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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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Single-cycle infection assays.Cells were infected at a low M.O.I., to reduce possible effects of saturation. Virus stocks were first titered by serial dilution and infection of CRFK cells (Figure S1), and equivalent infectious units of HIV-1 and SIV were used for parallel infections of macaque PBMC. All experiments were performed in triplicate. A. Infection of activated PBMC from 23 rhesus macaques, including 19 G/G, 1 T/T and 3 G/T individuals, with VSV-pseudotyped HIV-1. B. Same as in A, but using VSV-pseudotyped SIVmac239. C. Single cycle HIV and SIV infection of BLCL derived from a rhesus macaque homozygous for the TRIM5-CypA allele, in the absence and presence of cyclosporine A. D. Single cycle infectivity on immortalized BLCL lines from seven pig-tailed macaques. The BLCL line from each individual animal was tested in triplicate with each of the two viruses. Bars indicate mean infectivity +/−SEM for all seven cell lines.
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ppat-1000003-g002: Single-cycle infection assays.Cells were infected at a low M.O.I., to reduce possible effects of saturation. Virus stocks were first titered by serial dilution and infection of CRFK cells (Figure S1), and equivalent infectious units of HIV-1 and SIV were used for parallel infections of macaque PBMC. All experiments were performed in triplicate. A. Infection of activated PBMC from 23 rhesus macaques, including 19 G/G, 1 T/T and 3 G/T individuals, with VSV-pseudotyped HIV-1. B. Same as in A, but using VSV-pseudotyped SIVmac239. C. Single cycle HIV and SIV infection of BLCL derived from a rhesus macaque homozygous for the TRIM5-CypA allele, in the absence and presence of cyclosporine A. D. Single cycle infectivity on immortalized BLCL lines from seven pig-tailed macaques. The BLCL line from each individual animal was tested in triplicate with each of the two viruses. Bars indicate mean infectivity +/−SEM for all seven cell lines.

Mentions: To begin to test the effects of TRIM5 polymorphisms on viral infectivity, fresh blood samples were obtained from 22 rhesus macaques that were in the process of undergoing routine veterinary examination at the New England Primate Research Center. In addition, whole blood was obtained from animal 173-02, the previously identified T/T homozygote described above. PHA-activated, IL-2 stimulated lymphocytes were prepared from these samples and used as target cells for single-cycle infectivity measurements, using VSV-pseudotyped, HIV-1 and SIV particles carrying a transducible EGFP reporter construct (Figure 2). Uninfected PBMC aliquots from each animal were used to prepare genomic DNA. The genotypes of the donor animals were then determined, and found to include 19 wild-type homozygotes (G/G) and three heterozygotes (G/T), in addition to the previously typed animal 173-02 (T/T). Mean infectivity (% GFP-positive cells) was significantly different between PBMC of wild type homozygotes (G/G) and PBMC from animals carrying at least one copy of the T allele (G/T and T/T) (0.04%+/−0.007% vs 0.12%+/−0.05; p = 0.0068; unpaired, two-tailed t test). The difference remained significant even if the single T/T individual was excluded (0.04%+/−0.007 vs 0.13%+/−0.07; p = 0.0080). Infectivity of VSV-pseudotyped SIV was measured in parallel, and no significant difference in infectivity was found for the different genotypes, consistent with evidence that TRIM5α has little or no restricting activity against SIV (1.36% for G/G vs 1.66% for G/T+T/T; p = 0.3490). However, even in cells from individuals bearing a T allele (G/T and T/T), the mean infectivity of HIV-1 was still substantially lower than that of SIV. This may indicate that sufficient TRIM5α is expressed in these cells, or alternatively, that other post-entry blocks to HIV-1 infection are present.


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)

Single-cycle infection assays.Cells were infected at a low M.O.I., to reduce possible effects of saturation. Virus stocks were first titered by serial dilution and infection of CRFK cells (Figure S1), and equivalent infectious units of HIV-1 and SIV were used for parallel infections of macaque PBMC. All experiments were performed in triplicate. A. Infection of activated PBMC from 23 rhesus macaques, including 19 G/G, 1 T/T and 3 G/T individuals, with VSV-pseudotyped HIV-1. B. Same as in A, but using VSV-pseudotyped SIVmac239. C. Single cycle HIV and SIV infection of BLCL derived from a rhesus macaque homozygous for the TRIM5-CypA allele, in the absence and presence of cyclosporine A. D. Single cycle infectivity on immortalized BLCL lines from seven pig-tailed macaques. The BLCL line from each individual animal was tested in triplicate with each of the two viruses. Bars indicate mean infectivity +/−SEM for all seven cell lines.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000003-g002: Single-cycle infection assays.Cells were infected at a low M.O.I., to reduce possible effects of saturation. Virus stocks were first titered by serial dilution and infection of CRFK cells (Figure S1), and equivalent infectious units of HIV-1 and SIV were used for parallel infections of macaque PBMC. All experiments were performed in triplicate. A. Infection of activated PBMC from 23 rhesus macaques, including 19 G/G, 1 T/T and 3 G/T individuals, with VSV-pseudotyped HIV-1. B. Same as in A, but using VSV-pseudotyped SIVmac239. C. Single cycle HIV and SIV infection of BLCL derived from a rhesus macaque homozygous for the TRIM5-CypA allele, in the absence and presence of cyclosporine A. D. Single cycle infectivity on immortalized BLCL lines from seven pig-tailed macaques. The BLCL line from each individual animal was tested in triplicate with each of the two viruses. Bars indicate mean infectivity +/−SEM for all seven cell lines.
Mentions: To begin to test the effects of TRIM5 polymorphisms on viral infectivity, fresh blood samples were obtained from 22 rhesus macaques that were in the process of undergoing routine veterinary examination at the New England Primate Research Center. In addition, whole blood was obtained from animal 173-02, the previously identified T/T homozygote described above. PHA-activated, IL-2 stimulated lymphocytes were prepared from these samples and used as target cells for single-cycle infectivity measurements, using VSV-pseudotyped, HIV-1 and SIV particles carrying a transducible EGFP reporter construct (Figure 2). Uninfected PBMC aliquots from each animal were used to prepare genomic DNA. The genotypes of the donor animals were then determined, and found to include 19 wild-type homozygotes (G/G) and three heterozygotes (G/T), in addition to the previously typed animal 173-02 (T/T). Mean infectivity (% GFP-positive cells) was significantly different between PBMC of wild type homozygotes (G/G) and PBMC from animals carrying at least one copy of the T allele (G/T and T/T) (0.04%+/−0.007% vs 0.12%+/−0.05; p = 0.0068; unpaired, two-tailed t test). The difference remained significant even if the single T/T individual was excluded (0.04%+/−0.007 vs 0.13%+/−0.07; p = 0.0080). Infectivity of VSV-pseudotyped SIV was measured in parallel, and no significant difference in infectivity was found for the different genotypes, consistent with evidence that TRIM5α has little or no restricting activity against SIV (1.36% for G/G vs 1.66% for G/T+T/T; p = 0.3490). However, even in cells from individuals bearing a T allele (G/T and T/T), the mean infectivity of HIV-1 was still substantially lower than that of SIV. This may indicate that sufficient TRIM5α is expressed in these cells, or alternatively, that other post-entry blocks to HIV-1 infection are present.

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