Claudio Murgia

Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2–3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21ΔGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential.

A Phosphatidylinositol 3-Kinase Docking Site in the Cytoplasmic Tail of the Jaagsiekte Sheep Retrovirus Transmembrane Protein Is Essential for Envelope-Induced Transformation of NIH 3T3 Cells

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between JSRV and the JSRV-related endogenous retroviruses of sheep (enJSRVs) and assessed them in transformation assays. Chimeras containing the exogenous JSRV SU region and the enJSRV TM region were unable to transform NIH 3T3 cells. Additional chimeras containing only the carboxy-terminal portion of TM (a region that we previously identified as VR3) of the endogenous envelope with SU and the remaining portion of TM from the exogenous JSRV were also unable to transform NIH 3T3 cells. The VR3 region includes the putative membrane-spanning region and cytoplasmic tail of the JSRV TM glycoprotein; this suggested that the cytoplasmic tail of the JSRV Env mediates transformation, possibly via a cell signaling mechanism. Mutations Y590 and M593 in the cytoplasmic tail of the JSRV envelope were sufficient to inhibit the transforming abilities of these constructs. Y590 and M593 are part of a Y-X-X-M motif that is recognized by the phosphatidylinositol 3-kinase (PI-3K). PI-3K initiates a cell signaling pathway that inhibits apoptosis and is required for a number of mitogens during the G1-to-S-phase transition of the cell cycle. PI-3K activates Akt by phosphorylation of threonine 308 and serine 473. We detected by Western blot analysis phosphorylated Akt in serum-starved MP1 cells (NIH 3T3 cells transformed by JSRV) but not in the parental NIH 3T3 cells. These data indicate that the cytoplasmic tail of the JSRV TM is necessary for cell transformation and suggest a new mechanism of retroviral transformation. In addition, the ability to dissociate the function of the JSRV envelope to mediate viral entry from its transforming capacity has direct relevance for the design of JSRV-based vectors that target the differentiated epithelial cells of the lungs.

Molecular Cloning and Functional Analysis of Three Type D Endogenous Retroviruses of Sheep Reveal a Different Cell Tropism from That of the Highly Related Exogenous Jaagsiekte Sheep Retrovirus

ABSTRACT Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSVs) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRVs in vivo was studied by in situ hybridization. All of the threeenJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of thegag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV21. Phylogenetic analysis distinguished five ovine type D retroviruses:enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the threeenJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.

Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.

The Long Terminal Repeat of Jaagsiekte Sheep Retrovirus Is Preferentially Active in Differentiated Epithelial Cells of the Lungs

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a contagious bronchioloalveolar carcinoma of sheep known as sheep pulmonary adenomatosis (SPA; ovine pulmonary carcinoma). JSRV is unique among retroviruses because it transforms the alveolar type II cells and the nonciliated bronchiolar cells (Clara cells) of the lungs; these cells are where JSRV is specifically expressed in both naturally and experimentally SPA-affected sheep. In this study, we investigated the cell specificity of JSRV expression. By transient-transfection assays of 23 different cell lines with a reporter plasmid driven by the JSRV long terminal repeat (LTR), pJS21-luc, we found that the JSRV LTR is preferentially active in cell lines derived from type II pneumocytes and Clara cells (MLE-15 and mtCC1-2 mouse cell lines). Reporter assays using progressive 5′ deletions of pJS21-luc allowed us to establish that the JSRV enhancers are able to activate the JSRV proximal promoter in MLE-15 and mtCC1-2 cells, but they have very low activity in mouse cells of other lineages (e.g., NIH 3T3). The JSRV enhancers are able to activate heterologous promoters in both MLE-15 and 3T3 cells, although optimal activity is achieved in MLE-15 cells only with the homologous JSRV promoter. Thus, JSRV cell-specific LTR activity appears to result from an interaction between the enhancer elements and the JSRV proximal promoter elements. By mutation analysis, we established that an upstream NF-κB-like element appears to be responsible for approximately 50% of the JSRV LTR transcriptional activity in MLE-15 cells. Electrophoretic mobility shift assays showed evidence of a factor(s) that binds to this sequence. Antibody supershift experiments indicated that the factor(s) is not related to NF-κB component p50 or p52. This factor also appeared to be present in cells that do not support a high level of JSRV expression. Finally the JSRV21LTR contains putative enhancer binding motifs for transcription factors such as hepatocyte nuclear factor 3 (HNF-3) that are involved in lung-specific gene expression. Cotransfection experiments demonstrated that exogenous HNF-3 is able to enhance the expression of pJS21-luc in NIH 3T3 cells, which normally show minimal enhancer activity for the JSRV LTR.

Envelope-Induced Cell Transformation by Ovine Betaretroviruses

ABSTRACT Ovine betaretroviruses include Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). JSRV and ENTV represent a unique class of oncogenic retroviruses that induce tumors of the respiratory tract. JSRV and ENTV are highly related but induce different diseases. Expression of the JSRV envelope (Env) induces transformation of rodent fibroblasts in vitro and phosphorylation of Akt, a central player in the phosphatidylinositol 3-kinase (PI-3K)/Akt signal transduction pathway. However, little information is available on the molecular biology of ENTV. In this study, we initially assessed whether the ENTV Env has the same properties as the homologous JSRV protein. We performed entry and interference assays using retroviral vectors pseudotyped with either the JSRV or the ENTV Env and sheep choroid plexus cells, choroid plexus cells stably expressing the JSRV Env protein, human 293T cells, mouse NIH 3T3 cells, or NIH 3T3 cells expressing human hyaluronidase 2 (HYAL2), the cellular receptor for JSRV. The results obtained indicated that ENTV and JSRV share the same receptor in sheep cells and that they can use human HYAL2 as a cellular receptor in mouse cells. The ENTV Env induces transformation of rodent fibroblasts in vitro. As with the JSRV Env, the tyrosine at position 590 is critical for ENTV Env-induced cell transformation, and Akt is phosphorylated in ENTV Env-transformed cells but not in the parental cell lines. Thus, ovine betaretroviruses share a common mechanism of cell transformation. We further investigated the relevance of Akt activation in cells transformed by ovine betaretroviruses. A PI-3K inhibitor blocked Akt phosphorylation in JSRV Env-transformed cells, suggesting a possible involvement of PI-3K in JSRV and ENTV Env-induced cell transformation. In addition, phosphorylated Akt was detected in a cell line derived from a lung tumor of a sheep with naturally occurring ovine pulmonary adenocarcinoma.

The Signal Peptide of a Simple Retrovirus Envelope Functions as a Posttranscriptional Regulator of Viral Gene Expression

ABSTRACT 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.

Lung Adenocarcinoma Originates from Retrovirus Infection of Proliferating Type 2 Pneumocytes during Pulmonary Post-Natal Development or Tissue Repair

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer.

The Signal Peptide of a Recently Integrated Endogenous Sheep Betaretrovirus Envelope Plays a Major Role in Eluding Gag-Mediated Late Restriction

ABSTRACT The exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with highly related and biologically active endogenous retroviruses (enJSRVs). The endogenous enJS56A1 locus possesses a defective Gag polyprotein which blocks the late replication steps of related exogenous and endogenous retroviruses by a mechanism known as JSRV late restriction (JLR). Conversely, enJSRV-26, which most likely integrated into the sheep genome less than 200 years ago, is able to escape JLR. In this study, we demonstrate that the ability of enJSRV-26 to escape JLR is due to a single-amino-acid substitution in the signal peptide (SP) of its envelope glycoprotein. We show that enJSRV-26 SP does not localize to the nucleolus, unlike the functional SPs of related exogenous and endogenous sheep betaretroviruses. In addition, enJSRV-26 SP function as a posttranscriptional regulator of viral gene expression is impaired. enJSRV-26 JLR escape relies on the presence of the functional enJS56A1 SP. Moreover, we show that the ratio between enJSRV-26 and enJS56A1 Gag is critical to elude JLR. Interestingly, we found that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus. These data further reinforce the notion that transdominant enJSRV proviruses have been positively selected in domestic sheep, and that the coevolution between endogenous and exogenous sheep betaretroviruses and their host is still occurring.

Host Species Barriers to Jaagsiekte Sheep Retrovirus Replication and Carcinogenesis

ABSTRACT 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.