Independent genesis of chimeric TRIM5-cyclophilin proteins in two primate species

Abstract

The host range of retroviruses is influenced by antiviral proteins such as TRIM5, a restriction factor that recognizes and inactivates incoming retroviral capsids. Remarkably, in Owl monkeys (omk), a cyclophilin A (CypA) cDNA has been transposed into the TRIM5 locus, resulting in the expression of a TRIM5-CypA fusion protein (TRIMCyp) that restricts retroviral infection based on the retroviral capsid-binding specificity of CypA. Here, we report that the seemingly improbable genesis of TRIMCyp has, in fact, occurred twice, and pigtailed macaques (pgt) express an independently generated TRIMCyp protein. The omkTRIMCyp and pgtTRIMCyp proteins restrict infection by several lentiviruses, but their specificities are distinguishable. Surprisingly, pgtTRIMCyp cannot bind to or restrict HIV-1 capsids as a consequence of a point mutation close to the Cyp:capsid-binding interface that was acquired during or after transposition of pgtCypA. However, the same mutation confers on pgtTRIMCyp the ability to restrict FIV in the presence of cyclosporin A, a drug that normally abolishes the interaction between pgtTRIMCyp or omkTRIMCyp and lentiviral capsids. Overall, an intuitively unlikely evolutionary event has, in fact, occurred at least twice in primates and represents a striking example of convergent evolution in divergent species.

Fig. 1.

TRIM5-Cyp fusion protein (pgtTRIMCyp) expressed by pigtailed macaques. (A–D) Infection of pgtF cells by varying amounts of GFP-expressing VSV-G-pseudotyped vectors or reporter viruses based on HIV-1(A), SIV_AGMTan (B), HIV-2_ROD (C), or FIV (D), in the presence or absence of 5 μM CsA. (E) PCR products generated using pgtF cDNA template along with primers derived from the 5′ and 3′ ends of the rhTRIM5α coding sequence (lane 1) or from the 5′ end of rhTRIM5α and 3′ end of CypA (lane 2). Arrows indicate bands from which pgtTRIM5η (lane 1) and pgtTRIMCyp (lane 2) cDNAs were cloned. (F) Configuration of the TRIM5 and CypA domains and the linker peptide derived from the CypA mRNA 5′ noncoding sequence in pgtTRIMCyp, as compared with omkTRIMCyp. (G) Protein sequence differences in the Cyp domains of pgtTRIMCyp and omkTRIMCyp, as compared with human and pgt CypA proteins, amino acid postions are numbered relative to the methionine at the N terminus of each Cyp domain or CypA protein.

Fig. 2.

Pigtailed macaque TRIMCyp restricts infection by some lentiviruses, but not HIV-1. (A–D) Infection of CHO cells expressing C-terminally HA-tagged omkTRIMCyp or pgtTRIMCyp or by HIV-1(A), SIV_AGMTan (B), HIV-2_ROD (C) or FIV (D), in the presence or absence of 5 μM CsA. (E) Western blot analysis (αHA) of omkTRIMCyp or pgtTRIMCyp expression in the CHO cells used throughout the manuscript. (F and G) Infection of unmanipulated CHO cells, or CHO cells expressing pgtTRIMCyp or omkTRIMCyp by SIV_AGMTan (F) or FIV (G) in the presence of varying (0–20 μM) concentrations of CsA. (H) Same as F and G, except that pgtF cells were used as targets.

Fig. 3.

Differential restriction specificity of pgtTRIMCyp and omkTRIMCyp is governed by the Cyp domain. (A) Nomenclature and schematic representation of chimeric TRIMCyp proteins used herein. (B) Western blot analysis (αHA) of intact and chimeric TRIMCyp protein expression in CHO cells. (C–F) Infection of CHO cells expressing intact and chimeric TRIMCyp proteins by HIV-1 (C), SIV_AGMTan (D), HIV-2 (E) or FIV (F), in the presence (white bars) or absence (black bars) of 5 μM CsA.

Fig. 4.

Yeast two-hybrid analysis of interactions between HIV-1 Gag and TRIMCyp or CypA proteins. (A) β-Galactosidase activity [expressed in optical density units at 590 nm (OD₅₉₀)] in lysates of Y190 yeast cells expressing GAL4 DNA-binding domains either alone or fused to HIV-1 Gag, along with HA-tagged VP16 activation domains fused to omkTRIMCyp or pgtTRIMCyp. (B) Same as A, except that the VP16 activation domain was fused to the indicated CypA proteins or the Cyp domains from omkTRIMCyp [omkTC(Cyp)] or pgtTRIMCyp [pgtTC(Cyp)]. (C) Western blot analysis (αHA) of VP16-TRIMCyp and VP16-CypA protein expression in yeast lysates.

Fig. 5.

A single amino acid mutation in pgtTRIMCyp proteins alters recognition of lentiviral capsids. (A) Nomenclature and schematic representation of the mutant TRIMCyp proteins used herein, position numbering is relative to the methionine at the N terminus of the Cyp domain of each protein. (B) Infection of CHO cell clones expressing wild-type or mutant TRIMCyp proteins by HIV-1, in the presence (white bars) or absence (black bars) of 5 μM CsA. (C) β-Galactosidase activity (expressed in optical density units at 590 nm [OD₅₉₀)] in lysates of Y190 yeast cells expressing GAL4 DNA-binding domains either alone or fused to HIV-1 Gag, along with HA-tagged VP16 activation domains fused to wild-type or mutant TRIMCyp proteins. (D) Western analysis (αHA) of wild-type and mutant TRIMCyp protein expression in CHO cells. (E) Western analysis (αHA) of wild-type and mutant VP16-TRIMCyp fusion protein expression in yeast cells. (F–H) Infection of CHO cells expressing C-terminally HA-tagged wild-type or mutant TRIMCyp proteins by SIV_AGMTan (F), HIV-2_ROD (G), or FIV (H), in the presence (white bars) or absence (black bars) of 5 μM CsA. (I) Structure of an HIV-1 capsid monomer in complex with huCypA (identical in sequence to pgtCypA), indicating the amino acid resides (white) that differ in pgtTRIMCyp and influence capsid recognition. Also indicated is an HIV -1 capsid residue (P85, yellow) that is closely opposed to the R69 residue in CypA.