Human-specific integrations of the HERV-K endogenous retrovirus family

Abstract

Several distinct families of endogenous retrovirus-like sequences (HERVs) exist in the genomes of humans and other primates. One of these families, the HERV-K group, contains members that encode functional proteins and that have been implicated in the etiology of insulin-dependent diabetes mellitus (IDDM). Because of potential functional and disease relevance, it is important to determine if there are HERV-K-associated genetic differences between individuals. In this study, we have investigated the divergence and evolutionary age of HERV-K long terminal repeats (LTRs). Thirty-seven LTRs, taken primarily from random human clones in GenBank, were aligned and grouped into nine clusters with decreasing sequence divergence. Cluster 1 sequences are 8.6% divergent, on average, whereas cluster 9 LTRs, represented by the LTRs of the fully sequenced HERV-K10 clone, show an average of only 1.1% divergence from each other. The evolutionary age of 18 LTRs from different clusters was then investigated by genomic PCR to determine presence or absence of the retroviral element in different primate species. LTRs from clusters of higher divergence were detected in monkeys and apes, whereas LTRs in clusters with lower divergence were acquired later in evolution. Notably, LTRs of cluster 9 were found only in humans at all nine loci examined. Genomic Southern analysis with an oligonucleotide probe specific for cluster 9 LTRs suggests that HERV-K elements with this type of LTR expanded independently in the genomes of humans and the great apes. This is the first report of endogenous retroviral integrations that are specific to humans and indicates that some HERVs have amplified much later than previously thought. These elements may still be actively transposing and may therefore represent a source of genetic variation linked to disease development.

FIG. 1

Dendrogram derived from 37 HERV-K LTR sequences. The names of each sequence refer to the GenBank accession numbers from which the HERV-K LTRs were identified. The names of previously described HERV-K elements are shown in parentheses, where 5′ and 3′ refer to the LTRs flanking the proviral element. GenBank entries containing more than one LTR sequence are indicated by an a or a b following the accession number. All LTRs used were complete sequences of approximately 970 bp, except for the IDDM-associated element, for which only 809 bp of LTR sequence is available in GenBank (accession no. AF012335). The values at the nodes indicate the percentage of the bootstrapped trees supporting the branching pattern, and the numbers in parentheses indicate the clusters referred to in the text. The length of the bar corresponds to a nucleotide sequence divergence of 2%.

FIG. 2

Examples of PCR amplification results with locus-specific primers flanking the integrated LTR of the sequences indicated. Numbers in parentheses following GenBank accession numbers refer to the sequence clusters in Fig. 1. Numbers to the left correspond to the comigrating DNA size markers of each primate panel. N, New World monkey; O, Old World monkey; Gi, gibbon; Or, orangutan; G, gorilla; C, chimpanzee; H, human. Expected amplification products were always obtained for DNA with integrated LTRs or without an integrated retroviral sequence. A slightly larger product than expected was observed in the gibbon, using primers flanking sequence AF001550.

FIG. 3

Southern hybridization with an oligonucleotide specific for cluster 9 LTRs with a panel of primate DNAs digested with EcoRI (see legend to Fig. 2 for abbreviations). To the left are sizes of the comigrating DNA size markers (in kilobases), and to the right an arrow indicates a hybridizing DNA fragment common to the gorilla, chimpanzee, and human within the 1.5- to 3.5-kb region indicated with a bracket. Fragments larger than 3.5 kb were too numerous to analyze.

FIG. 4

Approximate integration times of HERV-K elements. Arrows indicate the lineage in which a particular LTR was first detected, and numbers refer to the cluster as identified in Fig. 1. Time estimates for divergence of the different primate lineages were taken from Bailey et al. (5).