All three variable regions of the TRIM5alpha B30.2 domain can contribute to the specificity of retrovirus restriction

Abstract

Recent studies have revealed the contribution of TRIM5alpha to retrovirus restriction in cells from a variety of primate species. TRIM5alpha consists of a tripartite motif (the RBCC domain) followed by a B30.2 domain. The B30.2 domain is thought to be involved in determination of restriction specificity and contains three variable regions. To investigate the relationship between the phylogeny of primate TRIM5alpha and retrovirus restriction specificity, a series of chimeric TRIM5alpha consisting of the human RBCC domain followed by the B30.2 domain from various primates was constructed. These constructs showed restriction profiles largely consistent with the origin of the B30.2 domain. Restriction specificity was further investigated with a variety of TRIM5alphas containing mixed or mutated B30.2 domains. This study revealed the importance of all three variable regions for determining restriction specificity. Based on the molecular structures of other PRYSPRY domains solved recently, a model for the molecular structure of the B30.2 domain of TRIM5alpha was developed. The model revealed that the variable regions of the B30.2 domain are present as loops located on one side of the B30.2 core structure. It is hypothesized that these three loops form a binding surface for virus and that evolutionary changes in any one of the loops can alter restriction specificity.

FIG. 1.

Amino acid sequences of primate TRIM5α exon8. Amino acid sequences were predicted from their nucleotide sequences and aligned using Clustal W. The amino acid positions are shown above the human TRIM5α sequence. All TRIM5α constructs studied herein have the RBCC domain of the human TRIM5α, so that the start position of all exon8 sequences is 299. The primate species of origin are shown to the left of the sequences, which are grouped as apes, OWM, and NWM. A dot indicates that the amino acid is identical to that of the human exon8 sequence, and a dash indicates an amino acid deletion. Amino acids substituted or deleted in this study were indicated by boxes, and their positions were shown above each amino acid. The boundaries of three variable regions were indicated on the basis of the previous study of Song and collaborators (49). The double line between positions 396 and 397 indicates the site at which the N- and C-terminal halves of the B30.2 domains were exchanged.

FIG. 2.

Phylogenetic tree of TRIM5α exon8 from various primates and their retrovirus restriction profiles. The tree was constructed on the basis of the amino acid sequence alignment shown in Fig. 1 with the maximum-likelihood method in PHYLIP software. Values to the right of the tree indicate the restriction activities against HIV-1, SIVmac, and N-tropic (N-MLV) and B-tropic (B-MLV) MLV; the restriction activity is the ratio of the percentage of TRIM5α-positive cells to that of TRIM5α-negative cells that were infected. A ratio larger than 0.7 indicates the absence of restriction (white box), whereas a ratio less than 0.3 indicates the presence of restriction (black box). A ratio between 0.3 and 0.7 indicates a restriction activity of an intermediate level (gray box). Each value is shown as an average of the ratios obtained from three independent experiments followed by the standard error of the mean. Agm, African green monkey.

FIG. 3.

Importance of the V1 region of the B30.2 domain of ape and NWM TRIM5αs for retrovirus restriction. Structures of the chimeras (a and c) and the altered amino acids on the B30.2 domain (b and d) made in this study are shown on the left, while their restriction properties are shown on the right. (a) White and black boxes indicate chimpanzee and gorilla sequences, respectively. (b and d) The amino acid positions corresponding to the human and NWM TRIM5α proteins are indicated above the constructs. (c) White boxes indicate tamarin-derived sequences, while black boxes indicate marmoset-derived sequences. Restriction assays were scored as described in the legend to Fig. 2. Hsa, Homo sapiens; Ggo, Gorilla gorilla; Soe, Saguinus oedipus; Cgo, Callimico goeldii.

FIG. 4.

Importance of the V2 region of the B30.2 domain of orangutan TRIM5α for HIV-1 and SIVmac restriction. Structures of the B30.2 domain mutants are shown on the left, while the restriction properties of the variants on HIV-1, SIVmac, and N-MLV are shown on the right. The amino acid positions corresponding to the orangutan TRIM5α protein are indicated above the constructs. Restriction assays were scored as described in the legend to Fig. 2. Ppy, Pongo pygemateus.

FIG. 5.

Importance of the V3 region of NWM B30.2 domain for retrovirus restriction. Schematic representations of the chimeras between chimpanzee and Goeldi's marmoset (a) and of their variants with an insertion and point mutations (b) are presented on the left, while their ability to restrict HIV-1, SIVmac, and N-MLV is shown on the right. (a) Chimpanzee sequences are represented by black boxes, whereas those of marmoset are represented by white boxes. (b) A dot indicates that the amino acid is identical to that in the chimpanzee exon8 sequence, and a dash indicates an amino acid deletion. The amino acid positions corresponding to the chimpanzee TRIM5α protein are indicated above the constructs. Restriction assays were scored as described in the legend to Fig. 2. Ptr, Pan troglodytes; Cgo, Callimico goeldii.

FIG. 6.

Protein expression levels of the MC chimera and the Cgo-V3 mutant. Expression levels in extracts of control cells and cells transduced with vectors encoding huTRIM5, chimera MC, and mutant Cgo-V3 were examined by Western blotting using sera specific for TRIM5α (top) and α-tubulin (bottom). The arrow indicates the TRIM5-specific band. Cgo, Callmico goeldii.

FIG. 7.

Model for the structure of the B30.2 domain of primate TRIM5α based on the molecular structure of other SPRY and PRYSPRY domains reported previously (61). (a) Ribbon model showing β-sheets. (b) Filled model showing a potential binding surface. Red, green, and blue colors indicate the V1, V2, and V3 regions, respectively. Amino acids that were found to be important for retrovirus restriction are highlighted.