BASE, INRA Centre de Tours, 37380 Nouzilly, France
Correspondence
Caroline Denesvre
denesvre{at}tours.inra.fr
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ABSTRACT |
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The accession numbers for the sequences described are AY157601 for ev/J 4.1 Rb and AY158693 for env ALV-J INRA-7.
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MAIN TEXT |
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We generated an ev/J 4.1 env expression plasmid. For this, total genomic DNA was isolated from the blood of a meat-type strain chicken and the env gene was amplified by PCR with ev/J-specific primers under conditions similar to those described by Ruis and colleagues using the Herculase DNA polymerase (Stratagene) (Ruis et al., 1999). The expected 2·2 kbp PCR product was purified (QIAquick gel extraction kit; Qiagen) and sequenced on both strands in the region corresponding to the env gene. This PCR product was then cloned into a pGEM-T easy vector (Promega). One clone, ev/J 4.1 Rb, was selected and sequenced. The ev/J 4.1 Rb sequence matched that of the PCR product and was found to be different from the published ev/J 4.1 prototype env gene cloned from DF-1 cells (ev/J 4.1 DF; GenBank accession no. AF125528) (Benson et al., 1998
). The amino acid sequence deduced from ev/J 4.1 Rb probably corresponds to the natural parental isolate since it was closer in identity to the Env protein of two ALV-J reference strains, HPRS-103 (96·74 %) and Hc1 (95·65 %), than was ev/J 4.1 DF (94·25 % and 93·45 %, respectively) (Fig. 1
). The 1828 bp SnaBIMluI fragment containing the complete ev/J 4.1 Rb env sequence was excised, blunt-ended and cloned into pcDNA3 at the EcoRV site. The junctions of this pEEJ4.1 expression vector, also expressing the neomycin selection marker, were verified by DNA sequencing. The expression of ev/J 4.1 Rb env in TelCeB6 cells was verified by RT-PCR with the previously reported primer pair EAV-SD and 103-ER (Sacco & Venugopal, 2001
) (data not shown).
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The pEEJ4.1 plasmid was used to generate DF-1 cells constitutively expressing the ev/J 4.1 Rb. After transfection of DF-1 cells with pEEJ4.1, G418-resistant cells were selected using 800 µg geneticin (Gibco) ml-1. G418-resistant clones were isolated and analysed for cell-surface expression of ev/J 4.1 Env by flow cytometry using a polyclonal chicken antiserum to HPRS-103. Two DF-1 clones, clones 2 and 4, were selected for their high level of Env cell-surface expression. In order to test the interference properties of the ev/J Env protein, the two DF-1 ev/J clones were challenged with LacZ virion pseudotypes as described above. The number of LacZ-positive cells was counted 48 h post-infection (Table 2). No blue cells were detected after challenging infection with LacZ (Hc1) or Lac Z (ev/J 4.1 Rb), thus demonstrating that overexpression of ev/J 4.1 Rb Env alone mediates complete auto- and cross-interference with ev/J 4.1 Rb Env and ALV-J Hc1 Env, respectively.
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ev/J 4.1 is the only EAV-HP or ev/J provirus identified from chicken genomes that carries a complete env gene. However, no protein expression of this provirus has been described to date. We have shown here that ev/J 4.1 Rb ORF expression leads to production of a functional Env product. Like other retroviral signal peptides, the signal peptides of ev/J 4.1 and ALV-J contain an N-terminal region rich in charged amino acids and a central hydrophobic core followed by small polar amino acids (Ellerbrok et al., 1992). Our observation of the rather low sequence identity between the ev/J 4.1 and ALV-J Env signal peptides confirms that conservation of these general features, rather than of strict amino acid sequences, can also predict the avian Env signal peptide functionality.
The fact that the endogenous ev/J 4.1 Rb and exogenous ALV-J Env exhibit complete and fully reciprocal interference to superinfection indicates that they share the same receptor(s) on DF-1 cells. On the basis of their interference properties and amino acid divergences with ALV-E endogenous envelope sequences, we propose to classify ev/J 4.1 endogenous virus Env into the ALV-J subgroup. This subgroup will therefore be the first ALV subgroup described that includes both endogenous sequences and exogenous viruses. The high sequence homologies and similar functional properties observed between ev/J 4.1 and ALV-J Env most likely indicate the recent emergence of exogenous infectious ALV-J viruses after the recombinational insertion of ev/J endogenous sequences.
The complete in vitro interference described here is reminiscent of the MLV interference observed with the murine ecotropic (Fv-4) and polytropic (rmcf) endogenous retroviral sequences (Ikeda & Sugimura, 1989; Lyu et al., 1999
). However, unlike the MLV ecotropic env-like Fv-4 resistance gene, ev/J 4.1 encodes an Env protein capable of driving virion infection similar to endogenous MLV polytropic sequences (Lavignon et al., 1994
). Whether the ev/J 4.1 loci confer in vivo resistance for ALV-J infection or ALV-J-induced disease is still not known and becomes of importance in view of the recent ALV-J epidemics. Previous attempts to detect ev/J 4.1 env mRNA either from an ev0 chicken embryo cDNA library by RT-PCR or by Northern blot did not succeed (Ruis et al., 1999
). Nevertheless, microarrays containing 1000 non-redundant selected cDNAs, used to examine host-cell gene expression after Marek's disease avian herpesvirus infection, revealed the expression of a mRNA encoding an ALV-J Env protein (Morgan et al., 2001
). This mRNA may have derived from the expression of an ev/J 4.1 sequence. Therefore, determining the conditions of expression of ev/J 4.1 sequences and their effect on further virus infection will help to elucidate the impact of this sequence in the chicken genome.
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ACKNOWLEDGEMENTS |
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Received 28 May 2003;
accepted 21 August 2003.