TRIMCyp expression in Old World primates Macaca nemestrina and Macaca fascicularis

Abstract

Primates have evolved a variety of restriction factors that prevent retroviral replication. One such factor, TRIM5alpha, mediates a postentry restriction in many Old World primates. Among New World primates, Aotus trivirgatus exerts a similar early restriction mediated by TRIMCyp, a TRIM5-cyclophilin A (CypA) chimera resulting from a CypA retrotransposition between exons 7 and 8 of the TRIM5 gene. Macaca nemestrina do not express TRIM5alpha; therefore, we asked whether these animals and related Old World primates express TRIMCyp. RT-PCR of total RNA from M. nemestrina and Macaca fascicularis yielded three TRIMCyp amplification products, one of which is predicted to encode a TRIMCyp chimera containing a full-length CypA. Unlike A. trivirgatus, genomic sequencing of M. nemestrina and M. fascicularis identifies a CypA retrotransposition in the 3' untranslated region of the TRIM5 locus. There is approximately 78% homology between the predicted protein sequences of Old World and New World primate TRIMCyp, with most of the differences found in the TRIM5-derived sequence. Notably, exon 7 is absent from both M. nemestrina and M. fascicularis TRIMCyp. Neither M. nemestrina nor M. fascicularis TRIMCyp could restrict HIV-1 or simian immunodeficiency virus SIVmac in an in vitro infectivity assay. The discovery of TRIMCyp in both M. nemestrina and M. fascicularis indicates that TRIMCyp expression may be more common among Old World primates than previously believed. Convergent evolution of TRIMCyp in both Old World and New World primates suggests that TRIMCyp may have provided evolutionary advantages.

Fig. 1.

RT-PCR amplification of TRIM5α and TRIMCyp from Old World primate total RNA. Total RNA was isolated from Owl monkey kidney (OMK) cells, M. mulatta PBMC, M. fascicularis PBMC, and M. nemestrina PBMC. Primers were designed to amplify either TRIM5α (odd lanes) or TRIMCyp (even lanes). Lane L, kb+ ladder; lane 1, dH₂O TRIM5α; lane 2, dH₂O TRIMCyp; lane 3, OMK TRIM5α; lane 4, OMK TRIMCyp; lane 5, M. mulatta TRIM5α; lane 6, M. mulatta TRIMCyp; lane 7, M. fascicularis TRIM5α; lane 8, M. fascicularis TRIMCyp; lane 9, M. nemestrina TRIM5; and lane 10, M. nemestrina TRIMCyp.

Fig. 2.

Alignment of A. trivirgatus, M. nemestrina, and M. fascicularis TRIMCyp predicted amino acid sequences. (A) Schematic diagram (not to scale) of the genomic organization of the TRIM5 and CypA loci on chromosome 14 (top). The shaded region indicates the exon 8 3′ UTR. Exons encoded by each TRIMCyp splice variant are indicated below. R, RING; B, B-Box; CC, coiled coil. (B) Alignment of TRIMCyp exon 6 predicted amino acid sequences from M. nemestrina and M. fascicularis and the published A. trivirgatus TRIMCyp sequence. The N-terminal methionine is omitted in this alignment because the start codon was replaced with a 5′ HA epitope tag during cDNA amplification. The RING, B-Box, coiled coil, and CypA domains are indicated by labeled bars over the sequence. Amino acid differences between M. nemestrina and M. fascicularis are indicated by bold letters.

Fig. 3.

Genomic characterization of Old World primate TRIMCyp. (A) PCR amplification of the TRIM5 gene from M. mulatta, M. nemestrina, and M. fascicularis PBMC genomic DNA using a forward primer specific for exon 6 and a reverse primer specific for exon 8. Lane 1, M. mulatta; lane 2, M. fascicularis; lane 3, M. nemestrina; and lane 4, kb+ DNA ladder. (B) Sequence of the M. fascicularis TRIM5 gene from intron 6 to exon 8. The underlined sequence indicates the location corresponding to the site of CypA retrotransposition in A. trivirgatus. (C) PCR amplification of TRIM5 exon 8 and CypA. M. nemestrina and M. fascicularis PBMC genomic DNA was PCR-amplified with a forward primer specific for TRIM5 exon 5 and a reverse primer specific for CypA. Lane 1, kb+ DNA ladder; lane 2, dH₂O; lane 3, M. fascicularis; and lane 4, M. nemestrina. (D) Sequence of the M. fascicularis TRIM5 3′ UTR and retrotransposed CypA. Lowercase letters indicate the TRIM5 3′ UTR. Italicized and underlined letters identify the conserved retrotransposition sequence. Capital letters identify the retrotransposed CypA sequence in TRIMCyp. Bold letters highlight the start and stop codons of the CypA coding sequence.

Fig. 4.

Expression of TRIMCyp by stably transformed CrFK cells. (A) Immunoblotting analysis of HA-tagged TRIMCyp proteins in CrFK whole-cell lysates. Lane 1, Protein Plus ladder; lane 2, M. nemestrina TRIMCyp; lane 3, M. mulatta TRIM5α; and lane 4, M. fascicularis TRIMCyp. (B) Analysis of stably transduced CrFK cells by flow cytometry (Left) and immunofluorescence assays (Right). The stably transduced TRIMCyp protein is indicated below each image. A monoclonal anti-HA Alexa Fluor conjugate 488 antibody was used in both assays to label the HA-tagged TRIMCyp in each cell line. The percentages of cells expressing M. nemestrina and M. fascicularis TRIMCyp are 85% and 94%, respectively.

Fig. 5.

Infectivity of HIV-1 and SIVmac in CrFK cells stably transduced with TRIMCyp. Serial dilutions of VSV-G pseudotyped HIV-1 pNL4–3-Δenv-eGFP (A) or env-minus SIVmac239-eGFP (B) were used to infect stably transduced CrFK cells. Forty-eight hours after infection, the percentage of GFP-positive cells was determined by flow cytometric analysis.