Human endogenous retroviral elements as indicators of ectopic recombination events in the primate genome

Abstract

HERV elements make up a significant fraction of the human genome and, as interspersed repetitive elements, have the capacity to provide substrates for ectopic recombination and gene conversion events. To understand the extent to which these events occur and gain further insight into the complex evolutionary history of these elements in our genome, we undertook a phylogenetic study of the long terminal repeat sequences of 15 HERV-K(HML-2) elements in various primate species. This family of human endogenous retroviruses first entered the primate genome between 35 and 45 million years ago. Throughout primate evolution, these elements have undergone bursts of amplification. From this analysis, which is the largest-scale study of HERV sequence dynamics during primate evolution to date, we were able to detect intraelement gene conversion and recombination at five HERV-K loci. We also found evidence for replacement of an ancient element by another HERV-K provirus, apparently reflecting an occurrence of retroviral integration by homologous recombination. The high frequency of these events casts doubt on the accuracy of integration time estimates based only on divergence between retroelement LTRs.

Figure 1.

Endogenous retrovirus evolution. (A) An integrated provirus. LTR sequences are represented by boxes flanking the internal viral sequence (straight line). Cellular DNA is depicted as wavy lines. Mutations are depicted as asterisks or dots. Two types of mutations can be distinguished: those that differentiate the 5′ and 3′ LTRs in all descendants (asterisks), which occur after integration but prior to speciation, and those that differentiate species, which occur subsequent to speciation (dots). (B) Tree that results from phylogenetic analysis of the 5′ and 3′ LTR sequences in four different species.

Figure 2.

PCR strategy used to amplify LTRs of specific HERV-K elements in primates. The positions of the primer pairs used to amplify the 5′ and 3′ LTR sequences are shown relative to that of the HERV-K element. One primer from each pair is located in the unique genomic sequence flanking the HERV so that only orthologous loci are amplified. An example of the results of this PCR strategy is shown, following agarose gel electrophoresis and ethidium bromide staining.

Figure 3.

Phylogenetic analysis of HERV-K elements that conform to predicted topology. Maximum-parsimony trees are shown for each element. Branch lengths are proportional to the number of changes occurring along a lineage. Bootstrap values taken from 100 replicates are shown. Nodes without indicated bootstrap values had very high support, ≥95%. Human 5′ and 3′ LTR sequences of closely related HERV-K elements were used as outgroups in each analysis and are indicated by element name. HERV-K3p25 is not full length in the chimpanzee and bonobo, but a solitary LTR, which is formed by homologous recombination between the 5′ and 3′ LTRs, was found at this locus and included in the analysis.

Figure 4.

Phylogenetic analysis of HERV-K elements that deviate from predicted topology. Analysis and labeling are the same as those in Figure 3.

Figure 5.

LTR-LTR gene conversion and sequence divergence in two HERV-K proviruses. (A) HERV-K20q11 sequences and (B) HERV-K9q34.3 sequences for the human, chimpanzee, bonobo, gorilla, and orangutan are shown. Only positions that differ between the 5′ and 3′ LTRs in at least one species are represented and the consensus base pair identities of these positions are indicated. 5′ LTR sequences are shown as open areas and 3′ LTR sequences are shown as shaded areas. Gene conversion events are evident by the transfer of a 5′ LTR sequence to the 3′ LTR or vice versa.

Figure 6.

Integration by homologous recombination generates chimeric LTRs in HERV-K6p21. (A) The LTRs of the newly integrating HERV-K element are depicted with shaded boxes and those of the ancient HERV-K element that it recombined into are depicted with cross-hatched boxes. The resultant recombinant element is shown. The chimeric LTRs generated by the recombination event are aligned for comparison. (B) The chimeric 5′ and 3′ LTR sequences in the human, chimpanzee, bonobo, and gorilla were aligned and the position of the base pair differences is plotted along the length of the LTR. The same analysis was done for HERV-K20q11 as a comparison and is shown below the 6p21 plot.

Figure 7.

6p21 replaced an ancient HERV-K provirus in the common ancestor of the hominids. (A) Kimura two-parameter corrected pairwise distances between species were plotted against divergence times for all available HERV-K20q11 and HERV-K6p21 sequences. The dotted line indicates the expected outcome for HERV-K6p21 if the sequences in the hominids and the rest of the species represented the same element, which had accumulated mutations at a constant rate over time. The discontinuity in the graph probably reflects the replacement of the original HERV-K element by a different, and somewhat divergent, HERV-K element. (B) The relative ages of HERV-K20q11 and HERV-K6p21 are demonstrated by comparing their sequences to a consensus HERV-K sequence. Dot matrix analysis is shown with the full-length (human-specific) HERV-K10 sequence along the y-axis and the sequence of the element under examination along the x-axis. The window size in the analysis was 30 and the minimum percentage score was 60.