Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl

Abstract

The EAV-HP group of chicken endogenous retrovirus elements was previously shown to be defective, with large deletions of the pol gene. In this report, we demonstrate that genomes of other Gallus species also maintain EAV-HP elements with similar deletions. The chicken EAV-HP1 locus was detected in both red (Gallus gallus gallus) and Sonnerat's (Gallus sonneratii) jungle fowl with identical integration sites, indicating that these elements had integrated before separation of the Gallus species. Furthermore, we demonstrate for the first time that the G. sonneratii genome carries EAV-HP elements with intact pol regions.The EAV-HP (also designated ev/J), the most recently identified members of the endogenous avian retrovirus (EAV) family, are present in Gallus species as 10 to 15 copies per genome (12). Four different structures of EAV-HP proviruses have been identified in the chicken genome. Three of these 4-kbplong provirus structures, designated types I, II, and III, show large deletions spanning the entire pol gene and parts of the gag and env regions. Each structure has a different gag-env deletion junction, with the type II and III provirus deletions extending an additional 57 and 86 bp, respectively, than the smallest (type I) provirus deletion size (9, 10). These proviruses are found at multiple loci, with the type I provirus appearing to be the most abundant, based on its frequency in clones randomly screened from separate chicken genomic DNA libraries (9, 10). A single clone, designated ev/J clone 4-1, forms the fourth type of proviral structure, comprising a cDNA of the env subgenomic transcript bounded by long terminal repeats (LTRs) (9). The EAV-HP proviruses are more than 97% identical to the newly emerged subgroup J avian leukosis virus (ALV-J) env gene and demonstrate Ͼ96% sequence identity to other EAV family members in the R and U5 regions, the 5Ј untranslated region, and portions of the gag gene (6, 9, 10).Because of their distribution in several Gallus species, the EAV family is considered an ancient group of retroviruses that integrated into a common ancestor. The genus Gallus is composed of four species: the Sonnerat's or grey jungle fowl (SJF; G. sonneratii), the green jungle fowl (GJF; G. varius), the Ceylonese jungle fowl (CJF; G. lafayettei), and the red jungle fowl (RJF; G. gallus), of which domesticated chickens are a subspecies (G. gallus domesticus) (4). EAV-HP proviruses have also been detected in both RJF and SJF by hybridization of env and LTR sequences and PCR amplification of the env region (10, 12). In this study, we have used a PCR approach to examine whether (i) the EAV-HP proviruses in RJF and SJF have the same pol region deletions as in chickens, (ii) the EAV-HP1 and EAV-HP2 loci, present in chickens, could be detected in the jungle fowls, and (iii) the genomic deletion and integration events occurred before or after the separation of Gallus species.The EMBL accession numbers for the sequences described here are AJ292966 for clone EAV-JF1 and AJ292967 for EAV-JF2, which includ...

Segregation of EAV-HP Ancient Endogenous Retroviruses within the Chicken Population

“…A previous attempt to PCR amplify the 5Ј ends of proviruses with intact env genes failed to amplify this provirus from a meat-type chicken (line 21), suggesting that this provirus might be absent from this chicken line and, consequently, segregating within the chicken population (12). To specifically amplify only provirus DNA with the structure of the spliced env subgenomic transcript, PCR was performed with primer EAV-SD (Table 1), which includes the splice donor sequence and the first six bases of the splice acceptor site, along with the env primer 103ER (Fig.…”

“…The ev/J clone 4-1 is the only EAV-HP provirus identified to date from the chicken genome carrying a complete env gene (9,12), making it a candidate as the source of env sequences involved in the generation of the ALV-J subgroup and the associated tolerance. A previous attempt to PCR amplify the 5Ј ends of proviruses with intact env genes failed to amplify this provirus from a meat-type chicken (line 21), suggesting that this provirus might be absent from this chicken line and, consequently, segregating within the chicken population (12).…”

“…All EAV-HP proviruses from chickens appear to be defective due to deletions of the pol gene; however, intact proviruses have been identified in Sonnerat's jungle fowl (Gallus sonneratii) (12). A single clone, designated ev/J clone 4-1, with the structure of a spliced env subgenomic transcript bounded by LTRs is the only chicken EAV-HP element described to date that possesses a complete env gene including the endoplasmic reticulum (ER) translocation signal coding sequence (9).…”

“…2, lower left panel). The pre-integration state PCR products were also amplified from Sonnerat's and red jungle fowl DNA previously shown to be positive for the proviral locus (12), indicating the EAV-HP1 proviral locus was hemizygous in these birds and is still segregating within these Gallus species (data not shown). The PCR products detected from BRL DNA, however, were approximately 300 bp larger than those of the pre-integration state.…”

“…The EAV-HP1 and EAV-HP2 clones were used as positive controls, and water was used for negative controls. Blots were hybridized as previously described (12), with the corresponding positive control PCR products used as probes; pre-integration state locus PCR products were hybridized simultaneously with the proviral locus right and left LTR probes, which include the flanking host sequences. …”