Figure 5. Binding of the Flavivirus Protease by rhTRIM5α Is Conformation-Dependent and Results in Proteasome-Dependent Degradation of NS2B/3.
(A) Stable HEK293 rhTRIM5α-HA (green) cells were transfected with plasmids coding for either NS2B/3-V5 or NS5-V5 (red) from LGTV and imaged by confocal microscopy. Scale bar, 10 μm.
(B) Relative intensity of TRIM5 aggregates were measured along vectors drawn in 3 fields of cells expressing LGTV NS2B/3 or NS5, with example vectors shown in Figure S3B (mean ± SD, ****p < 0.0001, 2-way ANOVA with Dunnett’s post-test).
(C) Western blot of LGTV NS2B/3-V5 or NS5-V5 in stable rhTRIM5α-HA or control HEK293 cells.
(D–F) Western blot analysis of HEK293 cells transfected with (D) increasing amounts of rhTRIM5α-HA and constant amounts of LGTV NS2B/3-V5, (E) increasing amounts of hTRIM5α-HA and constant amounts of LGTV NS2B/3-V5, and (F) increasing amounts of rhTRIM5α-HA and constant amounts of LGTV NS5-V5.
(G) Reciprocal coIP of rhTRIM5α-HA and LGTV NS2B/3-V5 following cotransfection and 4 h treatment with epoxomicin (200 nM). The asterisk indicates a non-specific band.
(H) Western blot of LGTV NS2B/3-V5, rhTRIM5α-HA, and endogenous p62 in HEK293 cells following 4 h treatment with DMSO (vehicle), Baf-A1 (200 nM), or epoxomicin (200 nM).
(I) Quantification of LGTV NS2B/3 expression with or without rhTRIM5α and treated with Baf-A1 or epoxomicin from 11 individual experiments (mean ± SD, **p < 0.01, 2-way ANOVA with Sidak’s post-test).
(J) LGTV NS2B/3-V5 and rhTRIM5α-FLAG were co-expressed with ubiquitin (Ub)-HA wild type (WT) or K48R or K63R mutants in HEK293 cells. Target proteins were immunoprecipitated using anti-V5 or anti-FLAG antibodies and blots probed with anti-HA to examine Ub conjugation.
(K) Domain structure of flavivirus NS2B/3 (PDB: 2vbc) and schematic representation of truncation mutants.
(L) Western blot analysis of HEK293 cells transfected with increasing amounts of rhTRIM5α-HA and constant amounts of LGTV NS3pro. Lysates were probed specifically for HA, V5 and b-actin.