Jaagsiekte sheep retrovirus envelope efficiently pseudotypes human immunodeficiency virus type 1-based lentiviral vectors.
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ABSTRACT:
SUBMITTER: Liu SL
PROVIDER: S-EPMC369219 | biostudies-literature | 2004 Mar
REPOSITORIES: biostudies-literature
Publications
Jaagsiekte sheep retrovirus envelope efficiently pseudotypes human immunodeficiency virus type 1-based lentiviral vectors.
Liu Shan-Lu SL Halbert Christine L CL Miller A Dusty AD
Journal of virology 20040301 5
Jaagsiekte sheep retrovirus (JSRV) infects lung epithelial cells in sheep, and oncoretroviral vectors bearing JSRV Env can mediate transduction of human cells, suggesting that such vectors might be useful for lung-directed gene therapy. Here we show that JSRV Env can also efficiently pseudotype a human immunodeficiency virus type 1-based lentiviral vector, a more suitable vector for transduction of slowly dividing lung epithelial cells. We created several chimeric Env proteins that, unlike the p ...[more]
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Project description: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.
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Project description:Jaagsiekte sheep retrovirus (JSRV) induces bronchioalveolar tumors in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse airway epithelial cells induces similar tumors, indicating that Env expression is sufficient for tissue-specific tumor formation. Enzootic nasal tumor virus (ENTV) is related to JSRV but induces tumors in the nasal epithelium of sheep and goats. Here we found that ENTV Env can also induce tumors in mice but, unexpectedly, with a phenotype identical to that of tumors induced by the JSRV Env, indicating that factors other than Env mediate the tissue specificity of tumor induction by ENTV.
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Project description:BackgroundAdenocarcinoma is the most common type of non-small cell lung cancer and is frequently observed in non-smoking patients. Adenocarcinoma in-situ (formerly referred to as bronchioloalveolar carcinoma) is a subset of lung adenocarcinoma characterized by growth along alveolar septae without evidence of stromal, vascular, or pleural invasion, that disproportionately affects never-smokers, women, and Asians. Adenocarcinoma in-situ is morphologically and histologically similar to a contagious lung neoplasm of sheep called ovine pulmonary adenocarcinoma (OPA). OPA is caused by infection with the exogenous betaretrovirus, jaagsiekte sheep retrovirus (JSRV), whose envelope protein (Env) is a potent oncogene. Several studies have reported that a proportion of human lung adenocarcinomas are immunopositive for an antigen related to the Gag protein of JSRV, however other groups have been unable to verify these observations by PCR.MethodsHere we examine human lung cancer tissue arrays (TA) for evidence of JSRV Env protein and DNA by immunohistochemical staining and PCR, respectively.ResultsOur results reveal that a subset of human lung cancers express an antigen that reacts with a JSRV Env-specific monoclonal antibody in immunohistochemistry and that exogenous JSRV-like env and gag sequences can be amplified from TA tumor samples, albeit inefficiently.ConclusionsWhile a causative role has not been established, these data suggest that a JSRV-like virus might infect humans. With next generation sequencing approaches, a JSRV-like virus in human lung cancers may be identified which could have profound implications for prevention, diagnosis and therapy.
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Project description:A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known.
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Project description:Jaagsiekte sheep retrovirus (JSRV) causes a lethal lung cancer in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse lung, by using a replication-defective adeno-associated virus type 6 (AAV6) vector, induces tumors resembling those seen in sheep. However, the mouse and sheep tumors have not been carefully compared to determine if Env expression alone in mice can account for the disease features observed in sheep, or whether additional aspects of virus replication in sheep are important, such as oncogene activation following retrovirus integration into the host cell genome.We have generated mouse monoclonal antibodies (Mab) against JSRV Env and have used these to study mouse and sheep lung tumor histology. These Mab detect Env expression in tumors in sheep infected with JSRV from around the world with high sensitivity and specificity. Mouse and sheep tumors consisted mainly of well-differentiated adenomatous foci with little histological evidence of anaplasia, but at long times after vector exposure some mouse tumors did have a more malignant appearance typical of adenocarcinoma. In addition to epithelial cell tumors, lungs of three of 29 sheep examined contained fibroblastic cell masses that expressed Env and appeared to be separate neoplasms. The Mab also stained nasal adenocarcinoma tissue from one United States sheep, which we show was due to expression of Env from ovine enzootic nasal tumor virus (ENTV), a virus closely related to JSRV. Systemic administration of the AAV6 vector encoding JSRV Env to mice produced numerous hepatocellular tumors, and some hemangiomas and hemangiosarcomas, showing that the Env protein can induce tumors in multiple cell types.Lung cancers induced by JSRV infection in sheep and by JSRV Env expression in mice have similar histologic features and are primarily characterized by adenomatous proliferation of peripheral lung epithelial cells. Thus it is unnecessary to invoke a role for insertional mutagenesis, gene activation, viral replication, or expression of other viral gene products in sheep lung tumorigenesis, although these processes may play a role in other clinically less important sequelae of JSRV infection such as metastasis observed with variable frequency in sheep.
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Project description:Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a neoplastic lung disease of sheep. OPA is an important economic and welfare issue for sheep farmers and a valuable naturally occurring animal model for human lung adenocarcinoma. Here, we used RNA sequencing to study the transcriptional response of ovine lung tissue to infection by JSRV. We identified 1,971 ovine genes differentially expressed in JSRV-infected lung compared to noninfected lung, including many genes with roles in carcinogenesis and immunomodulation. The differential expression of selected genes was confirmed using immunohistochemistry and reverse transcription-quantitative PCR. A key finding was the activation of anterior gradient 2, yes-associated protein 1, and amphiregulin in OPA tumor cells, indicating a role for this oncogenic pathway in OPA. In addition, there was differential expression of genes related to innate immunity, including genes encoding cytokines, chemokines, and complement system proteins. In contrast, there was little evidence for the upregulation of genes involved in T-cell immunity. Many genes related to macrophage function were also differentially expressed, reflecting the increased abundance of these cells in OPA-affected lung tissue. Comparison of the genes differentially regulated in OPA with the transcriptional changes occurring in human lung cancer revealed important similarities and differences between OPA and human lung adenocarcinoma. This study provides valuable new information on the pathogenesis of OPA and strengthens the use of this naturally occurring animal model for human lung adenocarcinoma.IMPORTANCE Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined the changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer.
| S-EPMC6803282 | biostudies-literature
Project description:Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The details of early events in the pathogenesis of OPA are not fully understood. For example, the identity of the JSRV target cell in the lung has not yet been determined. Mature OPA tumors express surfactant protein-C (SP-C) or Clara cell-specific protein (CCSP), which are specific markers of type II pneumocytes or Clara cells, respectively. However, it is unclear whether these are the cell types initially infected and transformed by JSRV or whether the virus targets stem cells in the lung that subsequently acquire a differentiated phenotype during tumor growth. To examine this question, JSRV-infected lung tissue from experimentally infected lambs was studied at early time points after infection. Single JSRV-infected cells were detectable 10 days postinfection in bronchiolar and alveolar regions. These infected cells were labeled with anti-SP-C or anti-CCSP antibodies, indicating that differentiated epithelial cells are early targets for JSRV infection in the ovine lung. In addition, undifferentiated cells that expressed neither SP-C nor CCSP were also found to express the JSRV Env protein. These results enhance the understanding of OPA pathogenesis and may have comparative relevance to human lung cancer, for which samples representing early stages of tumor growth are difficult to obtain.
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Project description: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.
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Project description:Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.
| S-EPMC479065 | biostudies-literature
Project description:BackgroundSheep pulmonary adenocarcinoma (OPA) is a contagious lung cancer of sheep caused by the Jaagsiekte retrovirus (JSRV). OPA typically has a serious economic impact worldwide. A vaccine has yet to be developed, even though the disease has been globally spread, along with its complications. This study aimed to construct an effective multi-epitopes vaccine against JSRV eliciting B and T lymphocytes using immunoinformatics tools.ResultsThe designed vaccine was composed of 499 amino acids. Before the vaccine was computationally validated, all critical parameters were taken into consideration; including antigenicity, allergenicity, toxicity, and stability. The physiochemical properties of the vaccine displayed an isoelectric point of 9.88. According to the Instability Index (II), the vaccine was stable at 28.28. The vaccine scored 56.51 on the aliphatic index and -0.731 on the GRAVY, indicating that the vaccine was hydrophilic. The RaptorX server was used to predict the vaccine's tertiary structure, the GalaxyWEB server refined the structure, and the Ramachandran plot and the ProSA-web server validated the vaccine's tertiary structure. Protein-sol and the SOLPro servers showed the solubility of the vaccine. Moreover, the high mobile regions in the vaccine's structure were reduced and the vaccine's stability was improved by disulfide engineering. Also, the vaccine construct was docked with an ovine MHC-1 allele and showed efficient binding energy. Immune simulation remarkably showed high levels of immunoglobulins, T lymphocytes, and INF-γ secretions. The molecular dynamic simulation provided the stability of the constructed vaccine. Finally, the vaccine was back-transcribed into a DNA sequence and cloned into a pET-30a ( +) vector to affirm the potency of translation and microbial expression.ConclusionA novel multi-epitopes vaccine construct against JSRV, was formed from B and T lymphocytes epitopes, and was produced with potential protection. This study might help in controlling and eradicating OPA.
| S-EPMC9463060 | biostudies-literature