RESUMO
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.
Assuntos
Autofagia , Produtos do Gene env/metabolismo , Retrovirus Jaagsiekte de Ovinos/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenomatose Pulmonar Ovina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Produtos do Gene env/genética , Interações Hospedeiro-Patógeno , Immunoblotting , Imuno-Histoquímica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Retrovirus Jaagsiekte de Ovinos/genética , Retrovirus Jaagsiekte de Ovinos/fisiologia , Pulmão/metabolismo , Pulmão/virologia , Camundongos , Células NIH 3T3 , Fosforilação , Adenomatose Pulmonar Ovina/genética , Adenomatose Pulmonar Ovina/virologia , Ovinos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
UNLABELLED: The native envelope gene (env) of Jaagsiekte sheep retrovirus (JSRV) also acts as an oncogene. To investigate the mechanism of transformation, we performed yeast 2-hybrid screening for cellular proteins that interact with Env. Among several candidates, we identified mouse or rat zinc finger protein 111 (zfp111). The interaction between Env and Zfp111 was confirmed through in vivo coimmunoprecipitation assays. Knockdown of endogenous Zfp111 caused a decrease in cell transformation by JSRV Env, while overexpression of Zfp111 increased overall Env transformation, supporting a role for Zfp111 in Env transformation. Knockdown of Zfp111 had no effect on the growth rate of parental rat 208F cells, while it decreased the proliferation rate of JSRV-transformed 208F cells, suggesting that JSRV-transformed cells became dependent on Zfp111. In addition, Zfp111 preferentially bound to a higher-mobility form of JSRV Env that has not been described previously. The higher-mobility form of Env (P70(env)) was found exclusively in the nuclear fraction, and size of its polypeptide backbone was the same as that of the cytoplasmic Env polyprotein (Pr80(env)). The differences in glycosylation between the two versions of Env were characterized. These results identify a novel cellular protein, Zfp111, that binds to the JSRV Env protein, and this binding plays a role in Env transformation. These results indicate that JSRV transformation also involves proteins and interactions in the nucleus. IMPORTANCE: The envelope protein (Env) of Jaagsiekte sheep retrovirus (JSRV) is an oncogene, but its mechanism of cell transformation is still unclear. Here we identified seven candidate cellular proteins that can interact with JSRV Env by yeast two-hybrid screening. This study focused on one of the seven candidates, zinc finger protein 111 (Zfp111). Zfp111 was shown to interact with JSRV Env in cells and to be involved in JSRV transformation. Moreover, coexpression of JSRV Env and Zfp111 led to the identification of a novel nuclear form of the JSRV Env protein that binds Zfp111. Nuclear Env was found to differ by glycosylation from the cytoplasmic Env precursor to the virion envelope proteins. These results suggest that JSRV Env transformation may involve nuclear events such as an alteration in transcription mediated by Env-Zfp111 interactions.
Assuntos
Transformação Celular Neoplásica , Transformação Celular Viral , Fibroblastos/virologia , Retrovirus Jaagsiekte de Ovinos/química , Proteínas do Envelope Viral/química , Dedos de Zinco/genética , Animais , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Vetores Genéticos , Glicosilação , Interações Hospedeiro-Patógeno , Humanos , Retrovirus Jaagsiekte de Ovinos/metabolismo , Lentivirus/genética , Camundongos , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ovinos , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
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.
Assuntos
Retrovirus Jaagsiekte de Ovinos , Ovinos , Animais , Humanos , Camundongos , Retrovirus Jaagsiekte de Ovinos/genética , Retrovirus Jaagsiekte de Ovinos/metabolismo , Produtos do Gene env , Transformação Celular Neoplásica , Autofagia , Glicoproteínas/metabolismoRESUMO
Many viruses use a pH-dependent pathway for fusion with host cell membrane, the mechanism of which is still poorly understood. Here we report that a subtle leucine (Leu)-valine (Val) change at position 501 in the envelope glycoproteins (Envs) of two related retroviruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), is responsible for their distinct low pH requirements for membrane fusion and infection. The Leu and Val residues are predicted to reside within the C-terminal heptad repeat (HR2) region of JSRV and ENTV Envs, particularly proximal to the hairpin turn of the putative six-helix bundle (6HB). Substitution of the JSRV Leu with a Val blocked the Env-mediated membrane fusion at pH 5.0, whereas replacement of the ENTV Val with a Leu rendered the ENTV Env capable of fusing at pH 5.0. A Leu-Val change has no apparent effect on the stability of native Env, but appears to stabilize an intermediate induced by receptor binding. These results are consistent with the existence of at least two metastable conformations of these viral glycoproteins, the native prefusion conformation and a receptor-induced metastable intermediate. Collectively, this work represents an interesting perhaps unique example whereby a simple Leu-Val change has critical impact on pH-dependent virus fusion and entry.
Assuntos
Substituição de Aminoácidos , Produtos do Gene env/metabolismo , Retrovirus Jaagsiekte de Ovinos/metabolismo , Fusão de Membrana , Estruturas Virais/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Produtos do Gene env/genética , Humanos , Concentração de Íons de Hidrogênio , Retrovirus Jaagsiekte de Ovinos/genética , Ovinos , Estruturas Virais/genéticaRESUMO
Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer in sheep. A unique feature is that JSRV envelope protein is also the oncogene for this virus. Previous studies have identified the cytoplasmic tail (CT) of the envelope transmembrane (TM) protein as critical for transformation although other regions of Env have also been implicated. In this study, the roles of other Env regions in transformation were investigated. Chimeras between JSRV Env and the Env of a related non-oncogenic endogenous retrovirus (enJSRV, 5F16) were used. A chimera containing the membrane-spanning region (MSR) of enJSRV inserted into JSRV Env showed substantially reduced transformation, indicating that the MSR plays a role in transformation. Transformation by this chimera was highly dependent on both Ras/Raf/MEK/MAPK and PI3K/Akt/mTOR signaling. A chimera containing the two amino acids in the TM ectodomain that distinguish JSRV and enJSRV showed modestly reduced transformation. Chimeras in the SU protein indicated that the amino terminal region of SU contributes to transformation, while the C-terminal part is not important. To test if Env trimerization is important for transformation, we mutated a leucine-rich sequence in the putative trimerization domain in the ectodomain of TM (Tri-M). This mutant could not transform cells and it did not oligomerize. However, Tri-M could complement a non-transforming mutant CT mutant (Y590F) so oligomerization is not necessary for at least some aspects of transformation. These experiments provide new insight into the regions and residues of JSRV Env protein necessary for oncogenic transformation.
Assuntos
Transformação Celular Viral , Retrovirus Jaagsiekte de Ovinos/genética , Adenomatose Pulmonar Ovina/virologia , Proteínas do Envelope Viral/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Retrovirus Jaagsiekte de Ovinos/metabolismo , Leucina/genética , Leucina/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Plasmídeos/genética , Plasmídeos/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Adenomatose Pulmonar Ovina/patologia , Ensaio de Radioimunoprecipitação , Ovinos/virologia , Relação Estrutura-Atividade , Proteínas do Envelope Viral/genéticaRESUMO
BACKGROUND: Endogenous Jaagsiekte sheep retrovirus envelope protein (enJSRV-Env) plays an important role in trophoblast cell fusion in sheep. However, the underlying mechanism remains unclear. METHODS: Primary endometrial luminal epithelial cells (LECs) were isolated from the sheep uterus and cocultured with sheep trophoblast cells (STCs). Giemsa staining was conducted to count multinucleated cells in the coculture system. Gain- and loss-of-function assays were performed to explore the role of enJSRV-Env in trophoblast cell fusion in the coculture system. Co-immunoprecipitation and mass spectrometry were carried out to identify the interacting partner of enJSRV-Env in the cocultures. Western blot analysis were conducted to determine the activation of protein kinase A (PKA)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. RESULTS: Primary LECs were identified by the expression of epithelial marker cytokeratin 18. Overexpression of enJSRV-Env promoted the formation of multinucleated cells in the coculture system. enJSRV-Env activated and physically interacted with PKA, along with the activation of MEK/ERK1/2 signaling. PKA inhibition completely reversed enJSRV-Env-induced MEK/ERK1/2 activation, and ERK1/2 inhibition abolished enJSRV-Env-induced formation of multinucleated cells in the coculture system. CONCLUSION: enJSRV-Env promotes trophoblast cell fusion in the sheep placenta by activating PKA/MEK/ERK1/2 signaling. This finding reveals a novel mechanism underlying the contribution of enJSRV-Env to trophoblast cell fusion during placental morphogenesis.
Assuntos
Retrovirus Jaagsiekte de Ovinos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Retrovirus Jaagsiekte de Ovinos/metabolismo , Queratina-18/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mitógenos/metabolismo , Placenta/metabolismo , Gravidez , Ovinos , Trofoblastos/metabolismoRESUMO
Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats, with significant animal health and economic consequences. The host range of JSRV is in part limited by species-specific differences in the virus entry receptor, hyaluronidase 2 (Hyal2), which is not functional as a receptor in mice but is functional in humans. Sheep are immunotolerant of JSRV because of the expression of closely related endogenous retroviruses, which are not present in humans and most other species, and this may facilitate oncogenesis. Here we show that expression of the JSRV envelope (Env) protein alone in lungs of mice, by using a replication-incompetent adeno-associated virus vector, results in tumours with a bronchiolo-alveolar localization like those seen in sheep. Whereas lethal disease was observed in immunodeficient mice, tumour development was almost entirely blocked in immunocompetent mice. Our results provide a rare example of an oncogenic viral structural protein, show that interaction of the viral Env protein with the virus entry receptor Hyal2 is not required for tumorigenesis, and indicate that immune recognition of Env can protect against JSRV tumorigenesis.
Assuntos
Produtos do Gene env/metabolismo , Retrovirus Jaagsiekte de Ovinos/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/virologia , Doenças dos Ovinos/patologia , Doenças dos Ovinos/virologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Transformação Celular Neoplásica , Modelos Animais de Doenças , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Genoma Viral , Humanos , Retrovirus Jaagsiekte de Ovinos/genética , Retrovirus Jaagsiekte de Ovinos/imunologia , Pulmão/patologia , Pulmão/virologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/veterinária , Camundongos , Receptores Virais/metabolismo , Ovinos/virologia , Especificidade da EspécieRESUMO
Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring retrovirus-induced transmissible lung cancer in sheep. Lungs and associated (bronchial and mediastinal) lymph nodes of seven sheep with OPA were examined. Lungs had few multifocal consolidated slightly elevated gray to white masses ranging from 0.5 to 3 cm in diameter. Histopathologically, these masses appeared as well-differentiated acinar adenocarcinoma with little evidence of anaplasia. The acini composed of well-differentiated cuboidal to low columnar epithelium with clear or vacuolated cytoplasm and low mitotic index. No metastases were observed in the bronchial and mediastinal lymph nodes of any animal. The presence of Jaagsiekte sheep retrovirus (JSRV) was demonstrated in the lungs by immunohistochemistry. JSRV protein was detected in all tumor epithelial cells, histologically normal alveolar type II cells, and few bronchiolar epithelial cells, alveolar macrophages, lymphocytes, and plasma cells. This study is the first to confirm the presence of natural OPA in Egypt.
Assuntos
Adenocarcinoma Bronquioloalveolar/veterinária , Proteínas do Capsídeo/metabolismo , Retrovirus Jaagsiekte de Ovinos/patogenicidade , Pneumopatias/patologia , Pulmão/metabolismo , Adenomatose Pulmonar Ovina/patologia , Proteínas dos Retroviridae/metabolismo , Células Acinares/metabolismo , Células Acinares/patologia , Células Acinares/virologia , Adenocarcinoma Bronquioloalveolar/metabolismo , Adenocarcinoma Bronquioloalveolar/virologia , Animais , Antígenos Virais/metabolismo , Egito , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Retrovirus Jaagsiekte de Ovinos/metabolismo , Pulmão/patologia , Pneumopatias/virologia , Linfonodos/metabolismo , Linfonodos/patologia , Linfonodos/virologia , Linfócitos/metabolismo , Linfócitos/virologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Plasmócitos/metabolismo , Plasmócitos/virologia , Adenomatose Pulmonar Ovina/virologia , Ovinos , Carneiro DomésticoRESUMO
Retroviruses use different strategies to regulate transcription and translation and exploit the cellular machinery involved in these processes. This study shows that the signal peptide of the envelope glycoprotein (Env) of Jaagsiekte sheep retrovirus (JSRV) plays a major role in posttranscriptional viral gene expression. Expression of the JSRV Env in trans increases viral particle production by mechanisms dependent on (i) its leader sequence, (ii) an intact signal peptide cleavage site, (iii) a cis-acting RNA-responsive element located in the viral genome, (iv) Crm1, and (v) B23. The signal peptide of the JSRV Env (JSE-SP) is 80 amino acid residues in length and contains putative nuclear localization and export signals, in addition to an arginine-rich RNA binding motif. JSE-SP localizes both in the endoplasmic reticulum and in the nucleus, where it colocalizes with nucleolar markers. JSE-SP is a multifunctional protein, as it moderately enhances nuclear export of unspliced viral mRNA and considerably increases viral particle release by favoring a posttranslational step of the replication cycle.
Assuntos
Regulação Viral da Expressão Gênica/genética , Processamento de Proteína Pós-Traducional/genética , Sinais Direcionadores de Proteínas/genética , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Nucléolo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Genoma Viral/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Retrovirus Jaagsiekte de Ovinos/genética , Retrovirus Jaagsiekte de Ovinos/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vírion/metabolismo , Proteína Exportina 1RESUMO
Retroviruses have played profound roles in our understanding of the genetic and molecular basis of cancer. Jaagsiekte sheep retrovirus (JSRV) is a simple retrovirus that causes contagious lung tumors in sheep, known as ovine pulmonary adenocarcinoma (OPA). Intriguingly, OPA resembles pulmonary adenocarcinoma in humans, and may provide a model for this frequent human cancer. Distinct from the classical mechanisms of retroviral oncogenesis by insertional activation of or virus capture of host oncogenes, the native envelope (Env) structural protein of JSRV is itself the active oncogene. A major pathway for Env transformation involves interaction of the Env cytoplasmic tail with as yet unidentified cellular adaptor(s), leading to the activation of PI3K/Akt and MAPK signaling cascades. Another potential mechanism involves the cell-entry receptor for JSRV, Hyaluronidase 2 (Hyal2), and the RON receptor tyrosine kinase, but the exact roles of these proteins in JSRV Env transformation remain to be better understood. Recently, a mouse model of lung cancer induced by JSRV Env has been developed, and the tumors in mice resemble those seen in sheep infected with JSRV and in humans. In this review, we summarize recent progress in our understanding the molecular mechanisms of oncogenic transformation by JSRV Env protein, and discuss the relevance to human lung cancer.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Produtos do Gene env/metabolismo , Retrovirus Jaagsiekte de Ovinos/metabolismo , Animais , Transformação Celular Neoplásica/genética , Produtos do Gene env/química , Produtos do Gene env/genética , Humanos , Retrovirus Jaagsiekte de Ovinos/química , Retrovirus Jaagsiekte de Ovinos/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Internalização do VírusRESUMO
BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Adenocarcinoma/veterinária , Produtos do Gene env/metabolismo , Retrovirus Jaagsiekte de Ovinos/patogenicidade , Neoplasias Pulmonares/patologia , Adenomatose Pulmonar Ovina/patologia , Doenças dos Ovinos/patologia , Adenocarcinoma/patologia , Adenocarcinoma/virologia , Animais , Retrovirus Jaagsiekte de Ovinos/metabolismo , Pulmão/patologia , Pulmão/virologia , Neoplasias Pulmonares/virologia , Camundongos , Camundongos Endogâmicos C57BL , Adenomatose Pulmonar Ovina/virologia , Ovinos/virologia , Doenças dos Ovinos/virologiaRESUMO
The HERV-K(HML-2) family is the most recent addition to the collection of human endogenous retroviruses. It comprises proviruses that encode functional proteins that can assemble into replication defective particles carrying the envelope protein. Using a reconstituted HERV-K113 envelope sequence, we have analyzed its ability to mediate entry into a set of 33 cell lines from 10 species. Of these, 30 were permissive, demonstrating an amphotropism consistent with a broad expression of receptor protein(s). In an initial effort to identify a receptor for HERV-K(HML-2) we investigated whether transferrin receptor 1 and hyaluronidase 2, known cellular receptors of the closely related betaretroviruses mouse mammary tumor virus (MMTV) and Jaagsiekte sheep retrovirus (JSRV), could facilitate HERV-K(HML-2) entry. However, neither of these proteins could serve as a receptor for HERV-K(HML-2). Moreover, during attempts to further characterize the tropism of HERV-K(HML-2), we identified a cellular activity that inhibits infection at a post-entry, pre-integration step.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Retrovirus Endógenos/metabolismo , Hialuronoglucosaminidase/metabolismo , Receptores da Transferrina/metabolismo , Proteínas do Envelope Viral/metabolismo , Tropismo Viral/fisiologia , Células 3T3 , Animais , Betaretrovirus/metabolismo , Células COS , Gatos , Linhagem Celular Tumoral , Chlorocebus aethiops , Cães , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Células HeLa , Humanos , Retrovirus Jaagsiekte de Ovinos/metabolismo , Vírus do Tumor Mamário do Camundongo/metabolismo , Camundongos , Receptores Virais , Células Vero , Internalização do VírusRESUMO
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.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/genética , Hialuronoglucosaminidase/genética , Retrovirus Jaagsiekte de Ovinos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Adenomatose Pulmonar Ovina/enzimologia , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Hialuronoglucosaminidase/metabolismo , Retrovirus Jaagsiekte de Ovinos/genética , Pulmão/enzimologia , Pulmão/virologia , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenomatose Pulmonar Ovina/virologia , Ovinos , Transdução de Sinais , TransfecçãoRESUMO
The endogenous retroviruses are inherited elements transmitted trough the germline of most animal species and their biological role is still controversial. Ovine Pulmonary Carcinoma (OPC) represents a good model for studying the interactions of endogenous retroviruses with their exogenous counterparts. The type D exogenous retrovirus known as Jaagsiekte Sheep Retro-Virus (JSRV) is necessary and sufficient to cause OPC in domestic and wild sheep, but both affected and unaffected animals host in their genome 15 to 20 copies of related endogenous retroviruses named endogenous JSRV (enJSRV). In this study we evaluated the expression of enJSRV gag sequences in ovine foetal and placental tissues. RNA in situ hybridisation was performed on tissue sections of thymi, lymph nodes and lungs from ovine foetuses and related placentas, taken at a late stage of development. Reverse transcriptase-in situ polymerase chain reactions were also carried out on placental samples to better define the involved cells. In foetal tissues, specific signals were observed in the thymus medulla, lymph nodes and, at a lesser extent, in foetal bronchiolar cells. In the placental tissues, positive areas were detected in various cell types in the sincythium-and cyto-trophoblast. These data demonstrate that en JSRV RNA is largely expressed in a broad spectrum of cells including tissues which are critical for the development of the immune system.
Assuntos
Feto/metabolismo , Retrovirus Jaagsiekte de Ovinos/metabolismo , Placenta/metabolismo , RNA Viral/biossíntese , Animais , Feminino , Hibridização In Situ , Linfonodos/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , OvinosRESUMO
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.
Assuntos
Adenocarcinoma/virologia , Retrovirus Jaagsiekte de Ovinos/metabolismo , Neoplasias Pulmonares/virologia , Proteínas Virais/metabolismo , Adenocarcinoma/patologia , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Humanos , Imuno-Histoquímica , Retrovirus Jaagsiekte de Ovinos/genética , Neoplasias Pulmonares/patologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Ovinos , Análise Serial de Tecidos , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Betaretrovirus/genética , DNA Viral/genética , Regulação Viral da Expressão Gênica , Provírus/genética , Infecções por Retroviridae/veterinária , Doenças dos Ovinos/virologia , Infecções Tumorais por Vírus/veterinária , Adenocarcinoma/veterinária , Adenocarcinoma/virologia , Animais , Sequência de Bases , Betaretrovirus/metabolismo , Técnicas de Cultura de Células/veterinária , Linhagem Celular , Sequência Conservada , DNA Viral/metabolismo , Feminino , Vetores Genéticos/genética , Retrovirus Jaagsiekte de Ovinos/genética , Retrovirus Jaagsiekte de Ovinos/metabolismo , Masculino , Dados de Sequência Molecular , Doenças Nasais/veterinária , Doenças Nasais/virologia , Filogenia , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Adenomatose Pulmonar Ovina/virologia , Infecções por Retroviridae/virologia , Ovinos , Sequências Repetidas Terminais , Infecções Tumorais por Vírus/virologiaRESUMO
JSRV, a simple beta-retrovirus, is the etiologic agent of ovine pulmonary adenocarcinoma, a form of non-small cell lung cancer in sheep and goats. It has been shown that the envelope protein alone is sufficient to induce tumorigenesis in the lungs of mice when delivered via an adeno-associated viral vector. Here, we tested the hypothesis that JSRV envelope-induced tumors are maintained by a small population of tumor-initiating cells, termed cancer stem cells. To test this hypothesis, dissociated cancer cells were sorted from envelope-induced tumors in mouse lung based on the putative stem cell markers Sca-1, CD34, and CD133, the pluripotency-associated transcription factor Oct4, and the level of Wnt signaling. No association with increased tumor-initiating capacity was found with any of the cell-surface markers. In addition, we were unable to detect any evidence of Oct4 expression in tumor-bearing mouse lung. However, tumor cells possessing an active Wnt signaling pathway did show a significant correlation with increased tumor formation upon transplantation. Limiting dilution transplant analysis suggests the existence of a large fraction of cells with the ability to propagate tumor growth, with increasing tumor initiation potential correlating with activated Wnt signaling.
Assuntos
Adenocarcinoma/induzido quimicamente , Produtos do Gene env , Retrovirus Jaagsiekte de Ovinos/química , Neoplasias Pulmonares/induzido quimicamente , Células-Tronco Neoplásicas/fisiologia , Via de Sinalização Wnt/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Células Cultivadas , Progressão da Doença , Retrovirus Jaagsiekte de Ovinos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células NIH 3T3 , Células-Tronco Neoplásicas/patologia , Ovinos , Doenças dos Ovinos/induzido quimicamente , Doenças dos Ovinos/genética , Doenças dos Ovinos/patologia , Ativação Transcricional/fisiologia , Via de Sinalização Wnt/genéticaRESUMO
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.
Assuntos
Betaretrovirus/fisiologia , Produtos do Gene env/química , Retrovirus Jaagsiekte de Ovinos/fisiologia , Receptores Virais/metabolismo , Proteínas Virais de Fusão/química , Ligação Viral , Internalização do Vírus , Sequência de Aminoácidos , Animais , Betaretrovirus/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Proteínas Ligadas por GPI/metabolismo , Produtos do Gene env/metabolismo , Células HEK293 , Humanos , Hialuronoglucosaminidase/metabolismo , Concentração de Íons de Hidrogênio , Retrovirus Jaagsiekte de Ovinos/metabolismo , Fusão de Membrana , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Ovinos , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
The ovine beta-retroviruses enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) are the causative agent of enzootic nasal adenocarcinoma (ENA) and ovine pulmonary adenocarcinoma (OPA), respectively, characterized by neoplastic transformation of secretory epithelial cells. The Envelope (Env) proteins of these related betaretroviruses act as oncogenes, in that they can transform fibroblast and epithelial cell lines in culture. In addition, viral vector-mediated expression of the Env proteins for these viruses causes tumors in animals. Here, we investigated what signaling pathways are required for the ENTV transformation in vitro. We have previously found that Ras-MEK-MAPK and PI3k-Akt-mTOR are involved in JSRV transformation of fibroblast and epithelial cells. In this study, we found that the MEK inhibitor PD98059 and mTOR inhibitor Rapamycin inhibited ENTV transformation in RK3E rat kidney epithelial cells, but the p38 inhibitor SB203580 drastically enhanced transformation, which is quite similar to JSRV transformation. Small molecular inhibitors and dominant negative versions of H-ras and Rac1 indicated a role for both of these molecules in transformation by either virus. These results indicate that the signaling pathways for ENTV and JSRV transformation are quite similar, consistent with the notion that these proteins do not determine the tissue-specificity of the tumors for these viruses.
Assuntos
Betaretrovirus/metabolismo , Transformação Celular Viral , Produtos do Gene env/metabolismo , Retrovirus Jaagsiekte de Ovinos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , RatosRESUMO
Jaagsiekte sheep retrovirus (JSRV) uses hyaluronidase 2 (Hyal2) as a cell entry receptor. By making inactivating mutations to the catalytic residues of human Hyal2, we found that hyaluronidase activity was dispensable for its receptor function. The affinities of the JSRV envelope glycoprotein for Hyal2 and the Hyal2 mutant were similar, and hyaluronan did not block either high-affinity interaction or virus infection. While generating the Hyal2 mutant, we discovered that our previous analysis of the hyaluronidase activity of Hyal2 was affected by a contaminating hyaluronan lyase, which we have identified as the occlusion-derived baculovirus E66 protein of the recombinant baculovirus used to produce Hyal2. We now report that purified human Hyal2 is a weak acid-active hyaluronidase.