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Evolution of a TRIM5-CypA splice isoform in old world monkeys.

Newman RM, Hall L, Kirmaier A, Pozzi LA, Pery E, Farzan M, O'Neil SP, Johnson W - PLoS Pathog. (2008)

Bottom Line: The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform.HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A.Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts, United States of America.

ABSTRACT
The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5-10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.

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The macaque TRIM5 locus.A. Schematic depiction of the primate TRIM5 locus including the seven coding exons (grey shaded regions) and introns, and the nucleotide sequence in the region of the 3′ss G/T SNP at the terminus of intron 6. Sequencing analysis confirmed that an NsiI restriction site was linked to the G/T change, and PCR amplification followed by NsiI digestion was used as an allelic discrimination assay to survey multiple individuals from two species of macaque. B. PCR/NsiI allelic discrimination in rhesus macaques (M. mulatta). C. Pedigree depicting genotype of rhesus macaque 173-02 (homozygous T/T) along with its dam (220-97; heterozygous G/T) and sire (76-99; heterozygous G/T). D. PCR+NsiI screening of sixteen Pig-tailed macaques (M. nemestrina). E. A second PCR screen of genomic DNA samples for the presence of a CypA insertion downstream of TRIM5. A gel revealing the presence of the insertion in 173-02 (T/T), 220-97(G/T), and 76-99 (G/T) (lanes 2–4) is shown. The insertion was not found in two wild type (G/G) individuals (lanes 5 and 6), but was present in two pig-tailed macaques (both T/T) (lanes 7 and 8).
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ppat-1000003-g001: The macaque TRIM5 locus.A. Schematic depiction of the primate TRIM5 locus including the seven coding exons (grey shaded regions) and introns, and the nucleotide sequence in the region of the 3′ss G/T SNP at the terminus of intron 6. Sequencing analysis confirmed that an NsiI restriction site was linked to the G/T change, and PCR amplification followed by NsiI digestion was used as an allelic discrimination assay to survey multiple individuals from two species of macaque. B. PCR/NsiI allelic discrimination in rhesus macaques (M. mulatta). C. Pedigree depicting genotype of rhesus macaque 173-02 (homozygous T/T) along with its dam (220-97; heterozygous G/T) and sire (76-99; heterozygous G/T). D. PCR+NsiI screening of sixteen Pig-tailed macaques (M. nemestrina). E. A second PCR screen of genomic DNA samples for the presence of a CypA insertion downstream of TRIM5. A gel revealing the presence of the insertion in 173-02 (T/T), 220-97(G/T), and 76-99 (G/T) (lanes 2–4) is shown. The insertion was not found in two wild type (G/G) individuals (lanes 5 and 6), but was present in two pig-tailed macaques (both T/T) (lanes 7 and 8).

Mentions: We have previously reported that the TRIM5 coding sequence of old world monkeys is highly polymorphic [7]. In the course of genotyping the TRIM5 locus in a colony of captive bred rhesus macaques, we identified a single-nucleotide polymorphism in the terminal nucleotide of intron 6 (Figure 1). The SNP is the result of a G-to-T substitution that alters the canonical 3′ splice acceptor site (AG to AU) immediately upstream of exon 7. Initial sequence data revealed the presence of this mutation in 2 of 8 animals, including one homozygote (T/T) and one heterozygote (G/T). The cis-acting AG element at the end of introns is a highly conserved feature of 3′ splice sites, and the presence of such a mutation is predicted to interfere with mRNA splicing.


Evolution of a TRIM5-CypA splice isoform in old world monkeys.

Newman RM, Hall L, Kirmaier A, Pozzi LA, Pery E, Farzan M, O'Neil SP, Johnson W - PLoS Pathog. (2008)

The macaque TRIM5 locus.A. Schematic depiction of the primate TRIM5 locus including the seven coding exons (grey shaded regions) and introns, and the nucleotide sequence in the region of the 3′ss G/T SNP at the terminus of intron 6. Sequencing analysis confirmed that an NsiI restriction site was linked to the G/T change, and PCR amplification followed by NsiI digestion was used as an allelic discrimination assay to survey multiple individuals from two species of macaque. B. PCR/NsiI allelic discrimination in rhesus macaques (M. mulatta). C. Pedigree depicting genotype of rhesus macaque 173-02 (homozygous T/T) along with its dam (220-97; heterozygous G/T) and sire (76-99; heterozygous G/T). D. PCR+NsiI screening of sixteen Pig-tailed macaques (M. nemestrina). E. A second PCR screen of genomic DNA samples for the presence of a CypA insertion downstream of TRIM5. A gel revealing the presence of the insertion in 173-02 (T/T), 220-97(G/T), and 76-99 (G/T) (lanes 2–4) is shown. The insertion was not found in two wild type (G/G) individuals (lanes 5 and 6), but was present in two pig-tailed macaques (both T/T) (lanes 7 and 8).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279257&req=5

ppat-1000003-g001: The macaque TRIM5 locus.A. Schematic depiction of the primate TRIM5 locus including the seven coding exons (grey shaded regions) and introns, and the nucleotide sequence in the region of the 3′ss G/T SNP at the terminus of intron 6. Sequencing analysis confirmed that an NsiI restriction site was linked to the G/T change, and PCR amplification followed by NsiI digestion was used as an allelic discrimination assay to survey multiple individuals from two species of macaque. B. PCR/NsiI allelic discrimination in rhesus macaques (M. mulatta). C. Pedigree depicting genotype of rhesus macaque 173-02 (homozygous T/T) along with its dam (220-97; heterozygous G/T) and sire (76-99; heterozygous G/T). D. PCR+NsiI screening of sixteen Pig-tailed macaques (M. nemestrina). E. A second PCR screen of genomic DNA samples for the presence of a CypA insertion downstream of TRIM5. A gel revealing the presence of the insertion in 173-02 (T/T), 220-97(G/T), and 76-99 (G/T) (lanes 2–4) is shown. The insertion was not found in two wild type (G/G) individuals (lanes 5 and 6), but was present in two pig-tailed macaques (both T/T) (lanes 7 and 8).
Mentions: We have previously reported that the TRIM5 coding sequence of old world monkeys is highly polymorphic [7]. In the course of genotyping the TRIM5 locus in a colony of captive bred rhesus macaques, we identified a single-nucleotide polymorphism in the terminal nucleotide of intron 6 (Figure 1). The SNP is the result of a G-to-T substitution that alters the canonical 3′ splice acceptor site (AG to AU) immediately upstream of exon 7. Initial sequence data revealed the presence of this mutation in 2 of 8 animals, including one homozygote (T/T) and one heterozygote (G/T). The cis-acting AG element at the end of introns is a highly conserved feature of 3′ splice sites, and the presence of such a mutation is predicted to interfere with mRNA splicing.

Bottom Line: The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform.HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A.Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts, United States of America.

ABSTRACT
The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5-10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.

Show MeSH
Related in: MedlinePlus