Human endogenous retrovirus K solo-LTR formation and insertional polymorphisms: implications for human and viral evolution

Abstract

Human endogenous retroviruses (HERVs) are a potential source of genetic diversity in the human genome. Although many of these elements have been inactivated over time by the accumulation of deleterious mutations or internal recombination leading to solo-LTR formation, several members of the HERV-K family have been identified that remain nearly intact and probably represent recent integration events. To determine whether HERV-K elements have caused recent changes in the human genome, we have undertaken a study of the level of HERV-K polymorphism that exists in the human population. By using a high-resolution unblotting technique, we analyzed 13 human-specific HERV-K elements in 18 individuals. We found that solo LTRs have formed at five of these loci. These results enable the estimation of HERV solo-LTR formation in the human genome and indicate that these events occur much more frequently than described in inbred mice. Detailed sequence analysis of one provirus shows that solo-LTR formation occurred at least three separate times in recent history. An unoccupied preintegration site also was present at this locus in two individuals, indicating that although the age of this provirus is estimated to be approximately 1.2 million years, it has not yet become fixed in the human population.

Fig. 1.

Detection of HERV-K polymorphisms. (Upper) The position of the K10 oligonucleotide probe and putative Ase I sites relative to a typical HERV-K element. (Lower) Unblotting of a set of human genomic DNA samples digested with Ase I and probed with K10. Gray arrows indicate bands that are polymorphic within the sample. Bands predicted from the genomic database are plotted to the left of the blot, and the bands that appear to be polymorphic are indicated. Five of these elements have been identified: HERV-K108, 109, and 115 (9, 11); and HERV-K11q22 and 12q14 (29). The HERV-K element 3q24 is not full-length and was identified in the clone with GenBank accession no. AC069410 (Human Bacterial Artificial Chromosome Library, Roswell Park Cancer Institute, Buffalo, NY). The band marked 108b is the size expected for the central LTR of the tandem form of HERV-K 108 (20) (see Fig. 2).

Fig. 2.

Detection of HERV-K solo LTRs. Two primer sets were used for each polymorphic HERV-K locus identified in the unblotting analysis, labeled A and B. A similar PCR strategy was used to probe for polymorphisms at the nine other known human-specific HERV-K loci (data not shown), and only HERVK1p31 was found to be polymorphic. Primer set A was used to amplify the 5′ LTR of the full-length element, and primer set B was used to amplify the corresponding solo LTR or unoccupied preintegration site at each locus. Samples 1–10 are the same as in Fig. 1, and the remaining samples are labeled according to the population of origin of their donors. The primer set used in each reaction is indicated to the left of the unblots, and the identity of each product, confirmed by sequence analysis, is indicated the right of the unblots.

Fig. 3.

Multiple alleles of HERV-K11q22 and its solo LTRs. The 5′ and 3′ LTRs of the full-length element and the solo LTR contain six polymorphic sites in the 968 base pairs sequenced in the five heterozygous individuals studied. The base pair positions and the identities of these sites are indicated. The ancestral states were determined from character state reconstructions (304, 824, and 928) or assigned by homology to other, closely related HERV-K loci (32, 345, and 575), in cases of ambiguity. There are two alleles (A and B) of the full-length element and three alleles (1, 2, and 3) of the solo LTR. The polymorphic sites that differ from the inferred ancestral sequence are shown in boxes. Of the five heterozygous individuals, two had full-length element A and solo-LTR 2. The remaining individuals each had one of the following combinations: A and solo-LTR 1, B and solo-LTR 1, and B and solo-LTR 3.