Two surface-exposed elements of the B30.2/SPRY domain as potency determinants of N-tropic murine leukemia virus restriction by human TRIM5alpha

Abstract

Human TRIM5alpha (TRIM5alpha(hu)) potently restricts N-tropic (N-MLV), but not B-tropic, murine leukemia virus in a manner dependent upon residue 110 of the viral capsid. Rhesus monkey TRIM5alpha (TRIM5alpha(rh)) inhibits N-MLV only weakly. The study of human-monkey TRIM5alpha chimerae revealed that both the v1 and v3 variable regions of the B30.2/SPRY domain contain potency determinants for N-MLV restriction. These variable regions are predicted to be surface-exposed elements on one face of the B30.2 domain. Acidic residues in v3 complement basic residue 110 of the N-MLV capsid. The results support recognition of the retroviral capsid by the TRIM5alpha B30.2 domain.

FIG. 1.

Contribution of the TRIM5α_hu B30.2 domain to potent restriction of N-MLV. (A) The TRIM5α chimerae generated to examine the role of the B30.2 domain in N-MLV restriction are depicted. The residue numbers of TRIM5α_hu are shown, and TRIM5α_hu segments are colored black. (B) Steady-state levels of expression of TRIM5α variants in MDTF cells are shown. Lysates from MDTF cells transduced with either an empty LPCX vector or vectors expressing HA-tagged TRIM5α variants were subjected to Western blotting using an anti-HA antibody. The lysates were also Western blotted for β-actin to control for total protein. (C and D) MDTF cells transduced with LPCX vectors expressing TRIM5α variants were incubated with various amounts of N-MLV-green fluorescent protein (GFP) (C) or B-MLV-GFP (D). GFP-positive MDTF cells were counted by fluorescence-activated cell sorter. The results from a typical experiment are shown. Similar results were obtained in at least two independent experiments.

FIG. 2.

Potency determinants for N-MLV restriction in two B30.2 variable regions. (A) Differences between the primary sequences of TRIM5α_hu and TRIM5α_rh in the B30.2 domain v1 to v3 variable regions are marked with asterisks. The locations of the changes in the v1, v2, and v3 regions of the B30.2 domain of the TRIM5α_rh mutants are shown beneath the alignment. Residues 409 and 410 in the v3 region of the TRIM5α_rh B30.2 domain are underlined. These residues, which are both acidic in TRIM5α_hu, are studied in greater depth in the experiments shown in Fig. 3. (B) Steady-state levels of expression of the TRIM5α variants in MDTF cells were analyzed by Western blotting cell lysates, as in Fig. 1. (C and D) MDTF cells transduced with LPCX vectors expressing TRIM5α variants were incubated with various amounts of N-MLV-green fluorescent protein (GFP) (C) or B-MLV-GFP (D). GFP-positive cells were counted by fluorescence-activated cell sorter. Panels C and D show data from a typical experiment. Similar results were obtained in at least two independent experiments.

FIG. 3.

Contribution of charge to the potency determinant in the TRIM5α B30.2 v3 region. (A) Steady-state levels of expression of TRIM5α variants in MDTF cells were measured by Western blotting cell lysates with an anti-HA antibody, as described in the legend to Fig. 1. TRIM5α_rh mutants are designated Rh, with the introduced amino acid change in parentheses. The number refers to the residue number in TRIM5α_rh. (B to E) MDTF cells transduced with either an empty LPCX vector or the LPCX vectors expressing TRIM5α variants were incubated with N-MLV-green fluorescent protein (GFP) (B), B-MLV-GFP (C), NBNN-MLV-GFP (D), or BNBB-MLV-GFP (E). GFP-positive MDTF cells were counted by fluorescence-activated cell sorter. Panels B to E show the results of typical experiments. Similar results were obtained in at least two independent experiments.

FIG. 4.

Models to explain the potency of N-MLV restriction by TRIM5α variants. (A) The structure of the B30.2/SPRY domain of the human PRY-SPRY-19q13.4.1 protein (5) is shown, with the surface loops equivalent to the v1 to v4 variable regions of TRIM proteins colored (14). The strands corresponding to the v1 (blue), v2 (red), v3 (yellow), and v4 (purple) regions are labeled. The predicted locations of residues 409 and 410 on TRIM5α_rh are indicated. The asterisk marks a potential ligand-binding cleft (5). The N and C termini of the B30.2/SPRY domain are labeled. (B) Electrostatic interactions between the B30.2 domain v3 region of TRIM5α and the surface of the viral capsid may modulate the potency of MLV restriction. The trimeric TRIM5α variants, with either negative (red) or positive (blue) charges in the N terminus of the B30.2 domain v3 region, are depicted. The ribbon structure of the MLV capsid hexamers is shown in a lateral view (8). From this perspective, the surface of the assembled capsid faces upward, and the interior of the capsid faces downward. The surface-exposed side chain of residue 110 is shown (arginine [blue] in N-MLV and BNBB, glutamic acid [red] in B-MLV). The presence of a single negative charge in the TRIM5α v3 N terminus allows modest restriction of N-MLV and BNBB (thin arrow). N-MLV and BNBB restriction is potentiated (thick arrow) by the addition of a second negative charge in this v3 region [as in TRIM5α_hu or Rh(Q409E)]. Removal of these acidic v3 residues [in Rh(E410A)] or addition of a basic residue [in Rh(E410R), Rh(Q409R), and Rh(E410K)] completely abrogates TRIM5α restriction of N-MLV and BNBB. The negatively charged B-MLV capsid is resistant to TRIM5α-mediated restriction.