Endogenous type D retrovirus in a marsupial, the common brushtail possum (Trichosurus vulpecula)

Abstract

We have sequenced and characterized an endogenous type D retrovirus, which we have named TvERV(D), from the genome of an Australian marsupial, the common brushtail possum (Trichosurus vulpecula). Intact TvERV(D) gag, pro, pol, and env open reading frames were detected in the possum genome. TvERV(D) was classified as a type D retrovirus, most closely related to those of Old World monkeys, New World monkeys, and mice, based on phylogenetic analyses and genetic organization. Approximately 30 TvERV(D) proviruses are present in the genomes of possums, as detected by Southern hybridization. However, variability in fragment patterns between possums was observed and suggests recent (or ongoing) retrotranspositional activity.

FIG. 1

Nucleotide sequence and deduced amino acid sequences of pTvERV(D). Locations of regulatory regions within the nucleotide sequence, including the internal repeat (IR) at the 3′ end of U5, the PBS, and SD, are overlined. The deduced amino acid sequences of the gag, pro, and pol ORFs, as well as a short region with TM homology, are shown below the nucleotide sequence. The shaded regions within each protein correspond to conserved domains described in the text. A shill (/) indicates a frameshift.

FIG. 1

Nucleotide sequence and deduced amino acid sequences of pTvERV(D). Locations of regulatory regions within the nucleotide sequence, including the internal repeat (IR) at the 3′ end of U5, the PBS, and SD, are overlined. The deduced amino acid sequences of the gag, pro, and pol ORFs, as well as a short region with TM homology, are shown below the nucleotide sequence. The shaded regions within each protein correspond to conserved domains described in the text. A shill (/) indicates a frameshift.

FIG. 1

Nucleotide sequence and deduced amino acid sequences of pTvERV(D). Locations of regulatory regions within the nucleotide sequence, including the internal repeat (IR) at the 3′ end of U5, the PBS, and SD, are overlined. The deduced amino acid sequences of the gag, pro, and pol ORFs, as well as a short region with TM homology, are shown below the nucleotide sequence. The shaded regions within each protein correspond to conserved domains described in the text. A shill (/) indicates a frameshift.

FIG. 2

Nucleotide sequence and deduced amino acid sequences of pTvERV(D)-env. The putative SA is overlined. The deduced amino acid sequences of the 3′ end of the pol gene and the entire env gene are shown below the nucleotide sequence. The shaded regions within each protein correspond to the conserved domains described in the text. Sites where the Env polyprotein is cleaved are indicated by arrows (↔). Asparagine (N) residues which are within A-X-S/T motifs (where X is any amino acid except proline) and are therefore potentially glycosylated are underlined.

FIG. 3

Neighbor-joining trees based on the derived amino acid sequences of the Pol proteins of type B and D retroviruses (A) and the Env proteins of the type D retroviruses and retroviruses with related pol genes (B). Amino acid sequences were aligned using CLUSTAL X, and the aligned sequences were used to construct neighbor-joining trees. Bootstrap values for 1,000 replicated trees are indicated. The Pol tree was rooted using MMTV as the outgroup, whereas the Env tree was rooted using Gibbon ape leukemia virus (GALV) and Moloney murine leukemia virus (MoMLV) as the outgroup. The accession numbers of retroviral sequences are as follows: BaEV, D10032; ENTV, Y16627; GALV, M26927; JSRV, M80216; MMTV, M15122; MPMV, AF033815; MoMLV, AF033811; RD114 env/Env, X87829; REV env/Env, 228842; SERV 23.1, U85505; SERV 25.2; U85506; SMRV-H, M23385; SNV Env, VCFVAS; SRV-1, M11841; and SRV-2, M16605.

FIG. 4

Southern hybridization of restriction enzyme-digested genomic DNA from 12 possums with the TvERV(D) gag probe. (A) The gag probe detects junction fragments generated by PvuII cleavage 5′ to the 5′ LTR of TvERV(D) and at the 3′ end of the TvERV(D) gag gene. Thus, each TvERV(D) provirus will generate a junction fragment of a distinct size, depending on the distance between the provirus and the nearest PvuII site. (B) The gag probe detects internal TvERV(D) fragments generated by NheI cleavage within the 5′ LTR and env gene of TvERV(D) elements with intact env genes and within the 5′ and 3′ LTRs of TvERV(D) elements with disrupted env genes. Possums 1 through 6 were from the Wellington region and possums 7 through 12 were from the Waikato region of the North Island of New Zealand. Bands common to all 12 possums are indicated by arrows (right). DNA sizes are indicated at left.