Novel Class of Viral Ankyrin Proteins Targeting the Host E3 Ubiquitin Ligase Cullin-2

Abstract

Ankyrin repeat (ANK) domains are among the most abundant motifs in eukaryotic proteins. ANK proteins are rare amongst viruses, with the exception of poxviruses, which presumably acquired them from the host via horizontal gene transfer. The architecture of poxvirus ANK proteins is, however, different from that of their cellular counterparts, and this precludes a direct acquisition event. Here we combine bioinformatics analysis and quantitative proteomics to discover a new class of viral ANK proteins with a domain organization that relates to cellular ANK proteins. These noncanonical viral ANK proteins, termed ANK/BC, interact with host Cullin-2 via a C-terminal BC box resembling that of cellular Cullin-2 substrate adaptors such as the von Hippel-Lindau protein. Mutagenesis of the BC box-like sequence abrogates binding to Cullin-2, whereas fusion of this motif to an ANK-only protein confers Cullin-2 association. We demonstrated that these viral ANK/BC proteins are potent immunomodulatory proteins suppressing the activation of the proinflammatory transcription factors NF-κB and interferon (IFN)-responsive factor 3 (IRF-3) and the production of cytokines and chemokines, including interferon, and that association with Cullin-2 is required for optimal inhibitory activity. ANK/BC proteins exist in several orthopoxviruses and cluster into 2 closely related orthologue groups in a phylogenetic lineage that is separate from that of canonical ANK/F-box proteins. Given the existence of cellular proteins with similar architecture, viral ANK/BC proteins may be closely related to the original ANK gene acquired by an ancestral orthopoxvirus. These findings uncover a novel viral strategy to antagonize innate immunity and shed light on the origin of the poxviral ANK protein family.IMPORTANCE Viruses encode multiple proteins aimed at modulating cellular homeostasis and antagonizing the host antiviral response. Most of these genes were originally acquired from the host and subsequently adapted to benefit the virus. ANK proteins are common in eukaryotes but are unusual amongst viruses, with the exception of poxviruses, where they represent one of the largest protein families. We report here the existence of a new class of viral ANK proteins, termed ANK/BC, that provide new insights into the origin of poxvirus ANK proteins. ANK/BC proteins target the host E3 ubiquitin ligase Cullin-2 via a C-terminal BC box domain and are potent suppressors of the production of inflammatory cytokines, including interferon. The existence of cellular ANK proteins whose architecture is similar suggests the acquisition of a host ANK/BC gene by an ancestral orthopoxvirus and its subsequent duplication and adaptation to widen the repertoire of immune evasion strategies.

Keywords: Ankyrin proteins; Cullin ubiquitin system; immune evasion; poxvirus.

FIG 1

ECTV protein 010 is a noncanonical ANK protein that binds Cul-2. (A) Phylogenetic tree of OPV ANK proteins. Orthologue groups are indicated in roman numerals and highlighted in several colors. Protein designations without background color are those that could not be assigned to an orthologue group unambiguously. Branch length indicates relationship. (B) Alignment of the C-terminal terminations of ECTV ANK proteins in comparison to the consensus poxviral F-box sequence (top). Identical residues are highlighted in black. Similar residues are highlighted in gray. (C) SILAC proteomics analysis of FLAG-immunoprecipitated proteins from cells expressing FLAG-tagged 010 or the corresponding empty vector (EV). The mean log2 fold increase (010/EV) in expression of each protein identified in three biological replicates is presented in comparison to values representing the mean intensity levels of those proteins. The data point corresponding to Cul-2 is highlighted in red. (D) HEK293T cells were cotransfected with FLAG-tagged 010 or RIG-I together with myc-tagged Cul-2 or TAK-1. After 24 h, the cells were lysed and subjected to FLAG immunoprecipitation (IP). Cell lysates (2%) and IP samples were analyzed by immunoblotting against the indicated proteins. (E) HEK293T cells were transfected with FLAG-tagged 010 or GFP. After 24 h, the cells were lysed and subjected to FLAG IP. Cell lysates (1.25%) and IP samples were analyzed by immunoblotting against the indicated proteins.

FIG 2

ECTV 010 targets Cullin-2 via a C-terminal BC box. (A) HEK293T cells were transfected with FLAG-tagged 010 or EV. After 24 h, the cells were treated with 1 μM MLN4924 or vehicle (DMSO) for a further 16 h. Cell lysates were subjected to immunoblotting (IB) against the indicated proteins. (B) Normalized intensity for 010 protein bands after DMSO or MLN4924 treatment from 3 independent experiments obtained via Li-COR. (C) HEK293T cells were cotransfected with FLAG-tagged full-length 010 (WT), 010 lacking the last 36 C-terminal amino acids (ΔBC), or RIG-I together with myc-tagged Cul-2. After 24 h, the cells were lysed and subjected to FLAG immunoprecipitation (IP). Cell lysates (2%) and IP samples were analyzed by immunoblotting against the indicated proteins. (D) HEK293T cells were transfected with FLAG-tagged full-length 010 (WT), 010.L728A/C723F (mut2), 010.L728A/C723F/I736A (mut 3), or the ANK-only protein K1 together with myc-tagged Cul-2. After 24 h, the cells were lysed and subjected to FLAG IP and immunoblotting as described above. (E) HEK293T cells were transfected with FLAG-tagged 010, GFP, K1, or K1 fused to the last 37 C-terminal amino acids of 010, together with myc-tagged Cul-2. After 24 h, the cells were lysed and subjected to FLAG IP and immunoblotting as described above. Data are representative of results obtained from at least 3 biological experiments.

FIG 3

ANK/BC proteins are conserved among several OPV. (A) Conservation of the BC box sequence in the indicated ANK proteins. (B) HEK293T cells were cotransfected with FLAG-tagged 016, 019, or K1 together with myc-tagged Cul-2. After 24 h, the cells were lysed and subjected to FLAG immunoprecipitation (IP). Cell lysates (2%) and IP samples were analyzed by immunoblotting against the indicated proteins.

FIG 4

ECTV 010 inhibits NF-kB and IRF-3 activation downstream of multiple PRRs. (A) Immunoblot from a representative reporter assay in HEK293T cells transfected with 25 ng/well of 010 or the corresponding empty vector (EV). Lysates were cleared by centrifugation and subjected to immunoblotting against the indicated proteins. (B) Cells were transfected with 010 or EV together with 10 ng/well of pTK-Renilla and 70 ng/well of pIFN-β–Luc. After 24 h, the cells were stimulated with Sendai virus (SeV) for a further 24 h and luciferase activity was measured. Firefly luciferase (FLuc)/Renilla Luc ratios were normalized to the nonstimulated (n.s.) control and are presented as fold increase values. (C) Cells were transfected as described for panel B but with 70 ng/well of pISRE-Luc and including C6 as a positive control. The cells were stimulated 24 h after with 50 ng/ml of IFN-β for a further 8 h. (D to F) Cells were transfected as described for panel B together with (D) 30 ng/well of TRIF, (E) 5 ng/well of RIG-I-CARD, or (F) 20 ng/well each of cGAS and STING. (G to I) Cells were transfected as described for panels D to F but with 70 ng/well of pISG56.1-Luc. (J to L) Cells were transfected as described for panels D to F but with pNF-κB–Luc. (M) Cells were transfected as described for panel B but with 250 ng/well of pAP-1–Luc. For all assays, data are presented as means ± SD and represent results from one experiment that is representative of at least three, each performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student's t test).

FIG 5

CPXV 016 and 019 inhibit NF-κB and IRF-3 activation. HEK293T cells were transfected with 25 ng/well of 010 (black bars), 016 (red bars), 019 (black bars), or the corresponding empty vector (EV; white bars) together with 10 ng/well of pTK-Renilla and 70 ng/well of (A) pIFN-β–Luc or (B) pNF-κB–Luc, as well as 50 ng/well of RIG-I, 20 ng/well each of cGAS and STING, or 10 ng/well of TRIF. After 16 h, the cells were harvested and luciferase activity was measured. FLuc/Renilla Luc ratios were normalized to the nonstimulated EV control and are presented as fold increase values. In all assays, data are presented as means ± SD and represent results from one experiment that is representative of at least three, each performed in triplicate. n.s. nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student's t test).

FIG 6

ANK/BC proteins suppress the production of CXCL10, CCL5, and IFN. (A) HEK293T-REx cells were induced with 2 μg/ml of doxycycline (Dox) for 16 h and subjected to immunoblotting against the indicated proteins. (B to D) The cells were induced with Dox as described for panel A and subsequently stimulated with SeV for a further 24 h. RNA expression levels for (B) IFN-β, (C) CXCL10, and (D) CCL5 were measured by quantitative PCR (qPCR). Data were normalized to 18S expression levels; data represent fold increase over the levels seen under nonstimulated conditions. (E and F) The cells were treated as described for panel B, and the resulting cell culture medium was subjected to ELISA to detect soluble levels of (E) CXCL10 and (F) CCL5. In all assays, data are presented as means ± SD and represent results from one experiment that is representative of at least three, each performed in triplicate. **, P < 0.01; ***, P < 0.001 (unpaired Student's t test).

FIG 7

Association with Cul-2 is required for ANK/BC optimal inhibitory activity. (A) HEK293T cells were transfected with 5 ng/well of 010, 010dBC, 010.L728A/C723F (mut2), 010.L728A/C723F/I736A (mut 3), or the corresponding empty vector (EV) together with 10 ng/well of pTK-Renilla and 70 ng/well of pNF-κB–Luc. After 24 h, the cells were stimulated with TNF-α for a further 6 h. FLuc/Renilla Luc ratios were normalized to the nonstimulated (n.s.) EV control and are presented as fold increase values. (B) NF-κB activation was assessed as described above but in the presence of increasing doses of each 010 construct. Luciferase ratios were plotted as percentages relative to the extent of NF-κB activation observed in EV-transfected cells stimulated with TNF-α. (C) Cells were transfected as described for panel A but together with 20 ng/well of TRIF. After 16 h, the cells were harvested and luciferase activity was measured. FLuc/Renilla Luc ratios were normalized to the nonstimulated (n.s.) EV control and are presented as fold increase values. (D) IFN-β activation was assessed as described above but in the presence of increasing doses of each 010 construct. Luciferase ratios were plotted as percentages relative to the extent of IFN-β activation observed in EV-transfected cells stimulated with TRIF. In all assays, data are presented as means ± SD and represent results from one experiment that is representative of at least three, each performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student's t test).