Lineage-specific expansions of retroviral insertions within the genomes of African great apes but not humans and orangutans

Abstract

Retroviral infections of the germline have the potential to episodically alter gene function and genome structure during the course of evolution. Horizontal transmissions between species have been proposed, but little evidence exists for such events in the human/great ape lineage of evolution. Based on analysis of finished BAC chimpanzee genome sequence, we characterize a retroviral element (Pan troglodytes endogenous retrovirus 1 [PTERV1]) that has become integrated in the germline of African great ape and Old World monkey species but is absent from humans and Asian ape genomes. We unambiguously map 287 retroviral integration sites and determine that approximately 95.8% of the insertions occur at non-orthologous regions between closely related species. Phylogenetic analysis of the endogenous retrovirus reveals that the gorilla and chimpanzee elements share a monophyletic origin with a subset of the Old World monkey retroviral elements, but that the average sequence divergence exceeds neutral expectation for a strictly nuclear inherited DNA molecule. Within the chimpanzee, there is a significant integration bias against genes, with only 14 of these insertions mapping within intronic regions. Six out of ten of these genes, for which there are expression data, show significant differences in transcript expression between human and chimpanzee. Our data are consistent with a retroviral infection that bombarded the genomes of chimpanzees and gorillas independently and concurrently, 3-4 million years ago. We speculate on the potential impact of such recent events on the evolution of humans and great apes.

Figure 1. Identification and Sequence Analysis of PTERV1

(A) A graphical alignment of chimpanzee genomic sequence (AC097267) and an orthologous segment from human Chromosome 16 (Build 34) depicting an example of a PTERV1 (approximately 10 kb) insertion. Aligned sequences are shown in blue (miropeats) [47]. (B) The typical retroviral structure of the insert (gag, pol, env, and LTR) is compared to a baboon (Papio cynocephalus) endogenous retrovirus (PcEV). Regions of nucleotide homology are designated by black blocks and inter-sequence connecting lines. The location of probes (see Table S1) used in genomic library hybridizations, Southern blot analyses, and neighbor-joining tree analyses are shown (red).

Figure 2. Southern Hybridization of PTERV1 among Primates

Species represented include human (HSA), common chimpanzee (PTR), bonobo (PPA), gorilla (GGO), orangutan (PPY), siamang (HSY), white-handed gibbon (HLA), Abyssinian black-and-white colobus monkey (CGU), olive baboon (PHA), rhesus macaque (MMU), and Japanese macaque (MFU). Below each panel, a restriction map (chimpanzee sequence AC097267) is presented in relation to the hybridization probe: PstI (closed circles), PvuII (open circles), and HpaII/MspI (triangles) (see Figures S1 and S2 for additional details). (A) The absence of PTERV1 among Asian apes and humans is shown in contrast to a generally accepted catarrhine species phylogeny. Primate DNAs have been digested with PstI restriction enzyme, Southern-transferred to nylon membrane, and hybridized with PTERV1 gag probe number 1. (B) Multiple African great ape species are compared for both the gag probe number 1 and env probe number 3 (Figure 1). Proximity of probe number 1 to the VNTR, which is variable in length between copies (400 bp to 10 kb), reveals hundreds of insertion sites. (C) Multiple individuals from different subspecies of the olive baboon are compared for both gag probe number 1 and env probe number 3. The pattern of Southern hybridization shows limited intra-specific variation, indicative of either polymorphism in restriction enzyme sites or copy number variation.

Figure 3. PTERV1 Insertion Sites

Large-insert genomic clones that contained full-length endogenous retrovirus were identified by hybridization from four species: chimpanzee (PTR), gorilla (GGO), baboon (PAN), and rhesus macaque (MMU). End sequencing of large-insert clones (n = 1,467) and alignment against the human genome reference sequence identified 287 insertion sites (see Table S2). A total of 95.8% of these sites were non-orthologous when compared between species. chr, chromosome.

Figure 4. PTERV1 Phylogenetic Tree

Portions of the gag and env genes (about 823 bp) were resequenced from 101 PTERV1 elements from common chimpanzee (n = 42), gorilla (n = 25), rhesus macaque (n = 14), and olive baboon (n = 20). A neighbor-joining phylogenetic tree shows a monophyletic origin for the gorilla and chimpanzee endogenous retroviruses but a polyphyletic origin among the Old World monkey species. Bootstrap support (n = 10,000 replicates) for individual branches are underlined. Although the retroviral insertions have occurred after speciation, retroviral sequences show greater divergence than expected for a non-coding nuclear DNA element (see Table S4). Table S8 provides a clone key for number designation. Phylogenetic trees showing the gag, env, and LTR segments separately are presented in Figure S6. Sequences 11 and 30 (red) are mapped to one of the 12 ambiguous overlapping loci described in the text (see Table S3). They do not cluster in this phylogenetic tree, which indicates that they are unlikely to be true orthologs.

Figure 5. LTR Variation

A total of 101 loci that contained full-length PTERV1 elements were examined for the number of mismatches between left and right LTR flanks (295 bp). Different distributions were obtained for Old World monkeys (baboon, mean = 1.6 ± 1.4; macaque, mean = 1.6 ± 1.5) and great ape species (chimpanzee, mean = 3.4 ± 2.2; gorilla, mean = 2.9 ± 2.3).