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Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of ovine pulmonary adenocarcinoma (OPA) (4, 15, 25) and is a contagious lung cancer of sheep and goats that closely resembles human adenocarcinomas of the deep airways, including bronchioloalveolar carcinoma (BAC) (21). Human lung cancer is the leading cause of human cancer deaths worldwide (12).
Fifteen to 20 copies of endogenous betaretroviruses (enJSRV) are present in the genome of sheep and goats (3, 8, 9, 17, 20). Endogenous retroviruses (ERV) are vertically transmitted as stable mendelian genes in the germline of most eukaryotes and it is believed that they derive from integration of exogenous (horizontally transmitted) retroviruses in the germ cells of a specific host (24). In recent years, considerable effort has been directed toward understanding the biological significance of ERV, particularly those present in the human germ line (5, 22). Generally, endogenous proviruses are transcriptionally silent and are often defective, typically differing from the exogenous counterpart by deletions or point mutations that render them incapable of forming infectious virus (12). The biological effects of ERV are largely unknown. Given the long standing presence of ERV in the genome, evolutionary theory would predict they might have more beneficial effects than detrimental effects (12). The incidence of human lung adenocarcinoma has steadily risen and this neoplasm is now responsible for up to one quarter of lung cancer fatalities (2). Ovine pulmonary adenocarcinoma presents a unique opportunity to study the me chanisms of lung carcinogenesis in an animal model (2).
Palmarini et al isolated, sequenced and functionally characterized three complete enJSRV proviruses (enJS56A1, enJS5F16 and enJS59A1) derived from a sheep genomic DNA phage library (16). All three proviruses contained open reading frames (ORF) encoding at least one or more structural genes. However, subtle differences in a gag region and long terminal repeat (LTR) U3 and envelope transmembrane (TM) nucleotide sequences were found to be useful in differentiating between enJSRV and the pathogenic, exogenous form of JSRV (16). Palmarini et al. also observed that enJSRVs can block the exogenous JSRV replication by a novel two-step interference mechanism acting both early and late during the virus replication cycle (19). enJSRVs are highly active and they are abundantly and specifically expressed throughout the reproductive tract of an infected sheep (18). So the sheep enJSRVs system is a model that can be utilized to study many different aspects of ERVs and retrovirus biology (19).
In this study, dot blot hybridization of genomic DNA with novel DNA probes derived from JSRV U3 domains, JSRV env TM domains, enJSRV gag domains and enJSRV env TM domains were used for the first time to identify the enJSRVs provirus in China. Cloning and sequence analysis of a intact enJSRV proviruses are also described in this report. The results provide insight into the genomic structure of enJSRVs, its phylogeny and its relationship to known enJSRV and JSRVs.
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In this study, healthy domestic sheep were collected from flocks in Inner Mongolia and defined as "normal" animals if they appeared normal by macroscopic and histological examination of the lungs. OPA-infected sheep were defined as animals which showed typical clinical signs, particularly the production of abundant lung fluid from the nostrils when the rear limbs were elevated above the head. Diagnosis was confirmed by macroscopic and histological examination of the lungs. Sections of lungs from sheep with OPA were used as positive controls.
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Genomic DNA was extracted as described previously (17). Briefly, uterus and lung tissues from healthy domestic sheep were ground to a powder in liquid nitrogen, lysed by addition to TES (0.01 mol/L Tris, 0.1 mol/L EDTA, 0.5% SDS), digested with 100 μg of RNase per ml for an hour at 37℃, then 4 μL (20 mg/mL) of proteinase K was added and incubated at 55℃ overnight and extracted with phenol, pH 8.0. DNA was precipitated with sodium acetate and isopropanol, washed twice with 70% ethanol, dried, resuspended in TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0) and stored at 4℃. DNA was also isolated from SPA-affected sheep lung tumor tissues for use as a positive control. The integrity of DNA was examined by electrophoresis on 1% agarose.
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Four probes were designed within a highly variable area between enJSRV and JSRV. The positions of the probes relative to the genome are shown in Table 1. The four digoxigenin-labeled probes were synthesized by TaKaRa Biotechnology Co., Ltd. (TaKaRa, Dalian, China).
Table 1. Probes used in this study
The dot blot hybridization procedure used was performed essentially as described previously (23). Briefly, DNA was denatured by boiling at 100℃ for 10 min and immediately cooling on ice for 10 min. Then DNA specimens were dotted onto nylon membrane (TaKaRa, Dalian, China). Dotted nylon membranes were sealed in plastic bags with 10 mL of a prehybridization mixture containing 5×SSC, 50% formamide, 25 mmol/L Na2HPO4 (pH 6.5), 5× Denhardt solution, and 250 μg of sheared denatured salmon sperm DNA per mL. The membranes were incubated at 42℃ for 2 to 4 h in a shaking water bath. The prehybridization mixture was removed and replaced with 10 mL of hybridization mixture containing 5×SSC, 45% formamide, 25 mmol/L Na2HPO4 (pH6.5), l×Denhardt solution, 250 μg of sheared denatured salmon sperm DNA per mL, 10% dextran sulfate, and 1.5 μL of 20 μmol/L DNA probe. The plastic bags were resealed and incubated for 18 to 22 h at 42℃ in a shaking water bath. The hybridization mixture was removed and stored at -20 ℃ for reuse. The nylon membranes were washed in (a) 250 mL of 2×SSC-1.0% (wt/vol) SDS for 3 to 5 min at room temperature. (b) 250 mL of 0.2×SSC-1.0% (wt/vol) SDS for 3 to 5 min at room temperature, and (c) 250 mL of 0.16×SSC-1.0% SDS for 15 min at 50℃ twice and then were rinsed briefly in 2× SSC-0.1% SDS at room temperature. Finally these membranes were dried and radiographed.
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Sequencing of the complete genome of enJSRVNM was achieved using overlapping PCR amplification. The positions of the fragments relative to the genome are shown in Fig 1. The primers and annealing temperature used to generate these fragments are shown in Table 2. PCR was performed using 0.5 μg of DNA of Uterus tissues identified, 0.5 μmol/L concentrations of each primer, 200 μmol/L concentrations of each deoxynucleoside triphosphate, 10 mmol/L Tris-HCl (pH 8.8), 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 2.5 U of Taq polymerase (TaKaRa, Dalian, China) in a total volume of 100 μL. The PCR cycles were 95℃ for 5 min, then 30 cycles were performed of 60 s at 94℃ (denaturation), 60 s annealing (for temperature see Table 1) and 120 s at 72℃, with a final extension of 5 min at 72℃. The amplified mixture was electrophoresed on agarose gels, and the specific bands were cut and purified. Then fragments were ligated into PMD19-T vector (Promega) as specified by the manufacturer. At least three clones from two independent PCR reactions were sequenced for each PCR fragment. The fulllength sequence was assembled by using the DNASTAR software package. Finally, the full-length sequence was annotated according to enJS56A1 (AF153615) and submitted to GenBank.
Figure 1. enJSRV-NM sequencing scheme. The clones to obtain the complete sequence of enJSRV-NM are show.
Table 2. Primers and PCR condition for amplification used for sequencing of enJSRV-NM
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Sequence data was analyzed using the programs of Fig. 1. enJSRV-NM sequencing scheme. The clones to obtain the complete sequence of enJSRV-NM are show. the DNASTAR software package (Lasergene). Sequences were aligned by using the MEGALIGN program DNASTAR1.59. Three phylogenetic trees were constructed by using the Kimura method of the DNAMAN software package (version 6.0.3.48), and 1000 bootstraps were run to validate the three phylogenetic threes.
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The nucleotide sequence of enJSRV-NM has been deposited in GenBank under accession number DQ838493.
Sources and preparation of samples
DNA extraction
Dot blot hybridization
Sequence of the enJSRV-NM genome
Computer-assisted analysis
Nucleotide sequence accession number
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Quantities of DNA were denatured and spotted onto a nylon membrane in the indicated pg amounts. Denatured salmon sperm was spotted as a negative control (NC). The four probes show a clear positive reaction with 10 pg and is weakly reactive with 5 pg of target DNA. The endpoint was approximately 5 pg in every test and at this concentration the color was faintly visible. Therefore, the actual sensitivity limit of our probes with DNA extracted from uterus and lung tissues of healthy and infected domestic sheep was probably 7 to 12 pg. The negative control spot was included to ensure probe specificity (Fig not shown).
To determine whether the four DNA probes were sufficient for differentiation between enJSRVs and JSRVs, digoxigenin-labeled DNA probes of enJSRVspecific (probe 1 and 3) and JSRV-specific (probe 2 and 4) were tested by hybridization with a known amount of purified chromosomal DNA of enJSRVs and JSRVs (Fig. 2). JSRV-specific probes (probe 2 and 4) hybridized with homologous DNA of lung tumor tissues of OPA-affected sheep identified by the wheelbarrow test and histological examination of the lungs. No hybridization was obtained with Uterus and lungs tissues of healthy domestic sheep identified by macroscopic and histological examination of the lungs DNA (Fig. 2). Although the sequence of enJSRVs and JSRVs have higher homolog, the results indicated that the four DNA probes were adequate for determination of enJSRV and JSRV using this method with 70~120 pg of target DNA fixed onto the nylon membrane.
Figure 2. Autoradiographs after hybridization of DNA probes with purified viral DNA. A: DNA of uterus tissue from healthy domestic sheep. B: DNA of lung tissue from healthy domestic sheep. C: DNA of lung tissue from OPA-affected sheep. NC, negative control. Sections of lungs from sheep with OPA were used as positive controls. The probe name is indicated at the bottom of each column.
The complete genomic sequence of enJSRV-NM loci was obtained using a shotgun sequencing approach. The complete assembled nucleotide sequence is 7 942 bp in length and contains the four overlapping open reading frames of the gag, pro, pol, and env genes as well as an additional open reading frame (orf-x) that overlaps pol (Fig. 3). This is the first nucleotide sequence of enJSRVs reported in China. The G+C content of the enJSRV genome averages 41.68%, which is very similar to the 41.78% G+C content of enJS56A1.
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The LTRs comprises 435bp of sequence at each end of the enJSRV-NM loci. The enJSRV-NM LTRs are subdivided into three adjacent sequences blocks consisting of a 319 bp U3 region, a 15bp R region and a 101 bp U5 region. The upstream and downstream LTRs of enJSRV-NM and enJS5F16 were identical, while those of enJS56A1 and enJS59A1 displayed two-and four-base changes respectively. Thus, intragenomic variability of the LTRs can be used as a molecular clock to estimate time of integration (16). By this analysis, the integration events of enJS56A1 and enJS59A1 happened ca. 0.9 to 1.8 million years ago, and the enJSRV-NM and enJS5F16 might have integrated less than 500, 000 years ago, based on sequence identity of the upstream and downstream LTRs. These numbers are subject to a wide margin of error (16). and do not take into account the possibility of gene conversion (9). The entire LTR sequence were 85-98% identical among enJSRV-NM and other enJSRVs and JSRVs, particularly in the R and U5 regions. However, there was only 49.3% sequence identity between enJSRV-NM loci and JSRVs in the U3 region because of deletions and point mutations in JSRVs. This region of divergence in U3 has been widely exploited in distinguishing JSRV-infected cells and tissues from sheep enJSRV sequence by PCR and by southern hybridization. Another region of divergence in the TM region of env is discussed below.
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The enJSRV-NM gag (nt 583 to 2433) encodes a 617-amino-acid (aa) polypeptide. There are two sequences of Cys-X2-Cys-X4-His-X4-Cys, separated by 13 residues, in the nucleic acid gag protein. This is typical of a zinc finger domain and is thought to mediate nucleocapsid binding to the genomic RNA. The presence of the ScaⅠsite at position 1724~1729 in gag of JSRV21 which is a molecular marker for JSRVs, was not found in gag of enJSRV loci (10). Two short regions (VR1 and VR2) were analyzed in enJSRV-NM in the gag and found to contain major differences between endogenous JSRVs and exogenous JSRVs but are absent in the corresponding regions of the enJSRV proviruses.
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The pro open reading frame (nt 2325 to 3194) encodes a 326-residue polypeptide. The protein encoded by pro is thought to comprise two domains, a pseudo protease (or protease-like) domain which exhibits a dUTPase activity, as has been suggested for other retroviruses by both computer analysis and functional studies (19), and an active protease (Pr). Alignment of deduced amino acid sequences showed high homology between enJSRV-NM and known endogenous and exogenous JSRVs.
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The pol open reading frame (nt 3 440 to 5 785) encodes a 781-residue polypeptide. The pol gene of enJSRV-NM and known endogenous and exogenous JSRVs had greater than 97% sequence identity. However, there are only 95% deduced amino acid sequences identity between the enJSRV-NM and enJS56A1 in the pol region and because In enJS56A1 there was a 2 bp deletion at the 3' end of the pol gene, the last 33 aa have no similarity due to the frame shift (16).
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The second ORF in pol region was identical to the orf-x of unknown function previously described for the JSRV from South Africa (25). The orf-x extended from nt 4 938 to 5 354 and encoded 166 amino acids. The enJSRV-NM loci was different from the JSRV-SA strain in only two amino acids, but was different from JSRV21 in 13 amino acids indicating that the JSRV strain from South Africa was more closely related to ESRV within the orf-x region, which is in agreement with results of an earlier study (1).
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The env open reading frame (nt 5 682 to 7 517) encodes a peptide of 611a.a. giving a predicted molecular mass of 69 kDa. As shown in Table 3, the env of enJSRV-NM loci and other enJSRVs are very similar, with nucleotide and amino acid identities greater than 98%. Alignment of the env gene of enJSRV-NM loci with JSRVs (type Ⅰ and type) Ⅱ shows that they share 88%-93% amino acid identity. The TM region of env is the most divergent of the coding regions of enJSRV-NM loci and JSRVs. They are 96% identical in SU and 85% identical in TM at the amino acid level. The TM region is also the most divergent of the coding regions of other enJSRVs and JSRVs and for this reason was chosen for selection of dot hybridization probes. The enJSRV-NM env lacked the YXXM motif present in the VR3 region of the TM domain of the exogenous JSRV env that has been found to be critical for transformation (18).
Table 3. Comparison of enJSRV-NM and other known enJSRV /JSRV genes and proteins
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We generated rooted neighbor-joining phylogenetic trees to assess the phylogenetic relationships between the enJSRV-NM cloned in this study and with other known sequences of endogenous and exogenous type D retroviruses of sheep. We generated a tree for the U3 region, one for env and one for gag (Fig. 4). In each tree it was possible to distinguish two major branches: one for the endogenous loci, and one for the exogenous JSRV sequence, confirming previous analyses with limited gag sequences. The exogenous JSRVs could be further divided into two branches corresponding to sequences derived from Africa (typeⅠ) or from the United States and the United Kingdom (typeⅡ) as previously described. The endogenous loci also could be further divided into at least two phylogenetic groups, enJSRV-A and -B. These phylogenetic trees show that the enJSRV-NM loci is most closely related to enJSRVs, especially enJS56A1 (enJSRV-B) and enJS5F16 (enJSRV-B). So the enJSRV-NM loci belong to type-B.