Prolonged activity of HERV-K(HML2) in Old World Monkeys accounts for recent integrations and novel recombinant variants

Abstract

Around 8% of the human genome comprises Human Endogenous Retroviruses (HERVs) acquired over primate evolution. Some are specific to primates such as HERV-K, consisting of 10 HML subtypes and including the most recently acquired elements. Particularly, HML2 is the youngest clade, having some human-specific integrations, and while it has been widely described in humans its presence and distribution in non-human primates remain poorly characterized. To investigate HML2 distribution in non-human primates, the present study focused on the characterization of HML2 integrations in Macaca fascicularis and Macaca mulatta which are the most evolutionarily distant species related to humans in the Catarrhini parvorder. We identified overall 208 HML2 proviruses for M. fascicularis (77) and M. mulatta (131). Among them, 46 proviruses are shared by the two species while the others are species specific. Only 12 proviruses were shared with humans, confirming that the major wave of HML2 diffusion in humans occurred after macaques' divergence. Phylogenetic analysis confirmed structural variations between HML2 macaques' species-specific proviruses, and the ones shared between macaques and humans. The HML2 loci were characterized in terms of structure, focusing on potential residual open reading frames (ORFs) for gag, pol, and env genes for the latter being reported to be expressed in human pathological conditions. The analysis identified highly conserved gag and pol genes, while the env genes had a very divergent nature. Of the 208 HML2 proviral sequences present in Macaca species, 81 sequences form a cluster having a MER11A, a characteristic HML8 LTR sequence, insertion in the env region indicating a recombination event that occurred between the HML2 env gene and the HML8 LTR. This recombination event, which was shown to be present only in a subset of macaques' shared sequences and species-specific sequences, highlights a recent viral activity leading to the emergence of an env variant specific to the Old World Monkeys (OWMs). We performed an exhaustive analysis of HML2 in two species of OWMs, in terms of its evolutionary history, structural features, and potential residual coding capacity highlighting recent activity of HML2 in macaques that occurred after its split from the Catarrhini parvorder, leading to the emergence of viral variants, hence providing a better understanding of the endogenization and diffusion of HML2 along primate evolution.

Keywords: HERV-K; HERV-K11; HML2; Macaca; Old World Monkeys; endogenous retroviruses; recombination; viral evolution.

Figure 1

Phylogeny of HML2 integrations in Humans, Macaca fascicularis and Macaca mulatta. Phylogenetic trees generated for full-length or near full-length sequences for all the three species using Neighbor-joining method with a bootstrap of 500 replicates in MEGA 11 software. The human sequences are indicated in yellow, M. fascicularis in cyan, and M. mulatta in magenta. Supported phylogenetic clusters are highlighted with a light green shade.

Figure 2

Phylogeny of HML2 loci in Old World Monkeys Macaca fascicularis and Macaca mulatta. Both trees represent the HML2 sequences that are specific to individual species (green), some common among the species (blue), and some shared with humans (orange). Lastly, the reference sequences are highlighted in red. Only the full-length sequences were included to generate the phylogeny by applying Neighbor-joining method with 500 bootstrap values in MEGA 11 software.

Figure 3

Box plot representation of HML2 diffusion in macaques. The age for all the three categories of macaque HML2 sequences (human shared, macaque shared, and species specific) were calculated by considering divergence between the (i) 5′ and 3′ LTR of same HML2 loci; (ii) LTR vs. Consensus generated; (iii) gag and pol genes against the consensus of each gene. The values in the box plot are the mean values. The theoretical age ranges for the three categories based on evolutionary divergence times are indicated with colored boxes for Catarrhini/Platyrrhini (around 43 mya, orange), OWMs/hominoids (around 30 mya, green), and macfas/rhesus (around 5 mya, blue).

Figure 4

Schematic representation of HML2 proviruses in Human and Macaque genomes. Representative Consensus of HML2 proviruses (A) Shared between human and macaques. (B) Macaque-shared HML2 sequences. (C) Species-Specific Sequences.

Figure 5

Phylogeny of HML2 gag and pol genes of macaque species. The trees represent the conservative nature of gag and pol genes of HML2 in both the Macaca species with the major upper cluster of bootstraps 100% (gag) and 98% (pol). The major cluster is highlighted in pink while the ones highlighted in green are the outliers.

Figure 6

Phylogeny of the env gene of HML2 in Macaca fascicularis and Macaca mulatta. The clusters indicate the presence of 3 subtypes of env in Old World Monkeys. The type1 (pink) is similar to the human HML2 type2 (rec) env, while type 2 (cyan) has specific insertions in the transmembrane region, and type 3 (purple) which forms the largest cluster mostly species-specific and some shared between the two macaques having HERVK11 insertion.

Figure 7

Structural representation of gag and env genes HML2 with respect to the consensus generated from potential residual ORFs of Human and Macaque species (Macaca fascicularis and Macaca mulatta). (A) The variants 1, 2, and 3 are specified based on the variation in the env region. The solid black lines refer to the deletion in the regions while the dotted lines are the stretch based on the insertions in that specific region. (B) Alignment of potential coding capacity of the gag genes of both the macaque species (described in detail in Table 3). Characteristics indels in macaque gag proteins as compared to human ones are highlighted with yellow blocks. (C) Alignment of the consensus generated for each cluster in both M. fascicularis (Macfas) and M. mulatta (Rhesus), the alignment of the three variants in both species are according to that indicates in (A).

Figure 8

Test of recombination between macaque HML2 env variant3 and HML8 MER11A LTR The portions deriving from HML2 env gene (cyan) and MER11A LTR (green) are annotated along rhesus consensus for env variant3.