The U3 and Env Proteins of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Both Contribute to Tissue Tropism.

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are small-ruminant betaretroviruses that share high nucleotide and amino acid identity, utilize the same cellular receptor, hyaluronoglucosaminidase 2 (Hyal2) for entry, and transform tissues with their envelope (Env) glycoprotein; yet, they target discrete regions of the respiratory tract-the lung and nose, respectively. This distinct tissue selectivity makes them ideal tools with which to study the pathogenesis of betaretroviruses. To uncover the genetic determinants of tropism, we constructed JSRV-ENTV chimeric viruses and produced lentivectors pseudotyped with the Env proteins from JSRV (Jenv) and ENTV (Eenv). Through the transduction and infection of lung and nasal turbinate tissue slices, we observed that Hyal2 expression levels strongly influence ENTV entry, but that the long terminal repeat (LTR) promoters of these viruses are likely responsible for tissue-specificity. Furthermore, we show evidence of ENTV Env expression in chondrocytes within ENTV-infected nasal turbinate tissue, where Hyal2 is highly expressed. Our work suggests that the unique tissue tropism of JSRV and ENTV stems from the combined effort of the envelope glycoprotein-receptor interactions and the LTR and provides new insight into the pathogenesis of ENTV.

Transcriptional Response of Ovine Lung to Infection with Jaagsiekte Sheep Retrovirus.

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.

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.

Blocked expression of key genes of the angiogenic pathway in JSRV-induced pulmonary adenocarcinomas.

JSRV (Jaagsiekte Sheep Retrovirus) is a retrovirus inducing a transmissible lung adenocarcinoma in sheep and goats with predominantly lepidic and papillary lesions. This naturally occurring lung cancer in large animals shares many features with human pneumonic-type lung adenocarcinomas with predominant lepidic growth. The metastatic spread is rare in both human and animal cancers. This unique feature prompted us to decipher the angiogenesis pathway in these cancers. We focused on the levels of mRNA and proteins of genes implicated in the extension of JSRV-induced lung adenocarcinomas by studying their expression in lung cancers (n = 10) and normal lungs (n = 10) and in primary epithelial alveolar type II cells derived from cancers (n = 10) or normal lungs (n = 6). In parallel, we evaluated the levels of expression of key genes in lung tissues collected from lepidic (n = 13) or papillary (n = 5) human adenocarcinomas and, when available, adjacent normal lungs (n = 11). We measured the expression of the same key genes implicated in angiogenesis, lymphangiogenesis and degradation of the extracellular matrix. In ovine adenocarcinomas, VEGFR2 and VEGFD mRNA were downregulated in cancers; MMP9, TIMP1 and FGFR2 mRNA were overexpressed as compared to normal lungs. Importantly, VEGFA and VEGFR2 proteins were not expressed in JSRV-induced cancers. In human lepidic adenocarcinomas, VEGFA and VEGFR2 mRNA were weakly expressed and no VEGFR2 protein was detectable. Downregulation of key angiogenic players may contribute to the control of extra thoracic invasion of cancer cells in human and ovine pneumonic-type adenocarcinoma with predominant lepidic growth.

Stereological and biophysical characteristics of the ovine surfactant system and its changes caused by ovine pulmonary adenocarcinoma.

Surfactant covers the inner surface of lung alveoli and lowers the surface tension to prevent alveoli from collapsing. A lack of surfactant or its dysfunction causes dyspnea. The Jaagsiekte Sheep Retrovirus (JSRV) causes ovine pulmonary adenocarcinoma (OPA), whose typical clinical appearance is fluid running from nostrils. This fluid might contain surfactant as alveolar type II pneumocytes (AEII) are target cells for JSRV. Therefore, the progressive dyspnea during OPA might be caused partially by surfactant alterations. Bronchoalveolar and intracellular surfactant as well as the biophysical function of surfactant were analyzed in OPA sheep and controls. Transmission electron microscopy and stereological methods were used to characterize ultrastructure and distribution of surfactant subtypes in AEII and bronchoalveolar lavage fluid (BALF). Pulsating Bubble Surfactometry enabled studying the surface activity of the surfactant, while lung volumes were detected by computed tomography. The methods used are suitable to determine intraalveolar and intracellular surfactant subtypes in OPA sheep and controls. OPA sheep showed more lamellar body-like forms, multivesicular vesicles and tubular myelin in BALF compared to controls. These higher amounts of active surfactant subtypes might be a consequence of a higher surfactant production and release. Surfactant subtypes in AEII of OPA sheep showed smaller and more immature lamellar bodies compared to controls. The surfactant surface activity of OPA sheep does not show obvious defects. In conclusion, the general quality of surfactant in OPA appears to be equivalent to surfactant produced in controls, however, dyspnea of OPA might be triggered by quantity of fluid production.

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.

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.

Early Steps of Jaagsiekte Sheep Retrovirus-Mediated Cell Transformation Involve the Interaction between Env and the RALBP1 Cellular Protein.

UNLABELLED: Ovine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte sheep retrovirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (RalA binding protein 1; also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast two-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs and in tumor-derived alveolar type II cells. Through its inhibition using specific small interfering RNA (siRNA), we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR (mammalian target of rapamycin)/p70S6K pathway by the retroviral envelope. IMPORTANCE: JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens up a new hypothesis for the dysregulation observed upon virus infection in sheep.

The sheep tetherin paralog oBST2B blocks envelope glycoprotein incorporation into nascent retroviral virions.

UNLABELLED: Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the "tethering" mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A. IMPORTANCE: BST2 is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. JSRV, the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study, we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles, which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from those of BST2 proteins of other species.

A sorting signal suppresses IFITM1 restriction of viral entry.

The interferon-induced transmembrane proteins (IFITMs) broadly inhibit virus infections, particularly at the viral entry level. However, despite this shared ability to inhibit fusion, IFITMs differ in the potency and breadth of viruses restricted, an anomaly that is not fully understood. Here, we show that differences in the range of viruses restricted by IFITM1 are regulated by a C-terminal non-canonical dibasic sorting signal KRXX that suppresses restriction of some viruses by governing its intracellular distribution. Replacing the two basic residues with alanine (KR/AA) increased restriction of jaagsiekte sheep retrovirus and 10A1 amphotropic murine leukemia virus. Deconvolution microscopy revealed an altered subcellular distribution for KR/AA, with fewer molecules in LAMP1-positive lysosomes balanced by increased levels in CD63-positive multivesicular bodies, where jaagsiekte sheep retrovirus pseudovirions are colocalized. IFITM1 binds to cellular adaptor protein complex 3 (AP-3), an association that is lost when the dibasic motif is altered. Although knockdown of AP-3 itself decreases some virus entry, expression of parental IFITM1, but not its KR/AA mutant, potentiates inhibition of viral infections in AP-3 knockdown cells. By using the substituted cysteine accessibility method, we provide evidence that IFITM1 adopts more than one membrane topology co-existing in cellular membranes. Because the C-terminal dibasic sorting signal is unique to human IFITM1, our results provide novel insight into understanding the species- and virus-specific antiviral effect of IFITMs.

"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.

Cells infected with Jaagsiekte sheep retrovirus are detected in the bone marrow of asymptomatic sheep.

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer caused by Jaggsiekte sheep retrovirus (JSRV). It is difficult to identify animals infected with JSRV but are clinically healthy. The virus does not induce a specific antibody response and, although proviral DNA sequences of JSRV can be found in mononuclear blood cells, the detection is inconsistent. The aim of this study was to investigate the presence of JSRV in the bone marrow of infected sheep and develop a more consistent screening method. Immunohistochemical examination of bone marrow samples from 8 asymptomatic JSRV-infected sheep revealed the presence of positively labelled cells. However, JSRV could not be detected by a highly sensitive polymerase chain reaction (PCR) in bone marrow aspirates periodically collected from these animals. Results suggest that JSRV-infected cells may be present in the bone marrow of symptomless animals, but the number is below the detectable level for PCR. Therefore, this technique does not seem to be helpful for preclinical diagnosis of OPA.

L'adénocarcinome pulmonaire ovin (OPA) est un cancer pulmonaire transmissible causé par le rétrovirus ovin de Jaggsiekte (JSRV). Il est difficile d'identifier les animaux infectés par le JSRV mais qui sont cliniquement en santé. Le virus n'entraine pas la production d'anticorps spécifiques et, bien que des séquences d'ADN provirales de JSRV peuvent être retrouvées dans les mononucléaires du sang, la détection est inconstante. L'objectif de la présente étude était d'examiner la présence de JSRV dans la moelle osseuse de moutons infectés et de développer une méthode de tamisage plus constante. L'examen par immunohistochime d'échantillons de la moelle osseuse de huit moutons asymptomatiques mais infectés par JSRV a révélé la présence de cellules positivement marquées. Toutefois, le JSRV ne put être révélé par une épreuve d'amplification en chaine par la polymérase (PCR) très sensible à partir d'aspirations de la moelle osseuse récolées périodiquement à partir de ces animaux. Les résultats suggèrent que les cellules infectées par JSRV peuvent être présentes dans la moelle osseuse d'animaux asymptomatiques, mais le nombre se situe sous le seuil détectable pas PCR. Ainsi, cette technique ne semble pas utile pour le diagnostic préclinique d'OPA.(Traduit par Docteur Serge Messier).

Evolutionary dynamics of endogenous Jaagsiekte sheep retroviruses proliferation in the domestic sheep, mouflon and Pyrenean chamois.

The oncogenic exogenous Jaagsiekte sheep retrovirus (JSRV), responsible for ovine pulmonary adenocarcinoma, has several endogenous counterparts termed enJSRVs. Although many of these elements have been inactivated over time by the accumulation of deleterious mutations or internal recombination leading to solo long terminal repeat (LTR) formation, several members of enJSRVs have been identified as nearly intact and probably represent recent integration events. To determine the level of enJSRV polymorphism in the sheep population and related species, we have undertaken a study by characterizing enJSRVs copies and independent integration sites in six domestic sheep and two wild species of the sheep lineage. enJSRVs copies were detected by amplifying the env-LTR region by PCR, and for the detection of the insertion sites, we used two approaches: (1) an in silico approach based on the recently published Sheep Reference Genome Assembly (OARv3.0) and (2) an experimental approach based on PCR suppression and inverse PCR techniques. In total, 103 enJSRV sequences were generated across 10 individuals and enJSRV integrations were found on 11 of the 28 sheep chromosomes. These findings suggest that there are still uncharacterized enJSRVs, and that some of the integration sites are variable among the different species, breeds of the same species, subspecies and geographic locations.

Host species barriers to Jaagsiekte sheep retrovirus replication and carcinogenesis.

Understanding the factors governing host species barriers to virus transmission has added significantly to our appreciation of virus pathogenesis. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep that has rarely been found in goats. In this study, in order to further clarify the pathogenesis of OPA, we investigated whether goats are resistant to JSRV replication and carcinogenesis. We found that JSRV induces lung tumors in goats with macroscopic and histopathological features that dramatically differ from those in sheep. However, the origins of the tumor cells in the two species are identical. Interestingly, in experimentally infected lambs and goat kids, we revealed major differences in the number of virus-infected cells at early stages of infection. These differences were not related to the number of available target cells for virus infection and cell transformation or the presence of a host-specific immune response toward JSRV. Indeed, we also found that goats possess transcriptionally active endogenous retroviruses (enJSRVs) that likely influence the host immune response toward the exogenous JSRV. Overall, these results suggest that goat cells, or at least those cells targeted for viral carcinogenesis, are not permissive to virus replication but can be transformed by JSRV.

Single residues in the surface subunits of oncogenic sheep retrovirus envelopes distinguish receptor-mediated triggering for fusion at low pH and infection.

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are two closely related oncogenic retroviruses that share the same cellular receptor yet exhibit distinct fusogenicity and infectivity. Here, we find that the low fusogenicity of ENTV envelope protein (Env) is not because of receptor binding, but lies in its intrinsic insensitivity to receptor-mediated triggering for fusion at low pH. Distinct from JSRV, shedding of ENTV surface (SU) subunit into culture medium was not enhanced by a soluble form of receptor, Hyal2 (sHyal2), and sHyal2 was unable to effectively inactivate the ENTV pseudovirions. Remarkably, replacing either of the two amino acid residues, N191 or S195, located in the ENTV SU with the corresponding JSRV residues, H191 or G195, markedly increased the Env-mediated membrane fusion activity and infection. Reciprocal amino acid substitutions also partly switched the sensitivities of ENTV and JSRV pseudovirions to sHyal2-mediated SU shedding and inactivation. While N191 is responsible for an extra N-linked glycosylation of ENTV SU relative to that of JSRV, S195 possibly forms a hydrogen bond with a surrounding amino acid residue. Molecular modeling of the pre-fusion structure of JSRV Env predicts that the segment of SU that contains H191 to G195 contacts the fusion peptide and suggests that the H191N and G195S changes seen in ENTV may stabilize its pre-fusion structure against receptor priming and therefore modulate fusion activation by Hyal2. In summary, our study reveals critical determinants in the SU subunits of JSRV and ENTV Env proteins that likely regulate their local structures and thereby differential receptor-mediated fusion activation at low pH, and these findings explain, at least in part, their distinct viral infectivity.

Jaagsiekte sheep retrovirus and enzootic nasal tumor virus promoters drive gene expression in all airway epithelial cells of mice but only induce tumors in the alveolar region of the lungs.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats, while the closely related enzootic nasal tumor virus type 1 (ENTV-1) and ENTV-2 induce tumors in the nasal epithelium of sheep and goats, respectively. When expressed using a strong Rous sarcoma virus promoter, the envelope proteins of these viruses induce tumors in the respiratory tract of mice, but only in the distal airway. To examine the role of the retroviral long terminal repeat (LTR) promoters in determining tissue tropism, adeno-associated virus (AAV) vectors expressing alkaline phosphatase under the control of the JSRV, ENTV-1, or ENTV-2 LTRs were generated and administered to mice. The JSRV LTR was active in all airway epithelial cells, while the ENTV LTRs were active in the nasal epithelium and alveolar type II cells but poorly active in tracheal and bronchial epithelial cells. When vectors were administered systemically, the ENTV-1 and -2 LTRs were inactive in major organs examined, whereas the JSRV showed high-level activity in the liver. When a putative transcriptional enhancer from the 3' end of the env gene was inserted upstream of the JSRV and ENTV-1 LTRs in the AAV vectors, a dramatic increase in transgene expression was observed. However, intranasal administration of AAV vectors containing any combination of ENTV or JSRV LTRs and Env proteins induced tumors only in the lower airway. Our results indicate that mice do not provide an adequate model for nasal tumor induction by ENTV despite our ability to express genes in the nasal epithelium.

Endogenous retroviruses in trophoblast differentiation and placental development.

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and originated from infections of the germline of the host by exogenous retroviruses. ERVs have coevolved with their hosts for millions of years and are recognized to contribute to genome plasticity, protect the host against infection of related pathogenic and exogenous retroviruses, and play a vital role in development of the placenta. Consequently, some ERVs have been positively selected and maintained in the host genome throughout evolution. This review will focus on the critical role of ERVs in development of the mammalian placenta and specifically highlight the biological role of sheep JSRV-related endogenous betaretroviruses in conceptus (embryo and associated extraembryonic membranes) development.

Pathology and pathogenesis of ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA), also known as jaagsiekte, is a transmissible lung tumour of sheep caused by jaagsiekte sheep retrovirus (JSRV). JSRV induces neoplastic transformation of alveolar and bronchiolar secretory epithelial cells and the resulting tumours can grow to occupy a significant portion of the lung. Tumour growth is frequently accompanied by the overproduction of fluid in the lung, which further compromises normal respiration. The period between infection and the appearance of clinical signs may be several months or years and many JSRV-infected sheep do not exhibit clinical signs at all during their lifespan. This allows the spread of OPA into new flocks through contact with infected but apparently normal animals. OPA was first described in the early 19th century; however, it has still not been possible to devise effective methods for controlling its spread and it remains an important problem in most countries where sheep are farmed. This is due in part to the absence of an immunological response to JSRV in infected animals, which has hindered the development of serological diagnostic tests and vaccines. In addition to its veterinary importance, OPA is regarded as a potential large animal model for human lung adenocarcinoma and this has stimulated research into the pathogenesis of the ovine disease. This work has produced some significant results, including the finding that one of the JSRV structural proteins is directly involved in oncogenesis. The recent advances in understanding JSRV and the pathogenesis of OPA should lead to novel strategies for diagnosis and control of this disease and for its exploitation as a comparative model for human lung cancer.

Jaagsiekte sheep retrovirus encodes a regulatory factor, Rej, required for synthesis of Gag protein.

Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. For some retroviruses, transport of unspliced viral RNA to the cytoplasm is mediated by small regulatory proteins such as human immunodeficiency virus Rev, while other retroviruses contain constitutive transport elements in their RNAs that allow transport without splicing. In this study, we found that the betaretrovirus Jaagsiekte sheep retrovirus (JSRV) encodes within the env gene a trans-acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences from a JSRV proviral expression plasmid (pTN3) abolished its ability to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by cotransfection of an env expression plasmid (DeltaGP). Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and a signal peptide expression construct showed Rej activity. Two other betaretroviruses, mouse mammary tumor virus (MMTV) and human endogenous retrovirus type K, encode analogous factors (Rem and Rec, respectively) that are encoded from doubly spliced env mRNAs. Reverse transcriptase-PCR cloning and sequencing identified alternate internal splicing events in the 5' end of JSRV env that could signify analogous doubly spliced Rej mRNAs, and cDNA clones expressing two of them also showed Rej activity. The predicted Rej proteins contain motifs similar to those found in MMTV Rem and other analogous retroviral regulatory proteins. Interestingly, in most cell lines, JSRV expression plasmids with Rej deleted showed normal transport of unspliced JSRV RNA to the cytoplasm; however, in 293T cells Rej modestly enhanced export of unspliced viral RNA (2.8-fold). Metabolic labeling experiments with [(35)S]methionine indicated that JSRV Rej is required for the synthesis of viral Gag polyprotein. Thus, in most cell lines, the predominant function of Rej is to facilitate translation of unspliced viral mRNA.

Identification and mutational analysis of a Rej response element in Jaagsiekte sheep retrovirus RNA.

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5' end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3' end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.