The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

Abstract

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection.IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.

Keywords: Ebola virus; TRIM6; VP35; innate immunity; tripartite motif (TRIM) protein; ubiquitination; unanchored ubiquitin; viral RNA polymerase; virus-host interactions.

FIG 1

EBOV VP35 interacts with the C-terminal SPRY region of TRIM6 and is recruited to TRIM6 cytoplasmic dots. (A) TRIM6 interacts with EBOV-VP35 but not with LCMV-NP or Marbug-VP40. HEK293T cells were transfected with plasmids encoding VP35, NP, VP40, and HA-TRIM6. Cells were harvested and lysed, and whole-cell extracts (WCE) were used for FLAG immunoprecipitation (IP) (VP35, NP, or VP40) using anti-FLAG beads. Immunobloting (IB) was performed with the indicated antibodies. (B and C) The C-terminal SPRY domain of TRIM6 interacts with VP35. (B) TRIM6 deletion mutants used for co-IP (GST tagged). (C) HEK293T cells were transfected with plasmids encoding the indicated GST-TRIM6 deletion mutants together with VP35. Cells were harvested and lysed, and WCE were used for TRIM6 pulldown using GST beads. (D) VP35 colocalizes with TRIM6 in cytoplasmic bodies. HeLa cells were transfected with plasmids encoding HA-TRIM6 and VP35. Cells were fixed and stained with the indicated primary antibodies (anti-HA and anti-Flag) and secondary antibodies (488 and 555) as described in Materials and Methods, followed by confocal microscopy. (E) VP35 inhibits RIG-I-induced TRIM6-mediated IFN-β. HEK293T cells were transfected with a plasmid encoding firefly luciferase under the control of the IFN-β promoter and a control plasmid encoding Renilla luciferase in the presence or absence of plasmids encoding the constitutively active RIG-I (2CARD), HA-TRIM6, and VP35 WT or an RNA binding-defective mutant of VP35 (KRA). Empty vector was used to ensure that the same amounts of plasmids were transfected. Cells were lysed after 30 h for luciferase assay. Data were normalized by the nonstimulated sample to obtain fold induction. The luciferase reporter assay was performed using triplicate samples, and results are depicted as the means ± SE (n = 3). All experiments were repeated at least two times, and representatives of independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significant.

FIG 2

EBOV-VP35 is ubiquitinated on K309. (A) Mass spectrometry analysis of ubiquitinated VP35. HEK293T cells were transfected with plasmids encoding VP35 and HA-Ub. After 30 h, cells were harvested and lysed, and whole-cell extracts (WCE) were used for HA immunoprecipitation (IP) using anti-HA beads. Ubiquitinated proteins were eluted with HA peptide. Eluted fractions were digested with trypsin and analyzed by MS. Peptides corresponding to VP35 are shown in red. GlyGly at residue 4 indicates ubiquitination (blue). (B) VP35 is ubiquitinated on K309. HEK293T cells were transfected with plasmids encoding VP35 WT or a VP35 K309A mutant and HA-Ub. Cells were harvested and lysed, and WCE were used for IP using anti-HA beads. Immunoblotting (IB) was performed with the indicated antibodies. The bands corresponding to the different ubiquitinated forms of VP35 are indicated according to the predicted molecular mass of Ub (∼8.5 kDa) and VP35 (37 kDa). (C) Quantification of the different ubiquitinated forms of VP35 WT and K309A. Densitometry of each separate band representing the different forms of ubiquitinated VP35 shown in panel B was done using ImageJ software. To normalize by the efficiency of HA-Ub pulldown and the levels of VP35 expression, the values were normalized first by the total amount of HA-Ub (shown in the IP and IB rows for HA-Ub in panel B) and then by the amounts of VP35 (from the WCE). The values obtained from 3 independent experiments were used to obtain statistics shown in panel C (*, P < 0.05; n = 3). (D) Schematic representation of the possible ubiquitinated forms of VP35 detected in the co-IP shown in panel B. Poly-Ub chains of up to 6 ubiquitin molecules attached to VP35 were detected based on molecular mass. The VP35 K309A mutant retains at least one di-Ub chain or two monoubiquitinated sites. (D) The C-terminal region of VP35 associates with ubiquitin. HEK293T cells were transfected with vectors encoding the N-terminal (1 to 218 aa) or C-terminal (219 to 340 aa) regions of VP35 (Flag tagged). Cells were harvested and WCE were used for IP using anti-FLAG beads. Immunoblotting was performed using anti-Ub antibodies.

FIG 3

VP35 interacts with poly-Ub chains by covalent and noncovalent interactions. HEK293T cells, transfected with VP35 and His-tagged Ub, were subjected to His pulldown under denaturing conditions (urea washes) or nondenaturing conditions (RIPA washes). The amounts of His-Ub (lower left) and VP35 (upper left) used for the pulldown are shown (input samples). The upper right panel shows the amount of VP35 that associates with Ub by covalent interactions (urea wash) or noncovalent interactions (RIPA wash). The bottom right panel shows the efficiency of the ubiquitin pulldown.

FIG 4

TRIM6 overexpression enhances VP35 ubiquitination. (A) HEK293T cells were transfected with plasmids encoding FLAG-VP35 with or without plasmids encoding HA-TRIM6. Cells were harvested and whole-cell extracts (WCE) were used for FLAG IP using anti-FLAG beads. Ubiquitinated VP35 was detected by immunoblotting (IB) using a specific antibody against endogenous ubiquitin. (B) HEK293T cells were transfected with a plasmid encoding untagged VP35 with or without GST-TRIM6 and HA-Ub. Cells were harvested and whole-cell extracts (WCE) were used for HA IP using anti-HA beads, and the levels of ubiquitinated VP35 were assessed by IB with anti-VP35. Different exposures for the VP35 blots are shown. The bands corresponding to ubiquitinated VP35 are indicated based on the molecular masses of VP35 (approximately 37 kDa) and ubiquitin (8.5 kDa). Polyubiquitinated VP35 with more than 25 ubiquitin molecules can be detected (over 250 kDa).

FIG 5

TRIM6 enhances VP35-dependent polymerase activity at limiting amounts of VP35. (A) An Ebola minigenome assay was performed in the presence of different amounts of transfected vectors expressing TRIM6 or C15A (10, 50, and 100 ng) and vectors expressing either VP35 WT or VP35 K309A at limiting amounts (25 ng). The fold increase was determined by setting the value for empty vector (no VP35) as 1. (B) TRIM6 expression decreases the VP35-mediated minigenome activity at optimal amounts of VP35. The minigenome assay was performed as described for panel A but using an optimal amount of VP35 vectors of 125 ng. These experiments are representative of 2 independent experiments, each performed in triplicates. The error bars indicate means ± SE (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significant. (C) Representative samples from the minigenome assay shown in panels A and B (with TRIM6 at 100 ng only) were analyzed by immunoblotting. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 6

TRIM6 is required for efficient EBOV replication. (A to C) TRIM6 A549 knockout cell lines were infected with EBOV-GFP at different MOIs (5, 0.5, and 0.01). Immunoblotting of TRIM6 (A) and GFP images at different time points for an MOI of 5 (bright-field [BF] and GFP images for EBOV) (B) are shown. (C) Plaque assays of WT A549 and TRIM6 knockout cell lines infected at different MOIs. Each experiment was performed using triplicate samples, and results are depicted as the means ± standard errors (SE) (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Experiments shown are representative of at least 2 independent experiments. The limit of detection for the plaque assay was 1 × 10² PFU/ml.

FIG 7

EBOV RNA and IFN-β but not IL-6 are reduced in TRIM6 knockout EBOV-infected cells and correlate with reduced levels of ubiquitinated VP35 in TRIM6 KO-transfected cells. (A) WT and TRIM6 knockout A549 cells were infected with EBOV-GFP at an MOI of 0.5. Cells were lysed at the indicated time points and RNA was extracted for qPCR analysis. (B) WT and TRIM6 knockout A549 cells were infected with Sendai virus (SeV; 100 HA units), and cells were harvested at 8 h p.i. for RNA extraction and qPCR analysis. (C) VP35 ubiquitination is reduced in TRIM6 KO cells. A549 WT and TRIM6 KO (KO6) cells were transfected with empty vector or a vector expressing FLAG-VP35. Forty-eight hours posttransfection, cells were lysed and whole-cell extracts (WCE) were used for FLAG (VP35) immunoprecipitation (IP) using anti-FLAG beads. Immunoblots with antiubiquitin and anti-VP35 are shown. The ubiquitin bands of VP35 correlate to the smear observed with VP35 antibody. (D) WT and TRIM6 KO cells were stimulated with IFN-β (100 U), and after 4 h cells were lysed for RNA extraction and qPCR analysis. Samples were normalized by the 18S housekeeping gene. Data for IFN-β, IL-6, ISG54, and STAT1 were further normalized by mock-treated samples to give fold induction. The qPCR was performed using triplicate samples, and results are depicted as the means ± SE (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 8

Proposed model of regulation of VP35 function by ubiquitination. Upon virus recognition of EBOV infection, pattern recognition receptor (PRR) signaling promotes the synthesis of unanchored K48-linked polyubiquitin chains by the E3-ubiquitin ligase TRIM6. These polyubiquitin chains interact with IKKε and induce its oligomerization and downstream signaling to produce IFN-β (44). VP35 inhibits IFN-β at the level of RIG-I and the kinases TBK1 and IKKε (20). Ubiquitination of VP35 by TRIM6 promotes VP35-mediated polymerase activity and enhances virus replication.