HERV-K(OLD): ancestor sequences of the human endogenous retrovirus family HERV-K(HML-2)

Abstract

Sequences homologous to the human endogenous retrovirus (HERV) family HERV-K(HML-2) are present in all Old World primate species. A previous study showed that a central region of the HERV-K(HML-2) gag genes in Hominoidea species displays a 96-bp deletion compared to the gag genes in lower Old World primates. The more ancient HERV-K(HML-2) sequences present in lower Old World primates were apparently not conserved during hominoid evolution, as opposed to the deletion variants. To further clarify the evolutionary origin of the HERV-K(HML-2) family, we screened GenBank with the 96-bp gag-sequence characteristic of lower Old World primates and identified, to date, 10 human sequence entries harboring either full-length or partially deleted proviral structures, probably representing remnants of a more ancient HERV-K(HML-2) variant. The high degree of mutations demonstrates the long-time presence of these HERV-K(OLD) proviruses in the genome. Nevertheless, they still belong to the HML-2 family as deduced from dot matrix and phylogenetic analyses. We estimate, based on the family ages of integrated Alu elements and on long terminal repeat (LTR) divergence data, that the average age of HERV-K(OLD) proviruses is ca. 28 million years, supporting an integration time before the evolutionary split of Hominoidea from lower Old World primates. Analysis of HERV-K(OLD) LTR sequences led to the distinction of two subgroups, both of which cluster with LTRs belonging to an evolutionarily older cluster. Taken together, our data give further insight into the evolutionary history of the HERV-K(HML-2) family during primate evolution.

FIG. 1

Dot matrix comparisons between the most intact member of the HERV-K(HML2) family (20, 28) and the identified HERV-K(OLD) proviruses. Lines represent regions with at least 70% similarity in a window of 30 nt between the two proviral sequences. HERV-K(OLD) sequences are shown on the x axis, and the numbers give the position in the GenBank entry. The location of features like deletions and insertions are indicated in the matrices.

FIG. 2

Multiple sequence alignment of HERV-K(OLD) LTR sequences. Only the LTR regions harboring the diagnostic differences between the two subgroups are shown. Positions 612 to 668 of the human-specific HERV-K(HML-2.HOM) LTRs (20, 28) are shown for comparison. 5′ and 3′ indicates the respective LTRs in a given provirus. “solAL121932” indicates the solitary LTR present in HERV-K(OLD-AL121932).

FIG. 3

Unrooted neighbor-joining tree of HERV-K LTR sequences. HERV-K(OLD) LTRs were compared to previously reported LTR sequences from evolutionary older (LTRI) and younger (LTRII) clusters derived from chromosome 19 (11). HERV-K(OLD) LTRs without the diagnostic deletions of 8 and 23 bp are boxed, and LTRII sequences are circled. All HERV-K(OLD) LTRs cluster with the older LTRI sequences (not included in the circle), independent from the presence or absence of the 8- and 23-bp deletions (see text for further details). The phylogenetic distances were calculated using the Kimura two-parameter model with a transition/transversion ratio of 2. Support values (1,000 bootstrap replicates) are indicated on the corresponding branches. The scale bar represents a 10% evolutionary distance.

FIG. 4

Neighbor-joining tree of HERV-K(OLD) polymerase sequences and previously published HML polymerase sequences (21). The tree is rooted with a HML-6 sequence being the most distant of the six HML groups (21), but the HML6-3 and HML3-6 relation could not be resolved in this analysis. The distances were calculated using the Kimura two-parameter model with a transition/transversion ratio of 2. Support values (1,000 bootstrap replicates) are indicated on the corresponding branches. The scale bar represents a 10% evolutionary distance.

FIG. 5

Amino acid alignment of conserved dUTPase motifs. Residues in HERV-K proviral sequences differing from highly conserved residues are indicated in boldface. “HERV-K Cons” corresponds to an enzymatically active HERV-K(HML-2) dUTPase construct (7). “HERV-K(OLD) Cons” is the dUTPase consensus sequence derived from eight proviral sequences. Ambiguous amino acids are indicated below the sequence. The amino acid sequence of HERV-K(OLD-AL136419), displaying a prt ORF, is included for comparison.

FIG. 6

Presence of HERV-K(OLD-AL035587) in hominoids and lower Old World monkeys. PCR primers were specific for 3′-flanking sequences and the HERV-K(OLD) LTR. NWM (New World monkeys): Age, Ateles geoffroyi; Ase, Alouatta seniculus. OWM (Old World monkeys): Msp, Mandrillus sphinx; Mfa, Macaca fascicularis; Tge, Thercopithecus gelada; Pha, Papio hamadryas. Hominoids: Hla, Hylobates lar; Ptr, Pan troglodytes; Hsa, Homo sapiens. M, marker (size of marker bands is given in base pairs); N, PCR control without DNA.