Retroviruses are RNA viruses that, when infecting a host cell, produce a viral reverse transcriptase and a viral integrase that make a DNA copy of the viral genome and integrate it into the host genome, respectively . The family Retroviridae comprises a diverse range of animal viruses, including the viral genus Lentivirus. Lentiviruses have been isolated from primates, domestic and wild felids, and a variety of domestic ungulates (goat, sheep, cattle and horse) . Until recently, all known lentiviruses were classified as exogenous (transmitted horizontally from host to host) . However, in 2007, Katzourakis and colleagues  reported the first endogenous lentivirus identified in any species, the rabbit endogenous lentivirus type K (RELIK), present in the genome of the European rabbit (Oryctolagus cuniculus). RELIK has subsequently been reported in other leporid genera (Lepus, Sylvilagus and Bunolagus), establishing it as at least 12 million years (My) old [5, 6]. These striking observations demonstrate that lentiviruses are more widespread than previously thought, extending the host range to a different mammalian order, and demonstrate that lentiviruses can be endogenized [4–6].
The intense study of lentiviruses in the past 30 years, especially of human immunodeficiency viruses (HIV-1 and HIV-2), has been more recently accompanied by the study of antiretroviral restriction factors, like the TRIM5α protein, one of the members of TRIM family [7–11]. TRIM proteins contain three domains, which together constitute the canonical TRIpartite Motif, including an N-terminal RING domain, one or two B-Box domains and a long Coiled-Coil (CC) domain [9–11]. TRIM5, like most TRIM proteins, also contains a C-terminal PRYSPRY domain, composed of four "variable loops" [9–11]. TRIM5α is the largest isoform encoded by the TRIM5 gene and restricts infection by HIV-1 and other retroviruses, dependent on a species-specific sequence variation in the PRYSPRY domain, upon entry into the host cell cytoplasm and prior to reverse transcription [7, 8]. Each TRIM5α domain plays distinct roles in its antiviral restriction activity. The RING domain has been shown to confer E3 ubiquitin ligase activity crucial for anti-HIV restriction [12, 13]. The B-box 2 domain influences recognition of the viral capsid by the C-terminal PRYSPRY domain [13–15]. The CC domain plays an important role in the restriction of viral infectivity and it is required for trimerization [11, 16]. Particularly for HIV-1 and N-tropic murine leukemia virus (N-MLV) retroviruses, restriction specificity has been mapped to the PRYSPRY domain for HIV-1 and N-MLV restriction specificity is determined by both the CC and PRYSPRY domains [9, 17, 18]. Human TRIM5α is not effective against HIV-1 but does inhibit N-MLV, while rhesus monkey TRIM5α restricts both [19–22]. However, a single amino acid change (R332P) in the human TRIM5α PRYSPRY domain causes it to behave like rhesus TRIM5α with regard to HIV-1 restriction [17, 23]. The PRYSPRY domain binds to the viral capsid, and the domain sequence variation determines the restriction specificity [17, 18, 24–26]. Recently, evidence from several studies began elucidating the detailed mechanism of TRIM5α activity. Also, additional activities linked to viral restriction have been described, including a role in signal transduction, the promotion of innate immune signaling and recognition of the retroviral capsid lattice [27, 28]. It has been suggested that direct binding of TRIM5α to the viral capsid leads to disruption of specific inter-hexamer interfaces, causing structural damage to the capsid. TRIM5α spontaneously forms a hexagonal lattice complementary to the capsid lattice, a molecular signature of retroviruses, which greatly stimulates TRIM5α lattice formation [29, 30].
Evolutionary studies of primate TRIM5α revealed a high ratio of non-synonymous to synonymous changes in the PRYSPRY domain [25, 31–33]. The distribution of positively selected residues is not random, but falls in a very tight cluster at the beginning of the domain in a 13 amino acid "patch", essential for retroviral restriction and responsible, in part, for the species-specific restriction activity . The same domain has also undergone length variation and segmental duplications in different primate lineages . However, polymorphisms found in the TRIM5α coding sequence for multiple individuals from two divergent lineages of Old World monkeys (rhesus macaque and sooty mangabey), indicated that specificity varies not only between different species but also within species . Despite the geographic separation and the divergence time (> 8 My), both species presented a highly similar pattern of polymorphisms, which constitutes compelling evidence for long-term balancing selection at the TRIM5 locus . Similar evidence of selection has been recently reported for the first intron of human TRIM5 gene, which may affect transcription factor-binding sites and TRIM5 transcriptional activity .
Although evolutionary and functional studies of TRIM5α antiretroviral restriction activity have primarily focused on the primate lineage, TRIM5 orthologs have been reported in other mammalian genomes, e.g. mouse, rat, cow and European rabbit [36–39]. Active TRIM5 was identified in the European rabbit and the ability to restrict the replication of multiple unrelated retroviruses was also described . Besides this leporid, Fletcher and co-workers  reported the restriction of divergent retroviruses by European brown hare (Lepus europaeus) TRIM5α and also significant differences between both leporids' TRIM5α PRYSPRY domains. These authors suggested that retroviruses like RELIK may have driven the speciation of the Old World rabbit and hare TRIM5α orthologs. The order Lagomorpha is divided into two families, Ochotonidae and Leporidae, which diverged around 40 My ago [41–43]. Ochotonidae contains only one extant genus, Ochotona, while the family Leporidae includes 11 genera where, Lepus, Sylvilagus and Oryctolagus, the most well-studied leporid genera, diverged around 12 My ago [41, 42]. It has been suggested that the global development of temperate grasslands (7 to 5 My ago) and the formation of the west Antarctic ice sheet (6.5 My ago) promoted the development of land bridges and consequent dispersal of the genus Lepus from North America through Asia and into Africa . The New World Sylvilagus lineage initially remained in North America from which it more recently colonized South America . The genus Oryctolagus is the only leporid genus native to Europe and consists of two subspecies, O. cuniculus cuniculus and O. cuniculus algirus, which diverged around 2 My ago. The subspecies O. c. algirus is restricted to the southwest region of the Iberian Peninsula and a few Atlantic islands, whereas O. c. cuniculus has essentially a man-made worldwide distribution and includes all domestic breeds .
As previously suggested, considering that RELIK must already have been present in a common ancestor of these leporid genera, the hypothesis that lentiviruses might have been the driving force of the leporid TRIM5α conserved antiretroviral activity can be challenged by extending the study of evolutionary patterns of TRIM5α to other members of the Leporidae family, particularly to the New World Sylvilagus lineage. Therefore, in this study we have examined the TRIM5α gene of additional subspecies and species of the three leporid genera: European rabbit subspecies (Oryctolagus cuniculus algirus), the Old World Iberian hare (Lepus granatensis) and European brown hare, and the New World brush rabbit (Sylvilagus bachmani).