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

Virgen et al. 10.1073/pnas.0709258105.

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SI Figure 6
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SI Figure 6

Fig. 6. (A) Alignment of the pgtTRIMCyp and omkTRIMCyp amino acid sequences. Residues comprising the linker peptide that separates the N-terminal TRIM5 sequences from the C-terminal CypA sequences are highlighted. The pgtTRIMCyp sequence is the majority consensus derived from nine clones. Deviations (point mutations) from this sequence occurred in several clones. With one exception, mutations never occurred in more than one out of nine clones and were thus assumed to represent PCR induced errors. The exception was a polymorphism at amino acid position 71 (serine in four of nine clones and asparagine in the remaining five clones) This polymorphism had no apparent effect on pgtTRIMCyp restriction activity (data not shown). (B) Alignment of the pgtTRIM5h and rhTRIM5a amino acid sequences. The pgtTRIM5h sequence is representative of three independent clones, two of which were identical to each other (and to the presented sequence).

SI Figure 7

Fig. 7. Characterization of the TRIM5 genomic locus in Macaca nemestrina. (A) Schematic representation of the 3' portion of the Macaca nemestrina TRIM5 locus, depicted approximately to scale. A series of PCR assays using primers directed to TRIM5 coding exons, 6, 7, or 8, coupled with an primers directed to pgtCypA was initially used to deduce the position of a CypA-like insert. Thereafter, PCR primers based on rhesus macaque genomic TRIM5 DNA sequence (indicated by the horizontal arrows) were used to independently verify the presence of an insertion, specifically in pigtailed macaque genomic DNA. The sense PCR primer was directed to the coding portion of exon 8, (GTC CCC ATG ACT CTG TGC TCA CCA AGC) and antisense primer was directed to the 3' portion of the TRIM5 3' UTR (ACC AGG GTA ATT AGC ATA TCC ATC ACC TC). Exon skipping/splicing events that generate the pgtTRIM5h and pgtTRIMCyp mRNAs are shown using dashed lines. Also indicated, for comparison, (light type and arrow) is the site of insertion of a CypA cDNA in the analogous owl monkey (omk) TRIM5 locus. (B) Verification of an insertion in the pigtailed macaque TRIM5 locus as compared to rhesus macaque TRIM5. PCR reactions were done using primers (sequence given and position indicated by arrows in panel A) directed to TRIM5 sequences. Analysis of genomic DNA from two pigtail macaque fibroblast cell lines indicate the presence of an insertion of ~0.7 kb in both copies of the TRIM5 gene relative to genomic DNA from the rhesus macaque cell line, 221. (C) Sequence analysis of the 3' portion of the Macaca nemestrina TRIM5 genomic locus. PCR products generated from pigtailed macaque genomic DNA using the primers shown in panel A were sequenced (pgtGENOMIC) and compared to the rhesus genomic database sequence (rhGENOMIC). A 726-nucleotide CypA cDNA that includes CypA coding sequences as well as 5' and 3' UTRs is present in the pigtailed TRIM5 locus. A putative polyadenylation signal and modification in the insert are indicated. Note that inserted CypA coding and 5' noncoding sequence are precisely identical to Cyp and linker sequences, respectively, found in the pgtTRIMCyp cDNA [pgtTC (Cyp)], and are different to other CypA pseudogenes that occur in the rhesus macaque genome distal to the 3' end of the TRIM5 gene (data not shown). The CypA cDNA insert is flanked by 12-nucleotide repeats, which represent putative target site duplications (TSD) that likely arose as a consequence of LINE-mediated retrotransposition.

SI Figure 8

Fig. 8. The TRIM5a-like protein, pgtTRIM5h, does not restrict HIV-1, SIV_AGMTan or N-MLV infection. (A) Infection of single CHO cell clones expressing C-terminally HA-tagged pgtTRIM5h(#A, #B, and #C) or rhTRIM5a, as indicated, by HIV-1 or SIV_AGMTan. The blot below the left chart contains lysates of #A, #B, and #C cells probed with an aHA antibody. (B) Infection of #A, #B, and #C cells, or a CHO cell clone expressing huTRIM5aby N-MLV or B-MLV as indicated

SI Text

Molecular Construction. Total RNA was extracted from pgtF cells and pgtPBMC and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) to generate cDNA. This cDNA was used as a template in PCR reactions with primers derived from the 5' and 3' ends of the rhTRIM5a coding sequence, generating the pgtTRIM5h cDNA, or with primers directed to the 5' end of the rhTRIM5a coding sequence and the 3' end of the huCypA sequence, generating the collection of pgtTRIMCyp cDNAs. The 5' and 3' primers were appended with sequences corresponding to EcoRI and NotI restriction sites that were used for to insert the cDNAs into an LNCX2 (CLONTECH) derived retroviral vector, such that the C terminus of each expressed protein would be appended with a HA epitope tag. Similar plasmids expressing the corresponding tagged omkTRIMCyp protein, as well as human and rhesus macaque TRIM5a have been previously described. (1, 2) Mutant forms of these proteins were generated by recombinant PCR-based techniques.

For yeast two-hybrid assays, a previously described pGBKT7 (CLONTECH) derived plasmid, expressing a GAL4 DNA-binding domain fused to HIV-1 Gag was used (3). Additionally, the wild-type and mutant omkTRIMCyp and pgtTRIMCyp cDNAs were inserted into pVP16/HA using EcoRI and NotI restriction sites (4), thereby generating plasmids that expressed an HA-tagged VP16 activation domain fused to the N terminus of the TRIMCyp proteins in yeast. Similarly, CypA cDNAs were amplified from human and pgtF cDNAs, and the isolated Cyp domains were amplified from the cloned omkTRIMCyp and pgtTRIMCyp cDNAs, and each was inserted into pVP16/HA.

1. Perez-Caballero D, Hatziioannou T, Zhang F, Cowan S, Bieniasz PD (2005) J Virol 79:15567-15572.

2. Perez-Caballero D, Hatziioannou T, Yang A, Cowan S, Bieniasz PD (2005) J Virol 79:8969-8978.

3. Martin-Serrano J, Zang T, Bieniasz PD (2001) Nat Med 7:1313-1319.

4. Bogerd HP, Fridell RA, Blair WS, Cullen BR (1993) J Virol 67:5030-5034.

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