Truncation of TRIM5 in the Feliformia explains the absence of retroviral restriction in cells of the domestic cat

Abstract

TRIM5alpha mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5alpha. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

FIG. 1.

Conserved synteny and phylogenetic clustering indicate that feTRIM5 is a true TRIM5 orthologue. (A) Paralogues TRIM6, TRIM34, TRIM5, and TRIM22 are found in a cluster in the human and dog genomes and in the 1.9-fold cat genome project. In all of these cases, the cluster is surrounded by olfactory genes. Exons 2 (ex2) and 8 (ex8) of the pseudogenized dog TRIM5 sequence are shown. Other genes in cat as well as dog TRIM6 are predicted genes or regions of homology only and may not be expressed or functional. ch, chromosome. (B) Neighbor-joining tree of TRIM5 orthologues from several mammalian species. feTRIM5 clusters with other TRIM5 orthologues in preference to closely related paralogues TRIM34, TRIM6, and TRIM22 and the more distantly related paralogue TRIM21. Moreover, the TRIM5 phylogeny reflects established mammalian evolutionary relationships. Numbers indicate bootstrap values after 1,000 iterations, and branch length reflects the number of base substitutions per site. Nucleic acid sequences were codon optimized and aligned by using ClustalW with manual adjustment. All of the positions containing gaps were eliminated from the data set.

FIG. 2.

TRIM5 expression is maintained in the felids, and the truncation is conserved across the Feliformia. (A) TRIM5 transcripts are present in cells from diverse members of the Feliformia. In addition to the Felis catus cell lines CrFK and Mya-1, exon 2 to exon 8 TRIM5 transcripts were amplified from cDNAs derived from cheetah (Acinonyx jubatus), lion (Panthera leo), and European wildcat (F. sylvestris) T cells. CON, control. (B) The 5′ end of TRIM5 exon 8 was sequenced from the genomic DNAs of several species to discern at which point in carnivoran evolution the truncation occurred. The stop codon TGA was found in all of the feliforms examined but absent from both the dog and mink sequences. When the findings are imposed on an established phylogenetic tree of the carnivorans (12, 15), the truncation of TRIM5 is shown to have taken place before the split of the Felidae and Hyaenidae lineages. An independent TRIM5 disruption is proposed to have taken place in the Caniformia lineage after the Feliformia-Caniformia split 53.8 million years ago and is present in modern dogs.

FIG. 3.

Biological function of feTRIM5. (A) Mouse fibroblasts which lack restriction activity were transduced with domestic feTRIM5 or huTRIM5α. While huTRIM5α specifically restricts N-tropic but not B-tropic MLV, feTRIM5, which lacks a B30.2 domain, restricts neither MLV-N nor MLV-B. Nor is feTRIM5 able to restrict lentiviral vectors derived from HIV-1 and simian immunodeficiency virus from rhesus macaques. MDTF, M. dunni tail fibroblasts. (B) feTRIM5 acts as a dominant negative agent against huTRIM5α-mediated restriction. The TE671 cell line, which expresses endogenous TRIM5α, was stably transduced with feTRIM5 or with huTRIM5 P306STOP, which bears a stop codon at the residue corresponding to that in feTRIM5. Expression of feTRIM5 or huTRIM5 P306STOP rescued MLV-N infectivity. Error bars represent mean ± standard error (n = 3).