Fig 1.
NSs acts as E3 ubiquitin ligase on RIG-I CARDs.
Toscana virus NSs inhibits the IFN-β promoter activation through the RIG-I signalling pathway mediating its degradation. Lenti-X 293T cells were transfected (A) with IFN-β promoter-driven FireFly Luciferase (p125-FFLuc) reporter plasmid, expression plasmid encoding FLAG-RIG-I and wild-type (wt-) NSs, as well as deleted NSs expression plasmids, as indicated. In addition, pSV40-RenLuc plasmid was added as internal control. Luciferase activity was analyzed at 48h post-transfection by the Dual-Luciferase Reporter assay as described by the manufacturer (Promega). Relative luciferase activities were measured as fold induction (relative to the basal level of reporter genes in the presence of empty vector after normalization with co-transfected RenLuc activities). Values represent means of triplicate independent experiments ± standard deviations (SD). Representative western blot showing the protein expression levels in the reporter gene assay samples was done on whole cell extracts, resolved by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) and analyzed by immunoblotting with the FLAG-RIG-I, 6xHis-NSs and β-Actin specific antibodies and densitometric analysis (Supplement data 2). (B) Recombinant proteins were used in the in vitro ubiquitination assay using UbcH5b/c as E2 and wt-rNSs, rNSsΔC or rNSsΔN as source of E3 ubiquitin ligase. The negative controls were represented by rRIG-I CARDs tested with the ubiquitination reagents except for E3 Ub ligase or TOSV nucleoprotein (rN) in place of E3 Ub ligase. The ubiquitinated rRIG-I CARDs were detected with anti-ubiquitin (left panel) or anti-RIG-I (right panel) antibodies. The ubiquitinated forms of rCARDS (indicated by asterisk) are present only in the samples containing wt-rNSs, as demonstrated by mass-spectrometry (S4 Fig). The band indicated by arrowhead (left panel) corresponding to the ubiquitinated-E2 (Ub-E2) intermediate present in all the tested samples, comigrates with the monoubiquitinated-rRIG-I CARDs in presence of rNSs. (C) NSs does not affect p53 expression in cells transfected with HA-p53 and 6xHis-NSs expressing plasmids (left panel). Immunofluorescence was performed with indicated specific antibodies; positive cells were counted. The bars depict the average number of p53 positive cells in the presence or absence of wt-NSs. The mean ± SD of three independent experiments is shown. The specificity of the E3 ubiquitin ligase activity of wt-NSs was evaluated in the in vitro ubiquitination assay using UbcH5b/c as E2 and recombinant p53 protein as acceptor target for ubiquitination (right panel). Poly-ubiquitinated forms were detected by anti-6xHis tag and anti-ubiquitin antibodies. Higher molecular weight bands corresponding to ubiquitinated p53 were detected only in the reaction supplemented with Mdm2 E3 ubiquitin ligase, but not in the samples containing wt-rNSs or rN.
Fig 2.
C-terminus of TOSV NSs is linked to E3 ubiquitin ligase activity.
The fusion of TOSV NSs C-terminus to Sandfly Fever Naples Virus (SFNV) NSs conferred it a different behaviour. (A, left panel) The chimeric protein cSFNV NSs was tested for its degrading activity on RIG-I co-transfected cells, by immunofluorescence, using specific antibodies [RIG-I (□), NSs (■)]. Graphs are based on the mean values of three independent experiments ± SD. (A, right panel) A more accurate analysis of cellular RIG-I degradation was performed in co-transfected cells by immunoblotting on the whole cell lysates using anti-FLAG or anti-6xHis antibodies. The intensity of the RIG-I band was quantified by densitometry (Supplement data 3). (B) Lenti-X 293T cells were transfected with IFN-β reporter plasmid, FLAG-RIG-I expression plasmid along with TOSV wt-NSs, SFNV wt-NSs or chimeric cSFNV NSs expressing plasmids. Luciferase activities were measured after poly(I:C) treatment. Fold induction was calculated for each sample with respect to the basal empty plasmid transfected sample, after normalization of the signal with the pSV40-RenLuc internal control. The mean values of at least three sets of experiments ± SD are presented. C) cSFNV NSs showed E3 ubiquitin ligase activity in the biochemical reaction, in association with UbcH5b/c as E2. Poly-ubiquitinated bands of rRIG-I CARDs were detected by immunoblotting using anti-RIG-I or anti-Ub antibodies when cSFNV or TOSV NSs was used in the reaction in place of E3 Ub ligase. No ubiquitination activity was shown when SFNV NSs was used.
Fig 3.
Effects of TOSV NSs N-terminal domain on E3 ubiquitin ligase activity.
(A) The involvement of NSs Cystein27 in the ubiquitination of RIG-I was evaluated by immunofluorescence on cells co-transfected with RIG-I and NSs plasmids. Cells were stained with anti-FLAG antibody and RIG-I positive cells were counted on different fields. Mean values ± SD of more than three independent experiments were plotted (left panel). Results were validated by immunoblotting (right panel) using total cell lysates of co-transfected cells and semi-quantitative analysis was done by densitometry (supplement data 4). (B) Lenti-X 293T cells were transfected with IFN-β reporter plasmid (p125-FFLuc) along with RIG-I and pSV40-RenLuc plasmids in addition with the wild type-NSs, or C27G-NSs mutant or empty plasmids. After stimulation with poly(I:C), luciferase activity was analyzed. For each sample, luciferase was normalized to the RenLuc reporter activity. Data are representative of three independent experiments and are expressed as mean ± SD of normalized luciferase activity.
Fig 4.
Model for NSs E3 Ub ligase function.
Schematic model for TOSV NSs which carries out an unconventional E3 Ub ligase activity (RING between RING; RBR) by a RING-HECT-hybrid mechanism. The model proposes the transfer of ubiquitin from the charged E2 Ub conjugating enzyme, bound to the C-terminal of NSs, to Cysteine 27 located at the N-terminal of the protein. Then, ubiquitin is transferred to the lysine residues of RIG-I target protein, interacting with the central region of TOSV NSs, and leading to its proteasome-dependent degradation.