Balancing selection and the evolution of functional polymorphism in Old World monkey TRIM5alpha

Abstract

Retroviral restriction factor TRIM5alpha exhibits a high degree of sequence variation among primate species. It has been proposed that this diversity is the cumulative result of ancient, lineage-specific episodes of positive selection. Here, we describe the contribution of within-species variation to the evolution of TRIM5alpha. Sampling within two geographically distinct Old World monkey species revealed extensive polymorphism, including individual polymorphisms that predate speciation (shared polymorphism). In some instances, alleles were more closely related to orthologues of other species than to one another. Both silent and nonsynonymous changes clustered in two domains. Functional assays revealed consequences of polymorphism, including differential restriction of a small panel of retroviruses by very similar alleles. Together, these features indicate that the primate TRIM5alpha locus has evolved under balancing selection. Except for the MHC there are few, if any, examples of long-term balancing selection in primates. Our results suggest a complex evolutionary scenario, in which fixation of lineage-specific adaptations is superimposed on a subset of critical polymorphisms that predate speciation events and have been maintained by balancing selection for millions of years.

Fig. 1.

Common polymorphisms in OWM TRIM5α coding sequences cluster in the CC and SPRY domains. A diagram depicting the 497-aa TRIM5α protein and its functional domains is shown. Locations of frequent, nonsynonymous polymorphisms are shown above and below the TRIM5α schematic. Sites of common, synonymous polymorphisms are indicated by asterisks.

Fig. 2.

Phylogenetic analysis reveals unusual features of TRIM5α evolution in Old World primates. A neighbor-joining tree is depicted; a similar topology was obtained by using maximum parsimony. Tree was rooted by designating the human/chimpanzee/gorilla cluster as an outgroup. Alleles described in this study include Ceat-1 through Ceat-4 and Mamu-1 through Mamu-6. Additional sequences included Macaca assamensis (accession nos. AY899873.1–AY899879.1), Macaca fascicularis (AB210052), Macaca nemestrina (AY899887.1–AY899893.1), Homo sapiens (NM_033034), Gorilla gorilla (DQ298178), and Pan troglodytes (NM_001012650). The sequences at sites representing putative transspecies polymorphisms are shown.

Fig. 3.

Rhesus macaque TRIM5α alleles differ in their ability to restrict retroviral infection. Retroviral restriction was assayed by using a transient two-color (A–D) and infection of stable cell lines (E–F). For the two-color assay, permissive cells expressing human and various rhesus TRIM5α alleles (Mamu-1 to Mamu-6) and coexpressing ECFP were challenged with single-cycle HIV-1 (A), MLV-N (B), SIVmac (C), or MPMV (D) viruses carrying an EGFP reporter. The ratio of infected, TRIM5α containing cells to infected, TRIM5α-null cells was determined by FACS analysis. As a negative control for restriction, cells expressing ECFP alone were also challenged. Stable cell lines expressing rhesus alleles Mamu-1 to Mamu-6 as well as human TRIM5α (Hs), were challenged with either HIV-1 (E) or MLV-N (F) expressing an EGFP reporter. The number of infected (GFP-positive) cells was determined by FACS analysis. Error bars represent three independent infections. Representative data from one of three independent experiments is shown. The level of TRIM5α protein in transduced or stable cells was determined by Western blotting using antibody against the HA tag (Inset in B) or TRIM5α (Inset in F). Levels of β-actin are also shown to demonstrate equal loading of cell lysate.

Fig. 4.

Restriction properties of sooty mangabey TRIM5α alleles. Cells expressing sooty mangabey TRIM5α alleles Ceat-1 to Ceat-4 were tested for their ability to inhibit infection by HIV-1 (A), MLV-N (B), SIVmac (C), or MPMV (D). Two-color restriction assays were carried out as described in Fig. 3. Representative data from one of three independent experiments is shown. Levels of HA-tagged TRIM5α protein in transduced cells is shown (Inset in B).