1 Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
2 Laboratory of Infectious Disease, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
3 Laboratory of Animal Experiment for Disease Model, Institute for Genetic Medicine, Hokkaido University, Sapporo, 060-0815, Japan
Correspondence
K. Ochiai
k-ochiai{at}vetmed.hokudai.ac.jp
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Chick embryo fibroblasts (CEF) were prepared from 10-day-old SPF chicken embryos of the WL-M/O (C/O) strain. Fertile eggs of the commercial SPF White Leghorn strain WL-M/O were purchased from Nippon Institute for Biological Science. This strain lacks both chicken helper factor (chf) and group specific (gs)-antigen and is susceptible to ALV subgroups A-E (International registry of poultry genetic stocks, Bulletin 476, March 1988, Ralph G. Somes, Jr, PhD, University of Connecticut, USA). Cell culture and preparation of the viral suspension were performed using the methods described by Iwata et al. (2002a). For sequencing, a 100-fold concentrated culture supernatant was prepared as described previously (Bowles et al., 1996
). Total RNA was extracted from the CEF inoculated with this concentrated virus and uninfected CEF using the TRIZOL reagent (Invitrogen). The extracted RNA was treated with 40 U of RNase-free DNase (Roche Diagnostics). Reverse transcription was carried out using random hexamers (Takara) and SuperScript II RNase H- Reverse Transcriptase (Invitrogen). Primers for PCR were designed based on the nucleotide sequence of ALV-A (accession No. M37980) or sequences newly obtained by genome walking. The PCR amplification for sequences more than 1·2 kb in length was performed using LA Taq polymerase (Takara). To obtain short-PCR products of up to 1·2 kb, amplification was performed using AmpliTaq DNA polymerase (Applied Biosystems). The PCR amplifications were performed using temperature profiles within the following ranges, depending on expected product sizes and primer sequences: 94 °C for 3 min, followed by 35 cycles of 94 °C for 30-60 s, 50-63 °C for 30-60 s, 72 °C for 1-3 min, and a final extension at 72 °C for 10 min. The PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen) and cloned into the pGEM-T vector (Promega). More than five colonies containing an insert were selected and plasmid DNA was purified by a standard mini-prep method. Sequencing was performed using the BigDye terminator cycle sequencing kit (Applied Biosystems) and a model 310 genetic analyser (Applied Biosystems). All computational sequence analysis was done using the GENETYXMAC 10.1.2 package (Software Development) in combination with the BLAST program at GenBank. The sequences (with accession numbers) used in the alignments were: ALV-RSA (M37980), ALV-HPRS103 (Z46390), Rous sarcoma virus (RSV; Prague C; J02342), RSV (Schmidt-Ruppin B; AF052428, Schmidt-Ruppin A; U41731, Schmidt-Ruppin D; D10652), avian carcinoma Mill Hill virus 2 (K02082), avian sarcoma virus CT10 (Y00302), and Y73 sarcoma virus (J02027). The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ nucleotide sequence databases under the accession number AB112960.
The full cDNA sequence of the glioma-inducing virus was 7,448 nucleotides long with a genetic organization typical of replication-competent type C retroviruses lacking viral oncogenes. A schema of the structure of the virus and comparison with other strains mentioned in the following sections are shown in Fig. 1. The gag and pol genes of the ALV (bases 383 to 2485 and 2506 to 5193, respectively) were well conserved with those of other avian leukosis and sarcoma viruses (ALSVs) (which show 96 to 98 % overall identity to the sequences of subgroups A, B, C, D and J). The gag and pol genes of ALV-A and RSV-Pr-C were most closely related to those of the glioma-inducing virus. The sequence encoding the SU (gp85) domain of the virus isolate showed 82, 92, 83 and 87 % overall nucleotide identity to that of subgroup A, B, C and D, respectively, and was most closely related to that of RSV-SR-B (92 %). The SU-encoding sequences had only 47 % identity overall to the corresponding sequences of ALV-J. The sequence encoding the TM (gp37) of the virus isolate showed over 92 % identity to the corresponding sequences of subgroup A, B, C, and D, and 61 % identity to that of ALV-J.
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The results were normalized to a positive control containing the SV40 early gene promoter and enhancer (Fig. 3ac). Transcriptional activity levels of the LTRgli observed in all cells examined were almost the same as or higher than those of the positive control. The transcriptional activity of the LTRgli in CEF was twelve times of that of the positive control and was higher than that observed in the other cells examined. In addition, the transcriptional activity of the LTRgli was compared with that of LTRrav1 in CEF and U87MG. Promoter activities of these two LTRs were equivalent in CEF (Fig. 3d
), whereas the activity of the LTRgli was significantly lower than that of LTRrav1 in U87MG (P<0·001, Fig. 3e
).
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Since the envelope glycoprotein of ALV serves as a ligand for a receptor (Weiss, 1992), the env gene influences the types of cell targets (Brown & Robinson, 1988
). ALV-J, which has a distinct env gene (Bai et al., 1995
), causes myeloid leukosis rather than lymphoid leukosis (Payne et al., 1992
). The specific oncogenicity is suggested to be largely dependent on the viral envelope (Chesters et al., 2002
). Unlike ALV-J, the env gene of the glioma-inducing ALV has high homology with that of subgroup A, B, C and D. Also the virus was detected in various organs with the same distribution pattern as other ALVs, indicating little difference among them in terms of viral entry and infection (Iwata et al., 2002b
; Tomioka et al., 2003
). The SU sequence more closely resembles SR-B than subgroup A, although we have previously reported the ALV causing glioma belongs to subgroup A. Some minor mutations in the sequence may be related to the biological activity. The cell type-specific oncogenesis regulated by the LTR is dependent on subtle differences among the cellular transcriptional factors. Because high expression of c-myc by stable transcription may lead to the elimination of infected pre-B cells before developing lymphoma, the labile, low LTR-enhanced transcription could be important in ALV lymphomagenesis (Ruddell et al., 1989
; Bowers et al., 1994
). The promoter activity of the LTRgli was significantly lower than that of LTRrav1 in the astrocytic cell line. The subtle sequence differences may be related to the transcriptional activity, which cause the unique capability of transformation.
As involved proto-oncogenes influence the neoplastic phenotype induced by ALV (Hayward et al., 1981; Fung et al., 1983
; Kanter et al., 1988
), it is also possible that the unique neoplastic phenotype of the glioma-inducing ALV could depend on the integration site. Further examination of these subjects is necessary for elucidation of the determinant of the neurotropic oncogenicity of this virus.
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ACKNOWLEDGEMENTS |
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Received 4 November 2003;
accepted 14 November 2003.