Molluscum Contagiosum Virus Protein MC008 Targets NF-κB Activation by Inhibiting Ubiquitination of NEMO

Abstract

Molluscum contagiosum virus (MCV) is a human-adapted poxvirus that causes a common and persistent yet mild infection characterized by distinct, contagious, papular skin lesions. These lesions are notable for having little or no inflammation associated with them and can persist for long periods without an effective clearance response from the host. Like all poxviruses, MCV encodes potent immunosuppressive proteins that perturb innate immune pathways involved in virus sensing, the interferon response, and inflammation, which collectively orchestrate antiviral immunity and clearance, with several of these pathways converging at common signaling nodes. One such node is the regulator of canonical nuclear factor kappa B (NF-κB) activation, NF-κB essential modulator (NEMO). Here, we report that the MCV protein MC008 specifically inhibits NF-κB through its interaction with NEMO, disrupting its early ubiquitin-mediated activation and subsequent downstream signaling. MC008 is the third NEMO-targeting inhibitor to be described in MCV to date, with each inhibiting NEMO activation in distinct ways, highlighting strong selective pressure to evolve multiple ways of disabling this key signaling protein. IMPORTANCE Inflammation lies at the heart of most human diseases. Understanding the pathways that drive this response is the key to new anti-inflammatory therapies. Viruses evolve to target inflammation; thus, understanding how they do this reveals how inflammation is controlled and, potentially, how to disable it when it drives disease. Molluscum contagiosum virus (MCV) has specifically evolved to infect humans and displays an unprecedented ability to suppress inflammation in our tissue. We have identified a novel inhibitor of human innate signaling from MCV, MC008, which targets NEMO, a core regulator of proinflammatory signaling. Furthermore, MC008 appears to inhibit early ubiquitination, thus interrupting later events in NEMO activation, thereby validating current models of IκB kinase (IKK) complex regulation.

Keywords: MC008; NEMO; NF-κB; human; innate; innate immunity; molluscum contagiosum; ubiquitin; ubiquitination.

FIG 1

MC008 inhibits the cytosolic nucleic acid-sensing pathway and antiviral TLR activation of NF-κB. (A) HeLa cells were transduced with lentivirus containing Lenti-X Tet-One inducible MC008-FLAG. Following selection, 3 × 10⁵ cells/well were seeded into a 6-well plate and were left untreated (−) or stimulated with 100 ng/mL of doxycycline (Dox) (+) for 24 h to stimulate MC008 expression. Cells were subsequently lysed 24 h later, followed by immunoblotting. (B) A total of 1 × 10⁵ stable HeLa cells/mL were transfected with 1 μg/mL poly(dA-dT) for 24 h following MC008 induction. Serum-free medium and Lipofectamine 2000 were used as the vehicle control. The cell supernatants were subsequently harvested, and IL-6 levels were measured using an ELISA. (C) Localization of MC008 in HEK293T cells. Cells were transfected with 100 ng of the pCEP4-MC008-HA expression vector and fixed 24 h later. Cells were subsequently stained with DAPI (blue) or probed with anti-HA (red). (D) HEK293T cells were seeded at 2 × 10⁵ cells/mL and transfected with 80 ng of NF-κB and 40 ng of the TK Renilla reporter gene, along with the empty vector and 25 or 50 ng of MC008-FLAG and MC020-FLAG for 24 h. Cell lysates were subsequently harvested, and Western blotting was performed. (E to O) Similarly, HEK293T cells were seeded at 2 × 10⁵ cells/mL and transfected as follows: 80 ng of NF-κB luciferase and 40 ng of TK Renilla reporter genes; 50 ng of the pathway activators TAB2, NEMO K277A mutant or IKKα; or 10 ng of IKKβ, or 1 ng of p65; 25 or 50 ng of the indicated MCV ORFs; and the final volume was adjusted to 220 ng with empty vector. 25 ng of cGAS and 25 ng of STING; 10 ng of MAVS; 5 ng of IRF3-GAL4; 50 ng of MyD88, CD4TLR3, or TRIF; 1 μg/mL of poly(dA-dT); or Sendai virus (SeV) was used to activate the pathways. The cells were subsequently lysed after 24 h and assayed for NF-κB and ISRE, IRF3-GAL4, or IFN-β reporter activity. Data are presented as percentages of reporter activation compared to the positive control and are the combined means ± standard deviations for triplicate samples from representative experiments (n = 3). Statistical significance is indicated by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 2

MC008 inhibits TNF-α- and IL-1β-stimulated NF-κB activation. (A) A total of 1 × 10⁵ stable HeLa cells/mL were stimulated with 50 ng/mL of TNF-α for 24 h following MC008 induction with doxycycline, and IL-6 levels were measured using an ELISA. (B and C) Effect of MC008 on TNF- and IL-1-stimulated NF-κB activation. A total of 2 × 10⁵ cells/mL were transfected with 80 ng of the NF-κB reporter gene and 40 ng of the thymidine kinase promoter-Renilla luciferase (TK Renilla) reporter gene, along with 25 or 50 ng of the MCV ORFs indicated, for 24 h. Cells were then stimulated with 50 ng/mL TNF-α or IL-1β for 8 h, subsequently lysed, and assayed for NF-κB reporter activity. (D and E) Similar to panels B and C except that 50 ng of TRAF2 and TRAF6 was overexpressed to drive NF-κB signaling. (F) The activation of Elk1 by Ras was measured by transfecting 80 ng of the pFR-luciferase reporter along with 50 ng of Ras, 5 ng of pFA-Elk1, and the indicated MCV ORFs (25 and 50 ng). Data are presented as percentages of reporter activation compared to the positive control and are the combined means ± standard deviations for triplicate samples from representative experiments (n = 3). Statistical significance is indicated by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 3

MC008 inhibits at a point proximal to the IKK complex. (A) Schematic showing multiple signaling pathways involved in NF-κB activation expected to be triggered by poxvirus infection. All of these pathways are perturbed by MC008 expression, as demonstrated above. IL1R, interleukin-1 receptor; TNFR, tumor necrosis factor receptor. (B to F) Comparison of MC008, MC005, and MC132 inhibition of signal transduction surrounding IKK complex activation. HEK293T cells were seeded at 2 × 10⁵ cells/mL and transfected as follows: 80 ng of NF-κB luciferase and 40 ng of TK Renilla reporter genes; 50 ng of the pathway activators TAB2, NEMO K277A mutant or IKKα; or 10 ng of IKKβ, or 1 ng of p65; 25 or 50 ng of the indicated MCV ORFs; and the final volume was adjusted to 220 ng with empty vector. The cells were subsequently lysed after 24 h and assayed for NF-κB reporter activity. Data are presented as percentages of reporter activation compared to the positive control and are the combined means ± standard deviations for triplicate samples from individual experiments (n = 3). Statistical significance is indicated by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 4

MC008 interacts with NEMO. (A and B) Four micrograms of MC008 and 4 μg of the indicated signaling elements were transiently expressed in 4 × 10⁶ HEK293T cells per plate. The cells were subsequently lysed using ice-cold lysis buffer with a cocktail of phosphatase and protease inhibitors, and coimmunoprecipitation (IP) was performed. The resulting immunoprecipitants were then immunoblotted with the antibodies indicated. Representative blots (n = 3) are shown. (C) A total of 2 × 10⁶ HaCaT cells per plate were transiently transfected with 4 μg of MC008 but not NEMO. Coimmunoprecipitation was performed, and the interaction with endogenous levels of NEMO was confirmed using a NEMO-specific antibody during immunoblotting. An antibody against endogenous p65 was used as a negative control for the pulldown. (D) HEK293T and HaCaT cells were seeded at 70,000 and 30,00 cells per well in 8-well chamber slides, respectively. Twenty-four hours later, they were transfected with 100 ng of expression vectors for MC008-HA and NEMO-FLAG. Cells were then fixed 24 h later and stained with DAPI (blue), FLAG (green), and HA (red).

FIG 5

MC008 disrupts NEMO ubiquitination. (A) NEMO truncations were generated according to the schematic. (B) Four micrograms of MC008-FLAG and 4 μg of NEMO or the indicated truncations were transiently expressed in 4 × 10⁶ HEK293T cells per plate. The cells were subsequently lysed, and coimmunoprecipitation was performed. The resulting immunoprecipitants were then immunoblotted with anti-FLAG and anti-HA antibodies. A representative blot (n = 3) is shown. (C) HEK293T cells, stably expressing pMEP4-MC008-FLAG, were seeded at 6 × 10⁵/well into six-well dishes and were left untreated (−) or treated with 1 μm CdCl₂ to induce MC008 expression (+). Cells were stimulated 24 h later with 50 ng/mL of TNF-α for the times indicated, and lysates were immunoblotted with the antibodies indicated. A representative blot (n = 3) is shown. (D to F) The indicated proteins were transiently expressed in 4 × 10⁶ HEK293T cells per plate in the following ratios: 2 μg of NEMO-FLAG, 2 μg of NEMO-HA, 3 μg of IKKα-FLAG or IKKβ-FLAG, 1 μg of ubiquitin-HA, and 3 μg of MC008-HA. The cells were subsequently lysed, and coimmunoprecipitation was performed. Black lines indicate areas of ubiquitination. Representative blots (n = 3) are shown.

FIG 6

Model of MC008 inhibition of NF-κB signaling. PRR and cytokine signaling pathways drive signal transduction leading to the activation of E3 ligases such as TRAF proteins, which generate ubiquitin chains. NEMO is locked in an autoinhibited state by the CC1 domain until it is relieved by long ubiquitin chains binding to the UBAN domain. This binding event induces a conformational change, priming NEMO for downstream signaling. MC008 inhibits this ubiquitination event by binding to the CC1 and UBAN domains, thus preventing complex priming and downstream NF-κB signaling.