Modulation of autophagy affected tumorigenesis induced by the envelope glycoprotein of JSRV.

Ovine pulmonary adenocarcinoma (OPA), caused by the jaagsiekte sheep retrovirus (JSRV), is a chronic, progressive, and contagious lung tumor that seriously affects sheep production. It also represents a valuable animal model for several human lung adenocarcinomas. However, little is known about the role of autophagy in OPA tumorigenesis. Here, Western blotting combined with transmission electron microscopy examination and Cyto-ID dye staining was employed for evaluation of changes of autophagic levels. The results of the present study showed that expression of the autophagy marker proteins Beclin-1 and LC3 was decreased in OPA lung tissues, as well as in cells overexpressing the envelope glycoprotein of JSRV (JSRV Env). Reduced numbers of autophagosomes were also observed in cells overexpressing JSRV Env, although assessment of autophagic flux showed that JSRV Env overexpression did not block the formation of autophagosomes, suggesting increased degradation of autolysosomes. Last, mouse xenograft experiments indicated that inhibition of autophagy by 3-methyladenine suppressed both tumor growth and the epithelial-to-mesenchymal transition. In conclusion, JSRV, through JSRV Env, takes advantage of the autophagy process, leading to the development of OPA.

Clinical, molecular, and pathological investigations of ovine pulmonary adenocarcinoma in the middle of Iraq.

Background: Ovine pulmonary adenocarcinoma (OPA), caused by Jaagsiekte sheep retrovirus (JSRV), is a contagious neoplastic disease in sheep characterized by chronic respiratory signs, inducing the transformation of secretory epithelial cells of the distal respiratory tract. Aims: To perform clinical, epidemiological, and molecular studies with evaluation of some predisposing factors at the herd level of OPA infection in sheep in Al-Qadisiyah Province, Iraq. Methods: The first step of the study was undertaken to evaluate the clinical cases of OPA in naturally infected sheep and correlation with observing respiratory signs. Seventy-five sheep with chronic respiratory signs were examined clinically, and by molecular and sequences analysis. The second step was the epidemiological part that was carried out on 195 randomly selected animals from 30 flocks, with the prevalence rate based on PCR; sex, age, and size of flocks were assessed, as well as macroscopic and microscopic features of the neoplastic lung. Deep nasal swabs and nasal secretion were collected from all animals. RNA extraction and RT-PCR were also carried out. Results: The results showed that 12 (16%) samples were positive for OPA, based on env gene-specific primers. Nucleotide sequences of partial 545 bp of the env gene showed (0.07-0.12) variations from global strains presented in the NCBI database. The prevalence rate of OPA was 21/195 (10.76%) with PCR. The epidemiological factors analysis showed that there was no effect of sex and herd size on the prevalence rates (p ≥ 0.01), whereas age was significantly affected and the age of 2-4 years was more susceptible (p ≥ 0.01). Gross and microscopic examinations were discussed with the confirmation of an OPA infection. Conclusion: The current study provides useful data about the clinical and epidemiological features of JSRV that is circulating in sheep of Iraq, and concludes that epidemiological studies and disease control may require multi-diagnostic assays.

Insertion of an endogenous Jaagsiekte sheep retrovirus element into the BCO2 - gene abolishes its function and leads to yellow discoloration of adipose tissue in Norwegian Spælsau (Ovis aries).

BACKGROUND: The accumulation of carotenoids in adipose tissue leading to yellow fat is, in sheep, a heritable recessive trait that can be attributed to a nonsense mutation in the beta-carotene oxygenase 2 (BCO2) gene. However, not all sheep breeds suffering from yellow fat have this nonsense mutation, meaning that other functional mechanisms must exist. We investigated one such breed, the Norwegian spælsau. RESULTS: In spælsau we detected an aberration in BCO2 mRNA. Nanopore sequencing of genomic DNA revealed the insertion of a 7.9 kb endogenous Jaagsiekte Sheep Retrovirus (enJSRV) sequence in the first intron of the BCO2 gene. Close examination of its cDNA revealed that the BCO2 genes first exon was spliced together with enJSRV-sequence immediately downstream of a potential -AG splice acceptor site at enJSRV position 415. The hybrid protein product consists of 29 amino acids coded by the BCO2 exon 1, one amino acid coded by the junction sequence, followed by 28 amino acids arbitrary coded for by the enJSRV-sequence, before a translation stop codon is reached. CONCLUSIONS: Considering that the functional BCO2 protein consists of 575 amino acids, it is unlikely that the 58 amino acid BCO2/enJSRV hybrid protein can display any enzymatic function. The existence of this novel BCO2 allele represents an alternative functional mechanism accounting for BCO2 inactivation and is a perfect example of the potential benefits for searching for structural variants using long-read sequencing data.

A survey of jaagsiekte sheep retrovirus (JSRV) infection in sheep in the three northeastern provinces of China.

Ovine pulmonary adenomatosis (OPA) is caused by jaagsiekte sheep retrovirus (JSRV) and is a chronic, progressive, and infectious neoplastic lung disease in sheep, which causes significant economic losses to the sheep industry. Neither a vaccine nor serological diagnostic methods to detect OPA are available. We performed a JSRV infection survey in sheep using blood samples (n = 1,372) collected in the three northeastern provinces of China (i.e., Inner Mongolia, Heilongjiang, and Jilin) to determine JSRV infection status in sheep herds using a real-time PCR assay targeting the gag gene of JSRV. The ovine endogenous retrovirus sequence was successfully amplified in all sheep samples tested (296 from the Inner Mongolia Autonomous Region, 255 from Jilin province, and 821 from Heilongjiang province). Subsequently, we attempted to distinguish exogenous JSRV (exJSRV) and endogenous JSRV (enJSRV) infections in these JSRV-positive samples using a combination assay that identifies a ScaI restriction site in an amplified 229-bp fragment of the gag gene of JSRV and a "LHMKYXXM" motif in the cytoplasmic tail region of the JSRV envelope protein. The ScaI restriction site is present in all known oncogenic JSRVs but absent in ovine endogenous retroviruses, while the "LHMKYXXM" motif is in all known exJSRVs but not in enJSRVs. Interestingly, one JSRV strain (HH13) from Heilongjiang province contained the "LHMKYXXM" motif but not the ScaI enzyme site. Phylogenetic analysis showed that strain HH13 was closely related to strain enJSRV-21 reported in the USA, indicating that HH13 could be an exogenous virus. Our results provide valuable information for further research on the genetic evolution and pathogenesis of JSRV.

JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-mediated Transgene Expression in the Lung and Liver of Mice.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3' end of the JSRV env gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.

Old origin of a protective endogenous retrovirus (enJSRV) in the Ovis genus.

Sheep, the Jaagsiekte sheep retrovirus (JSRV) and its endogenous forms (enJSRVs) are a good model to study long-time relationships between retroviruses and their hosts. Taking advantage of 76 whole genome resequencing data of wild and domestic Ovis, we investigated the evolution of this relationship. An innovative analysis of re-sequencing data allowed characterizing 462 enJSRVs insertion sites (including 435 newly described insertions) in the Ovis genus. We focused our study on endogenous copies inserted in the q13 locus of chromosome 6 (6q13). Those copies are known to confer resistance against exogenous JSRV thanks to alleles bearing a mutation in the gag gene. We characterized (i) the distribution of protective and non-protective alleles across Ovis species and (ii) the copy number variation of the 6q13 locus. Our results challenged the previous hypothesis of fixation and amplification of the protective copies in relation with domestication, and allowed building a new model for the evolution of the 6q13 locus. JSRV would have integrated the 6q13 locus after the Ovis-Capra divergence (5-11 MYA) and before the Ovis diversification (2.4-5 MYA). The protective mutation in the enJSRV 6q13 copy appeared shortly after its insertion and was followed by genomic amplifications, after the divergence between Pachyform lineage on one side and the Argaliform and moufloniform lineages on the other (2.4-5 MYA). Considering the potential selective advantage of the protective mutation, its fixation in both sheep and its closest wild relative Ovis orientalis may be due to natural selection before domestication from O. orientalis populations.

Characterization of a transgenic mouse model exhibiting spontaneous lung adenocarcinomas with a metastatic phenotype.

Developing lung cancer in mouse models that display similarities of both phenotype and genotype will undoubtedly provide further and better insights into lung tumor biology. Moreover, a high degree of pathophysiological similarity between lung tumors from mouse models and their human counterparts will make it possible to use these mouse models for preclinical tests. Ovine pulmonary adenocarcinomas (OPAs) present the same symptoms as adenocarcinomas in humans and are caused by a betaretrovirus. OPAs have served as an exquisite model of carcinogenesis for human lung adenocarcinomas. In this study, we characterized the histopathology and transcriptome profiles of a jaagsiekte sheep retrovirus (JSRV)-envelope protein (Env) transgenic mouse model with spontaneous lung tumors, and associations of the transcriptome profiles with tumor invasion/metastasis, especially the phenomenon of the epithelial-mesenchymal transition (EMT). Genetic information obtained from an expression array was analyzed using an ingenuity pathways analysis (IPA) and human disease database (MalaCards). By careful examination, several novel EMT-related genes were identified from tumor cells using RT-qPCR, and these genes also scored high in MalaCards. We concluded that the JSRV-Env mouse model could serve as a spontaneous lung adenocarcinoma model with a metastatic phenotype, which will benefit the study of early-onset and progression of lung adenocarcinoma. In addition, it can also be a valuable tool for biomarkers and drug screening, which will be helpful in developing intervention therapies.

Modulation of autophagy in exJSRV-env-transfected cells through the Akt/mTOR and MAPK signaling pathway.

The envelope (Env) of Jaagsiekte sheep retrovirus (JSRV) is an oncoprotein of ovine pulmonary adenocarcinoma (OPA). Autophagy is involved in different cancers, but how it is carcinogenic in JSRV Env is unclear. Modulation of autophagy in exJSRV-env-NM-transfected cells through the Akt/mTOR and MAPK signaling pathway was studied, and we observed strong positive labeling of p-Akt, p-mTOR, p-MEK1/2, p-ERK1/2, p-p38 and p-JNK in tumor cells and typical type II pneumocytes in naturally infected OPA lung tissues, which was co-aligned with JSRV-Env positive cells as shown by immunohistochemical and microscopic analysis. Akt/mTOR and MAPK pathways were activated in OPA lung and JSRV-Env transfected NIH 3T3 cells. Decreased Beclin1 and LC3 II/I suggested that autophagy was inhibited in OPA lung and JSRV-Env transfected NIH 3T3 cells. Beclin1 and LC3 II/I increased in JSRV-Env transfected NIH3T3 cells treated with mTOR inhibitor (rapamycin), ERK1/2 inhibitor (PD 98059), p38 inhibitor (SB 203580) and JNK inhibitor (SP 600125), suggesting that Akt/mTOR and MAPK pathways were responsible for JSRV-Env decreased autophagy. In conclusion, JSRV Env decreased autophagy in JSRV-Env transfected NIH3T3 cells through Akt/mTOR and MAPK pathways, in particular, JNK and p38 pathways.

Lack of association between polymorphic copies of endogenous Jaagsiekte sheep retrovirus (enJSRVs) and Ovine Pulmonary Adenocarcinoma.

Ovine Pulmonary Adenocarcinoma (OPA) is a retrovirus-induced lung tumor of sheep, goat and mouflon, and its etiologic agent, Jaagsiekte sheep retrovirus (JSRV) is the only virus known to cause a naturally occurred lung adenocarcinoma. The oncogenic JSRV has several endogenous counterparts termed enJSRVs, some of which have been shown to interfere with JSRV replication at early and late stages of the retroviral cycle inhibiting JSRV exit from the cell, and thus, protecting sheep against the infection. In this work, Latxa sheep breed animals were classified depending on the presence/absence of OPA-characteristic clinical lesions in the lung. Using a PCR genotyping method and a logistic regression-based association study, five polymorphic enJSRV copies were analyzed in 49 OPA positive sheep and 124 control individuals. Our results showed that the frequency of the provirus enJSRV-16 is much higher in Latxa sheep breed than in other breeds, suggesting a recent proliferation of this provirus in the studied breed. However, no polymorphic enJSRV was found to be statistically associated with the susceptibility/resistance to OPA development.

[Changes in the Expression of AKT and ERK after the JSRV-Env-Induced Transfection of TC-1 and TC-1-Hyal2 Cells].

This study aims to explore the tumorigenic mechanism of the target cells following JSRV interaction with its receptor. We transfected mouse lung epithelial cells (TC-1) and mouse lung epithelial cells stably expressing sheep Hyal-2(TC-1-Hyal2)with JSRV-Env eukaryotic expression vector, measured the changes in the mRNA and protein expression of AKT(serine/threonine kinase)and ERK(extracellular signal-regulated kinase)in cellular signal transduction pathways, and analyzed the role of sheep Hyal-2in JSRV-Env-induced transformation of TC-1cells.First,TC-1and TC-1-Hyal2 cells were cultured in vitro and were each divided into pEGFP-C1-env transfection group,pEGFP-C1 transfection group, and untransfected group. The expression of key enzymes was determined by PCR and Western blotting. qPCR showed that, for both cell lines, compared with untransfected cells, the expression of AKT and ERK1/2mRNA was significantly increased in the pEGFP-C1-env transfected cells(P<0.05).Western blotting showed that, relative to untransfected cells, transfection with pEGFP-C1-env significantly increased p-Akt (S473)protein expression in both cell lines(P<0.05).Moreover, p-Akt (T308)and p-Erk1/2protein expression was increased significantly in the pEGFP-C1-env transfected TC-1cells(P<0.05),and very significantly in the pEGFP-C1-env transfected TC-1-Hyal2cells(P<0.01).Cells of each type transfected with the empty vector pEGFP-C1 and the untransfected cells did not show significant differences in their mRNA and protein levels of AKT and ERK(P >0.05).Thus, the expression of JSRV-Env in the cell lines TC-1and TC-1-Hyal2 activated the cellular signal transduction pathways Ras-Raf-MAPK and PI3K-Akt.The expression of AKT and ERK was significantly increased in pEGFP-C1-env transfected TC-1and TC-1-Hyal2 cells, but a greater increase was seen in the TC-1-Hyal2 cells.We speculate that Hyal2 plays a catalytic role in JSRV-Env-induced transformation of TC-1cells.

No evidence for viral sequences in five lepidic adenocarcinomas (former "BAC") by a high-throughput sequencing approach.

BACKGROUND: The hypothesis of an infectious etiology of the formerly named bronchiolo-alveolar carcinoma (BAC) has raised controversy. We investigated tumor lung tissues from five patients with former BAC histology using high-throughput sequencing technologies to discover potential viruses present in this type of lung cancer. Around 180 million single reads of 100 bases were generated for each BAC sample. RESULTS: None of the reads showed a significant similarity for Jaagsiekte sheep retrovirus (JSRV) and no other viruses were found except for endogenous retroviruses. CONCLUSIONS: In conclusion, we have demonstrated the absence of JSRV and other known human viruses in five samples of well-characterized lepidic adenocarcinoma.

[Pathological Diagnoses and Whole-genome Sequence Analyses of the Jaagsiekte Sheep Retrovirus in Xinjiang, China].

To carry out pathologic diagnoses and whole-genome sequence analyses of the Jaagsiekte sheep retrovirus (JSRV) in Xinjiang, China, we first observed sheep suspected to have the JSRV. Then, the extracted virus suspension was observed by transmission electron microscopy (TEM). Total RNAs from lungs of JSRV-infected sheep were extracted and reverse-transcribed using a cDNA synthesis kit. Six pairs of primers were designed according to the exogenous reference virus strain (AF105220). Reverse transcription-polymerase chain reaction was carried out from JSRV-infected tissue, and the whole genome of the JSRV sequenced. Our results showed: flow of nasal fluid ("wheelbarrow test"); different sizes of adenoma lesions in the lungs; papillary hyperplasia of alveolar epithelial cells; alveolar cavity filled with macrophages; dissolute nuclei in central lesions. TEM revealed JSRV particles with a diameter of 88 nm to 125. 4 nm. The full-length of the viral genome sequence was 7456 bp. BLAST analyses showed nucleotide homology of 96% and 95% compared with that of the representative strain from the USA (AF105220) and UK (AF357971). Nucleotide homology was 89.8% and 89.9% compared with the endogenous Jaagsiekte sheep retrovirus, Inner Mongolia strain (DQ838493) and USA strain (EF680300). The specific pathogenic amino-acid sequence "YXXM" was found in the TM district, similar to the exogenous JSRV: this gene has been reported to be oncogenic. This is the first report of the complete genomic sequence of the exogenous JSRV from Xinjiang, and could lay the foundation for study of the biological characteristics and pathogenic mechanisms of the pulmonary adenomatosis virus in sheep.

The use of endogenous retroviruses as markers to describe the genetic relationships among local Swedish sheep breeds.

The aim of this study was to describe the genetic relationships among five Swedish sheep breeds using insertional polymorphisms of six endogenous Jaagsiekte retroviruses of sheep. Although the Swedish breeds were found to have genomes of 'primitive' origin, there also are indications of the presence of more recently derived sheep breeds within the ancestries of three of the breeds.

"Ménage à Trois": the evolutionary interplay between JSRV, enJSRVs and domestic sheep.

Sheep betaretroviruses represent a fascinating model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with a variety of highly related endogenous JSRVs, referred to as enJSRVs. During evolution, some of them were co-opted by the host as they fulfilled important biological functions, including placental development and protection against related exogenous retroviruses. In particular, two enJSRV loci, enJS56A1 and enJSRV-20, were positively selected during sheep domestication due to their ability to interfere with the replication of related competent retroviruses. Interestingly, viruses escaping these transdominant enJSRVs have recently emerged, probably less than 200 years ago. Overall, these findings suggest that in sheep the process of endogenization is still ongoing and, therefore, the evolutionary interplay between endogenous and exogenous sheep betaretroviruses and their host has not yet reached an equilibrium.

Jaagsiekte sheep retrovirus detected in human lung cancer tissue arrays.

BACKGROUND: Adenocarcinoma 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. METHODS: Here we examine human lung cancer tissue arrays (TA) for evidence of JSRV Env protein and DNA by immunohistochemical staining and PCR, respectively. RESULTS: Our 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. CONCLUSIONS: While 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.

Diagnosis and phylogenetic analysis of ovine pulmonary adenocarcinoma in China.

Ovine pulmonary adenocarcinoma (OPA) is a lung tumor of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is common in sheep, and it is most commonly observed in China. Without preventative vaccines and serological diagnostic tools for assay of OPA, identification of JSRV based on reverse transcription polymerase chain reaction (RT-PCR) is very important for prevention and control measures for OPA in practice management. In this study, the diagnosis of OPA was made from analysis of clinical signs, pathological observations, JSRV-like particle discovery, and RT-PCR of the target env gene. The phylogenetic analysis showed that the China Shandong (SD) strain studied in this article belonged to exogenous JSRV, and it was very similar to 92k3, which was isolated from sheep in the Kenya (Y18305). The current study reported a severe outbreak of OPA in Shandong Province, China. The observations could offer a comparative view of the env gene of JSRV.

[Cloning and sequencing analyses of the complete genome of the provirus of the Inner-Mongolia pandemic strain of the Jaagsiekte sheep retrovirus].

To investigate the kinship between the Inner Mongolia pandemic strain and representative strains of the Jaagsiekte sheep retrovirus (JSRV), total DNA from the lung tissue of a JSRV-infected sheep in Inner Mongolia was used to clone fragments of gag, pro and pol genes. The recombinant plasmid pMD-JSRV (including complete genomic sequence of the JSRV strain isolated from Inner Mongolia) was constructed by linking all the cloned fragments with long terminal repeat (LTR) and env gene fragments (cloned previous and reserved by our research team). Sequence analyses revealed that the genome was 7690 bp in length and contained several typical molecular markers for exogenous form of JSRV. These included the Sca I restriction site in the gag gene, two predicted "CCHC" motifs of zinc finger in the encoded nucleocapsid protein and the predicted "YXXM" motif in the TM region of Env. Homology analyses showed that the virus strain belonged to the JSRV type II. pMD-JSRV and AF105220 strains shared a nucleotide identification of 95%. The full length genomic clone of JSRV could provide a molecular basis for an infectious JSRV molecular clone as well as an experimental platform to study the detection and pathogenesis of JSRV.

Genetic variability and in vitro transcriptional permissibility of primary ovine beta-retrovirus promoter isolates.

OBJECTIVE: To assess genomic sequence conservation and variation in the proviral promoter of enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) in tissue samples from 3 sheep with nasal adenocarcinoma associated with ENTV and 3 sheep with pulmonary adenocarcinoma associated with JSRV and to identify a cell culture system that supports transcriptional activity of the ENTV and JSRV viral promoters. ANIMALS: 6 adult sheep. PROCEDURES: Standard PCR procedures for detection of the ENTV and JSRV long terminal repeat (LTR) promoter region were performed on samples from the 3 nasal adenocarcinomas and 3 pulmonary adenocarcinomas, respectively. The LTRs were cloned into shuttle vectors, amplified, sequenced, and analyzed. The cloned LTR regions were transferred into reporter plasmids and multiple human and ruminant cell lines, and primary cells were transfected with the promoter-reporter plasmids. The viral promoter activity was evaluated by use of an in vitro ß-galactosidase reporter assay. RESULTS: Each isolate had a unique nucleotide sequence. Single nucleotide polymorphisms were the most common LTR mutation and rarely occurred at transcription factor binding sites. Relative to ENTV, the JSRV promoter isolates had a conserved 66-bp U3 insertion, including the lung-specific transcription factor HNF-3ß binding site. Among the cell lines used, human embryonic kidney (293T) and goat synovial membrane cells supported promoter transcription. CONCLUSIONS AND CLINICAL RELEVANCE: The LTRs of ENTV and JSRV have extensive blocks of sequence conservation. Human 293T and goat synovial membrane cell lines may be suitable in vitro cell culture systems for further research of viral promoter functions.

Betaretroviral envelope subunits are noncovalently associated and restricted to the mammalian class.

The structure of the transmembrane subunit (TM) of the retroviral envelope glycoprotein (Env) is highly conserved among most retrovirus genera and includes a pair of cysteines that forms an intramolecular disulfide loop within the ectodomain. Alpha-, gamma-, and deltaretroviruses have a third cysteine, adjacent to the loop, which forms a disulfide bond between TM and the surface subunit (SU) of Env, while lentiviruses, which have noncovalently associated subunits, lack this third cysteine. The Betaretrovirus genus includes Jaagsiekte sheep retrovirus (JSRV) and mouse mammary tumor virus (MMTV), as well as many endogenous retroviruses. Envelope subunit association had not been characterized in the betaretroviruses, but lack of a third cysteine in the TM ectodomain suggested noncovalently associated subunits. We tested the Env proteins of JSRV and MMTV, as well as human endogenous retrovirus K (HERV-K)108--a betaretrovirus-like human endogenous retrovirus--for intersubunit bonding and found that, as in the lentiviruses, the Env subunits lack an intersubunit disulfide bond. Since these results suggest that the number of cysteines in the TM loop region readily distinguishes between covalent and noncovalent structure, we surveyed endogenous retroviral TM sequences in the genomes of vertebrates represented in public databases and found that (i) retroviruses with noncovalently associated subunits have been present during all of anthropoid evolution and (ii) the noncovalent env motif is limited to mammals, while the covalent type is found among five vertebrate classes. We discuss implications of these findings for retroviral evolution, cross-species transmissions, and recombination events involving the env gene.

Adeno-associated virus vector mediated expression of an oncogenic retroviral envelope protein induces lung adenocarcinomas in immunocompetent mice.

Lung cancer is the most common cause of cancer-related death worldwide. A poor overall survival rate of 16% necessitates the need for novel treatment strategies. Mouse models of lung cancer are important tools for analyzing the significance of somatic mutations in the initiation and progression of lung cancer. Of additional importance, however, are animal models of virally induced cancers. JSRV is a simple betaretrovirus that causes contagious lung cancer in sheep known as ovine pulmonary adenocarcinoma and closely resembles human lung adenocarcinoma. Previously we showed that expression of the JSRV envelope (Env) from an AAV vector induced lung tumors in immunodeficient mice, but not in immunocompetent mice. Because of the importance of studying lung cancer in the context of an intact immune system we sought to improve our mouse model. In this report, we employed the use of a strong JSRV enhancer-promoter combination to express Env at high levels and demonstrate for the first time, lung tumor induction in immunocompetent mice. This occurred despite a robust Env-specific antibody-mediated immune response. The PI3K/Akt and MAPK pathways were activated in both immunocompetent and immunodeficient mice, however, differential activation of PTEN, GSKα, p70S6K, p38MAPK, ATF2 and STAT5 was observed. A JSRV Env lung tumor-derived cell line was shown to have a similar signal transduction activation profile as Env-induced lung tumors in C57BL/6 mice. Given the similarities between our model and pulmonary adenocarcinomas in humans, and the ease with which tumors can be induced in any transgenic mouse, this system can be used to uncover novel mechanisms involved lung tumorigenesis.