Cell Biological Function Of EnJSRV Envelope Proteins In Sheep Trophoblast Cells

BACKGROUND:Endogenous retroviruses （ERVs） comprise a significant portion of the genome of all mammals and have been implicated in placental development in multiple species. The ovine genome contains approximately 27 copies of endogenous betaretroviruses （enJSRVs） that are related to the exogenous Jaagsiekte sheep retrovirus （JSRV）, an oncogenic retrovirus tropic to the lung. enJSRVs have played several roles in the evolution of the domestic sheep as they are able to block the JSRV replication cycle and play a critical role in sheep conceptus development and placental morphogenesis. The enJSRV loci are abundantly expressed in the female reproductive tract and the conceptus, and they are essential to conceptus development and placental morphogenesis.OBJECTIVE:In this study, we focus on the biological roles of enJSRVs envelope proteins in the development of sheep trophoblast cells. We further discuss the existing controversy in the field and highlight areas of future research and the value of knowledge gained from enJSRVs envelope proteins studies.METHODS:1. The primary sheep trophoblast cells （STCs） were isolated and purified by using Percoll and specific immuno-affinity purification, respectively. The purified STCs were transfected with a plasmid carrying sequences of human telomerase reverse transcriptase （hTERT） to create immortalized sheep trophoblast cell line （hTERT-STCs）.2. RT-PCR technique was used to amplify enJSRV-env gene as well as the full-length gene of its receptor Hyal2. The genes were then directionally cloned into the eukaryotic expression vector pEGFP-C1. The electric transfection conditions of the chorionic trophoblast cells were optimized and the electrical transfer efficiency was measured. Designed and constructed the RNA interference vector that inhibited enJSRV-env gene, and detected its interference efficiency by quantitative RT-PCR and Western blot.3. The constructed eukaryotic expression plasmids pEGFP-C1/enJSRV-env and pEGFP-C1/Hyal2 were transfected into chorionic trophoblast cells by electroporation, and transient expressions of enJSRV envelope protein and its receptor Hyal2 were observed under a fluorescence microscope. The sheep trophoblast cells fusion activity, invasiveness, secretion of Chorionic Gonadotrophin beta subunit （CG-beta） and Placental Lactogen （PL） in sheep trophoblast cells were assessed after transfection with vectors that overexpressed or silenced enJSRV-env gene.4. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus （exJSRV-env） was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localization of exJSRV-env and enJSRV-env was also performed. The recombinant clone of pEGFP-C1/exJSRV-env was transfected into 293T cells by Lipofectamine（?） LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. The recombinant clone of pEGFP-C1/exJSRV-env and pEGFP-C1/enJSRV-env was transfected into and NIH3T3 cells by Lipofectamine（?） LTX. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. Cells in the logarithmic growth phase were collected and subjected to trypsinization. Cell suspension was subcutaneously injected into the right flanks of nude mice. After two months, the mice were decapitated, and dissected for subcutaneous tumor nodules. These were paraffin embedded and subjected to Hematoxylin and eosin （H&E） staining.5. High-molecular-weight genomic DNA was isolated from a lung tumor collected from a Mongolian sheep with naturally acquired ovine pulmonary adenocarcinoma. The genomic DNA was digested to completion with XbaI. High-molecular-weight genomic DNA was fractionated by twice CHEF gel electrophoresis. Digested DNA fragments in the range of 10kb～40kb were recovered by electro-elution.The eluted DNA was ligated to CopyControl pCC1BAC BamHI Cloning-Ready Vectors. After ligation, the dialysed ligation product was introduced into TransforMax EPI300 Electrocompetent E. coli cells with a Gene Pulser XcellTM Total System. Finally, the cells were spread on LB agar plates containing 12.5 μg chloramphenicol/mL,100 μg IPTG/mL and 40 μg X-gal/mL and incubated at 37 ℃ for 16 h. White clones were picked with sterile toothpicks and manually arrayed in 96-well plates filled with 80 μL freezing LB media, containing 12.5 μg chloramphenicol/mL. All 96-well plates were incubated at 37 ℃ overnight until the media became turbid. To reduce the workload of BAC library screening, we designed a descending-pool system for rapid screening by PCR. BAC clones containing enJSRV loci were screened from BAC library using the gag, pro, pol and env primers corresponding to a highly conserved region in genomic of the known endogenous and exogenous sheep betaretroviruses.RESULTS:1. Immortalized sheep trophoblast cell line （hTERT-STCs） showed a stable expression of hTERT gene, serially passaged for a year, and showed active proliferation without signs of senescence. Cytokeratin 7 （CK-7）, secreted human chorionic gonadotrophin subunit β （CG-β）, placental lactogen （PL）, and endogenous Jaagsiekte sheep retrovirus （enJSRVs） envelope genes were expressed in hTERT-STCs. Transwell cell invasion assay indicated that hTERT-STCs still possessed the same invasive characteristics as normal primary sheep trophoblast cells. HTERT-STCs could not grow in soft agar and did not develop into tumors in nude mice.2. The results showed that eukaryotic expression plasmids were successfully constructed and named as pEGFP-C1/enJSRV-env and pEGFP-C1/Hyal2, respectively. The best transfection conditions was pulse voltage 150 V, pulse time 5 ms, electric shock number for 2 times with 50 ms interval. The results showed that the five chimeras （pcDNA6.2-GW/EmGFPmiR-enJSRV-env-1～4 and Negative-shRNA） were successfully constructed. Using the enJSRV-env mRNA level in normal control cell group as reference, real time RT-PCR results showed that relative enJSRV-env mRNA level in the negative control was 0.985±0.015, and it was 0.581±0.167,0.179±0.018,0.346±0.095, and 0.403±0.051 in cells treated with different shRNAs, respectively. Western Blot results showed that the expression levels of enJSRV Env in the interference and empty carrier groups were significantly lower than that in the blank control group. The pcDNA6.2-GW/EmGFPmiR-enJSRV-env-2 showed the highest interference efficiency.3. The results showed significantly increased fusion efficiency of STCs that were transfected with pEGFP-C1/enJSRV-env or co-transfected with pEGFP-C1/enJSRV-env and pEGFP-C1/Hya12, averaging 20.44%±7.79% and 24.09%±10.65% multinucleated cells per field, respectively. Sheep trophoblast cell invasiveness was significantly intensified when transfected with pEGFP-C1/enJSRV-env, while it was significantly decreased in STCs transfected with pcDNA6.2-GW/EmGFP-miR/enJRSV-env-shRNA-2. There was no significant difference in secretion levels of CG-beta and PL by primary STCs transfected with pEGFP-Cl/enJSRV-env or pcDNA6.2-GW/EmGFP-miR/enJRSV-env-shRNA-2.4. DNA sequencing and restriction enzyme digestion with Kpn I and Hindâ…¢ indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. A YRNM motif was discovered at the cytoplasmic tail of exJSRV envelope gene, which is exclusively found in exogenous viruses. But YRNM motif wasn’t discovered at the cytoplasmic tail of enJSRV envelope gene. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. NIH3T3 cells transfected with enJSRV-env didn’t lose contact inhibition, and didn’t colony forming ability in soft agar. This study indicated that envelope protein of enJSRV can not induce malignant transformation of mouse fibroblast cell NIH3T3.5. The ovine pulmonary adenocarcinoma （OPA） tumors bacterial artificial chromosome （BAC） library has been constructed. The ovine pulmonary adenocarcinoma （OPA） tumors BAC library includes three sub-libraries （OPA-BAC₁0K, OPA-BAC₂5K, OPA-BAC₄0K）. This OPA tumors BAC library consists of 106,500 clones. The average insert size of the library is 20-25 kb, which was evaluated from analysis of 526 randomly selected BAC clones. The ratio of negative clone in this BAC library is 0.38%. Establish of polymerase chain reaction （PCR） screen system with high efficient. Using 4 enJSRV specific PCR primers to screen the BAC library. As a result,1 positive BAC clone was identified from the library.CONCLUSIONS:In this study, we established a strain of immortalized sheep trophoblast cell line which could be gainfully employed in future as an experimental model to study trophoblast cells with secretory function, invasive features and with the probable biological function of enJSRVs envelope genes. Three eukaryotic expression plasmids （pEGFP-C1/enJSRV-env, pEGFP-C1/Hyal2 and pEGFP-C1/exJSRV-env） were successfully constructed. We concluded that the enJSRV envelope protein promoted STC fusion and intensified the invasion of STCs. The ovine pulmonary adenocarcinoma （OPA） tumors bacterial artificial chromosome （BAC） library has been constructed and 1 positive BAC clone was identified from this library.