoculated with JSRV. Cells were enriched to high purity and then analyzed for JSRV proviral DNA by heminested PCR, and the proviral burden was quantitated by limiting dilution analysis. JSRV proviral DNA was found in all subsets examined but not in appropriate negative controls. In sheep naturally affected with SPA, JSRV proviral burden was greatest in the adherent cell population. In the nonadherent lymphocyte population, surface immunoglobulin-positive B cells contained the greatest proviral burden, while CD4(+) and CD8(+) T cells contained the lowest levels of JSRV proviral DNA. In most of the cases (5 of 8), provirus also could be detected in the peripheral blood mononuclear cell (PBMC) population. A kinetic study of JSRV infection in the mediastinal lymphocyte population of newborn lambs inoculated with JSRV found that JSRV proviral DNA could be detected as early as 7 days postinoculation before the onset of pulmonary adenomatosis, although the proviral burden was greatly reduced compared to adult natural cases. This was reflected in the levels found in PBMC since proviral DNA was detected in 2 of 13 animals. At the early time points studied (7 to 28 days postinoculation) no one subset was preferentially infected. These data indicate that JSRV can infect lymphoid and phagocytic mononuclear cells of sheep and that dissemination precedes tumor formation. Infection of lymphoid tissue, therefore, may play an important role in the pathogenesis of SPA. (+info)
(3/48) Sequence comparison of JSRV with endogenous proviruses: envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor.
Ovine pulmonary carcinoma, a contagious lung cancer of sheep, is caused by the oncogenic jaagsiekte sheep retrovirus (JSRV) that is closely related to a family of endogenous sheep retroviral sequences (ESRVs). By using exogenous virus-specific U3 oligonucleotide primers, the entire JSRV proviral genome or its 3' part was amplified from tumor DNA. Analysis of these proviral sequences revealed a novel open reading frame (ORF) within the pol coding region, designated ORF X, which was well conserved in ESRV and JSRV sequences. Deduced amino acids of ORF X showed similarity to a portion of the mammalian adenosine receptor subtype 3, a member of the G-protein-coupled receptor family. Comparison of deduced env amino acids of six JSRV strains from three continents identified 15 residues that defined two distinct genotypes of JSRVs. Sequence analysis identified two highly variable regions between JSRV and ESRV in the transmembrane domain of env (TM) and the 3' unique sequence (U3) of the long terminal repeat, from which JSRV-specific DNA probes were derived. By using these DNA probes in Southern hybridization, for the first time we successfully identified JSRV proviral sequences in tumor genomic DNA in the presence of multiple ESRV loci, validating the use of exogenous virus-specific DNA probes in the analysis of oncogenic proviral integration sites and identification of integrated exogenous proviral sequences. (+info)
(4/48) Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep.
Sheep pulmonary adenomatosis (SPA) is a contagious and experimentally transmissible lung cancer of sheep resembling human bronchiolo-alveolar carcinoma. A type D retrovirus, known as jaagsiekte sheep retrovirus (JSRV), has been associated with the etiology of SPA, but its exact role in the induction of the tumor has not been clear due to the lack of (i) a tissue culture system for the propagation of JSRV and (ii) an infectious JSRV molecular clone. To investigate the role of JSRV in the etiology of SPA, we isolated a full-length JSRV proviral clone, pJSRV21, from a tumor genomic DNA library derived from a natural case of SPA. pJSRV21 was completely sequenced and showed open reading frames in agreement with those deduced for the original South African strain of JSRV. In vivo transfection of three newborn lambs by intratracheal inoculation with pJSRV21 DNA complexed with cationic lipids showed that pJSRV21 is an infectious molecular clone. Viral DNA was detected in the peripheral blood mononuclear cells (PBMCs) of the transfected animals by a highly sensitive JSRV-U3 heminested PCR at various time points ranging from 2 weeks to 6 months posttransfection. In addition, proviral DNA was detected in the PBMCs, lungs, and mediastinal lymph nodes of two lambs sacrificed 9 months posttransfection, but no macroscopic or histological SPA lesion was induced. We prepared JSRV particles by transient transfection of 293T cells with a JSRV construct (pCMV2JS21) in which the upstream U3 was replaced with the cytomegalovirus early promoter. Four newborn lambs were inoculated with JSRV21 particles produced in this manner, and two of them showed the classical signs of SPA 4 months postinfection. The resulting tumors were positive for JSRV DNA and protein. Thus, JSRV21 is an infectious and pathogenic molecular clone and is necessary and sufficient to induce sheep pulmonary adenomatosis. (+info)
(5/48) In vitro infection of ovine cell lines by Jaagsiekte sheep retrovirus.
Sheep pulmonary adenomatosis (SPA), also known as jaagsiekte or ovine pulmonary carcinoma, is a contagious lung cancer of sheep, originating from type II pneumocytes and Clara cells. Previous studies have implicated a type D retrovirus (jaagsiekte sheep retrovirus [JSRV]) as the causative agent of SPA. We recently isolated a proviral clone of JSRV from an animal with a spontaneous case of SPA (JSRV(21)) and showed that it harbors an infectious and oncogenic virus. This demonstrated that JSRV is necessary and sufficient to induce SPA. A major impediment in research on JSRV has been the lack of an in vitro tissue culture system for the virus. The experiments reported here show the first successful in vitro infection with this virus, using the JSRV(21) clone. JSRV(21) virus was obtained by transiently transfecting human 293T cells with a plasmid containing the JSRV(21) provirus driven by the human cytomegalovirus immediate-early promoter. Virus produced in this manner exhibited reverse transcriptase (RT) activity that banded at 1.15 g/ml in sucrose density gradients. Infection of concentrated JSRV(21) into ovine choroid plexus (CP), testes (OAT-T3), turbinate (FLT), and intestinal carcinoma (ST6) cell lines resulted in establishment of infection as measured by PCR amplification. Evidence that this reflected genuine infection included the fact that heat inactivation of the virus eliminated it, the levels of viral DNA increased with passage of the infected cells, and the infected cells released active RT as measured by the sensitive product enhancement RT assay. The RT activity released from the infected cells banded at 1.15 g/ml, and JSRV(21) provirus was transmitted from infected cells to uninfected ones by cocultivation. However, the amount of virus released from infected cells was low. These results suggest that the JSRV receptor is present on many ovine cell types and that the observed restriction of JSRV expression in vivo to tumor cells might be controlled by factors other than the viral receptor. Finally we tagged the U3 of pJSRV(21) with the bacterial supF gene, an amber suppressor tRNA gene. The resulting clone, termed pJSRV(supF), is infectious in vitro. It may be a useful tool for future studies on viral DNA integration, since the normal sheep genome contains 15 to 20 copies of highly JSRV-related endogenous sequences that cross-react with many JSRV hybridization probes. (+info)
(6/48) Retrovirus vectors bearing jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3.
Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus associated with a contagious lung tumor of sheep, ovine pulmonary carcinoma. Other than sheep, JSRV is known to infect goats, but there is no evidence of human infection. Until now it has not been possible to study the host range for JSRV because of the inability to grow this virus in culture. Here we show that the JSRV envelope protein (Env) can be used to pseudotype Moloney murine leukemia virus (MoMLV)-based retrovirus vectors and that such vectors can transduce human cells in culture. We constructed hybrid retrovirus packaging cells that express the JSRV Env and the MoMLV Gag-Pol proteins and can produce JSRV-pseudotype vectors at titers of up to 10(6) alkaline phosphatase-positive focus-forming units/ml. Using this high-titer virus, we have studied the host range for JSRV, which includes sheep, human, monkey, bovine, dog, and rabbit cells but not mouse, rat, or hamster cells. Considering the inability of the JSRV-pseudotype vector to transduce hamster cells, we used the hamster cell line-based Stanford G3 panel of whole human genome radiation hybrids to phenotypically map the JSRV receptor (JVR) gene within the p21.3 region of human chromosome 3. JVR is likely a new retrovirus receptor, as none of the previously identified retrovirus receptors localizes to the same position. Several chemokine receptors that have been shown to serve as coreceptors for lentivirus infection are clustered in the same region of chromosome 3; however, careful examination shows that the JSRV receptor does not colocalize with any of these genes. (+info)
(7/48) The long terminal repeat of Jaagsiekte sheep retrovirus is preferentially active in differentiated epithelial cells of the lungs.
Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a contagious bronchioloalveolar carcinoma of sheep known as sheep pulmonary adenomatosis (SPA; ovine pulmonary carcinoma). JSRV is unique among retroviruses because it transforms the alveolar type II cells and the nonciliated bronchiolar cells (Clara cells) of the lungs; these cells are where JSRV is specifically expressed in both naturally and experimentally SPA-affected sheep. In this study, we investigated the cell specificity of JSRV expression. By transient-transfection assays of 23 different cell lines with a reporter plasmid driven by the JSRV long terminal repeat (LTR), pJS21-luc, we found that the JSRV LTR is preferentially active in cell lines derived from type II pneumocytes and Clara cells (MLE-15 and mtCC1-2 mouse cell lines). Reporter assays using progressive 5' deletions of pJS21-luc allowed us to establish that the JSRV enhancers are able to activate the JSRV proximal promoter in MLE-15 and mtCC1-2 cells, but they have very low activity in mouse cells of other lineages (e.g., NIH 3T3). The JSRV enhancers are able to activate heterologous promoters in both MLE-15 and 3T3 cells, although optimal activity is achieved in MLE-15 cells only with the homologous JSRV promoter. Thus, JSRV cell-specific LTR activity appears to result from an interaction between the enhancer elements and the JSRV proximal promoter elements. By mutation analysis, we established that an upstream NF-kappaB-like element appears to be responsible for approximately 50% of the JSRV LTR transcriptional activity in MLE-15 cells. Electrophoretic mobility shift assays showed evidence of a factor(s) that binds to this sequence. Antibody supershift experiments indicated that the factor(s) is not related to NF-kappaB component p50 or p52. This factor also appeared to be present in cells that do not support a high level of JSRV expression. Finally the JSRV(21) LTR contains putative enhancer binding motifs for transcription factors such as hepatocyte nuclear factor 3 (HNF-3) that are involved in lung-specific gene expression. Cotransfection experiments demonstrated that exogenous HNF-3 is able to enhance the expression of pJS21-luc in NIH 3T3 cells, which normally show minimal enhancer activity for the JSRV LTR. (+info)
(8/48) Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus.
Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSVs) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRVs in vivo was studied by in situ hybridization. All of the three enJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV(21). Phylogenetic analysis distinguished five ovine type D retroviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the three enJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs. (+info)