The Human Papillomavirus E6 Oncoprotein Targets USP15 and TRIM25 To Suppress RIG-I-Mediated Innate Immune Signaling

Abstract

Retinoic acid-inducible gene I (RIG-I) is a key pattern recognition receptor that senses viral RNA and interacts with the mitochondrial adaptor MAVS, triggering a signaling cascade that results in the production of type I interferons (IFNs). This signaling axis is initiated by K63-linked ubiquitination of RIG-I mediated by the E3 ubiquitin ligase TRIM25, which promotes the interaction of RIG-I with MAVS. USP15 was recently identified as an upstream regulator of TRIM25, stabilizing the enzyme through removal of degradative K48-linked polyubiquitin, ultimately promoting RIG-I-dependent cytokine responses. Here, we show that the E6 oncoprotein of human papillomavirus type 16 (HPV16) as well as of other HPV types form a complex with TRIM25 and USP15 in human cells. In the presence of E6, the K48-linked ubiquitination of TRIM25 was markedly increased, and in line with this, TRIM25 degradation was enhanced. Our results further showed that E6 inhibited the TRIM25-mediated K63-linked ubiquitination of RIG-I and its CARD-dependent interaction with MAVS. HPV16 E6, but not E7, suppressed the RIG-I-mediated induction of IFN-β, chemokines, and IFN-stimulated genes (ISGs). Finally, CRISPR-Cas9 gene targeting in human keratinocytes showed that the TRIM25-RIG-I-MAVS triad is important for eliciting an antiviral immune response to HPV16 infection. Our study thus identifies a novel immune escape mechanism that is conserved among different HPV strains and further indicates that the RIG-I signaling pathway plays an important role in the innate immune response to HPV infection.IMPORTANCE Persistent infection and tumorigenesis by HPVs are known to require viral manipulation of a variety of cellular processes, including those involved in innate immune responses. Here, we show that the HPV E6 oncoprotein antagonizes the activation of the cytoplasmic innate immune sensor RIG-I by targeting its upstream regulatory enzymes TRIM25 and USP15. We further show that the RIG-I signaling cascade is important for an antiviral innate immune response to HPV16 infection, providing evidence that RIG-I, whose role in sensing RNA virus infections has been well characterized, also plays a crucial role in the antiviral host response to small DNA viruses of the Papillomaviridae family.

Keywords: RIG-I; innate immunity; interferons; papillomavirus.

FIG 1

Interaction of the HPV E6 protein with TRIM25 and USP15. (A) HEK 293T cells were transfected with plasmids encoding FLAG-tagged E6 from HPV16 (16E6-FLAG) together with empty vector or Myc-tagged USP15 and/or V5-tagged TRIM25. Forty-eight hours later, whole-cell lysates (WCLs) were subjected to immunoprecipitation (IP) with anti-FLAG antibody, followed by immunoblotting (IB) with anti-V5, anti-Myc, and anti-FLAG antibodies. WCLs were further immunoblotted with anti-Myc, anti-V5, and anti-actin antibodies. (B) C33A cells stably expressing either empty vector, HA-tagged wild-type (WT) 16E6, or 16E6 I128T mutant were treated with 50 μM concentrations of the proteasome inhibitor MG132 for 5 h. WCLs were prepared and subjected to IP with anti-HA antibody, followed by IB with anti-USP15 and anti-TRIM25 antibodies. (C) HEK 293T cells were transfected with empty vector or HA-tagged E6 from HPV strain 16, 52, 33, or 6 (HA-16E6, HA-52E6, HA-33E6, or HA-6E6). Forty-eight hours later, cells were treated with 50 μM MG132 for 5 h. WCLs were prepared and subjected to pulldown (PD) with anti-HA, followed by IB with anti-TRIM25 and anti-HA antibodies. WCLs were further immunoblotted with anti-TRIM25 and anti-actin antibodies. (D) HEK 293T cells were transfected with empty vector or plasmids encoding the E6 proteins (FLAG tagged) from HPV strain 16, 11, 6, 18, 5, or 8 (16E6-FLAG, 11E6-FLAG, 6E6-FLAG, 18E6-FLAG, 5E6-FLAG, or 8E6-FLAG) or E7 from HPV16 (16E7-FLAG). Forty-eight hours later, cells were treated with 50 μM MG132 for 5 h. WCLs were prepared and subjected to PD with anti-FLAG, followed by IB with anti-TRIM25 and anti-FLAG antibodies. WCLs were further immunoblotted with anti-TRIM25 and anti-actin antibodies. (E) Schematic representation of TRIM25 domain structure as well as truncation constructs used for mapping studies. RING, really interesting new gene domain; BB1/2, B-box 1 and 2; CCD, coiled-coil domain; B30.2/SPRY, Spla and the ryanodine receptor domain. Numbers indicate amino acids. (F) HEK 293T cells were transfected with 16E6-FLAG together with GST or the indicated GST-TRIM25 fusion constructs. Forty-eight hours later, WCLs were prepared and subjected to PD with anti-GST, followed by IB with anti-FLAG and anti-GST antibodies. WCLs were further immunoblotted with anti-FLAG and anti-actin antibodies. (G) HEK 293T cells were transfected with empty vector or a plasmid encoding 16E6-FLAG together with GST-RING-BB, GST-BB, or GST-RING. Forty-eight hours later, cells were incubated with 50 μM MG132 for 5 h. WCLs were subjected to IP with anti-FLAG antibody, followed by IB with anti-GST and anti-FLAG antibodies. Efficient expression of GST-TRIM25 fusion proteins was confirmed in the WCLs by IB with anti-GST antibody.

FIG 2

Enhanced ubiquitination of TRIM25 induced by HPV16 E6. (A) HEK 293T cells were transfected with V5-tagged TRIM25 together with empty vector or a plasmid encoding 16E6-FLAG. Forty-eight hours later, WCLs were subjected to IP with anti-V5 antibody, followed by IB with anti-ubiquitin (Ub) and anti-V5. WCLs were further immunoblotted with anti-FLAG and anti-actin antibodies. (B) HEK 293T cells were transiently transfected with empty vector, HA-tagged HPV16 E6 WT, or mutant I128T together with TRIM25-V5 and Ub-FLAG. Forty-eight hours later, cells were treated with 50 μM MG132 for 5 h. WCLs were subjected to IP with anti-V5 antibody, followed by IB with anti-FLAG and anti-V5. WCLs were further immunoblotted with anti-FLAG, anti-HA, and anti-actin. (C) HEK 293T cells were transfected with a plasmid encoding 16E6-FLAG together with Myc-tagged USP15 WT or its mutant C783A or C269A. Forty-eight hours later, cells were treated with 50 μM MG132 for 5 h. WCLs were prepared and subjected to IP with anti-FLAG antibody, followed by IB with anti-Ub and anti-FLAG antibodies. WCLs were further immunoblotted with anti-Ub, anti-Myc, and anti-actin antibodies. IB of WCLs with anti-FLAG determined E6 protein levels stabilized by WT or mutant USP15. (D) HeLa cells stably overexpressing either empty vector or 16E6-HA were treated with 100 μM cycloheximide (CHX) for the indicated times. WCLs were subjected to SDS-PAGE, followed by IB with anti-TRIM25 antibody. IB of WCLs with anti-actin antibody served as a loading control. (E) SiHa cells were transfected with nontargeting control siRNA (siC) or siRNA targeting E6 (siE6). Ninety-six hours later, cells were treated with 100 μM CHX for the indicated times. (Left) WCLs were subjected to SDS-PAGE, followed by IB with anti-TRIM25 antibody. IB of WCLs with anti-actin antibody served as a loading control. (Right) Knockdown efficiency of E6 was assessed by qPCR, and values were normalized to those for GAPDH. Relative knockdown efficiency is shown compared to values for siC-transfected cells, set to 1. (F) HaCaT cells were mock treated or infected with HPV16 (MOI of 50) for 24, 48, and 72 h. (Upper) WCLs were subjected to SDS-PAGE, followed by IB with anti-TRIM25 and anti-actin antibodies. (Lower) The intensities of the bands for TRIM25 and actin were quantified by densitometry, and ratios of the abundance of TRIM25 protein relative to that of actin were calculated. Data shown are representative of two independent experiments.

FIG 3

HPV16 E6 suppresses the K63-linked ubiquitination of RIG-I and its binding to MAVS. (A) HEK 293T cells were transfected with GST or GST-RIG-I-2CARD and TRIM25-V5 together with empty vector or FLAG-tagged 16E6. Forty-eight hours later, WCLs were subjected to PD with anti-GST, followed by IB with anti-Ub and anti-GST antibodies. WCLs were further immunoblotted with anti-FLAG, anti-V5, and anti-actin antibodies. (B) HEK 293T cells were transfected with a plasmid encoding an HA-tagged Ub mutant in which all internal lysines are mutated, except K63 (K63only-Ub-HA), together with either empty vector or a plasmid encoding 16E6-FLAG. Twenty-four hours later, cells were infected with SeV (50 HA units/ml) for 18 h to induce RIG-I K63-linked ubiquitination or left uninfected. The extent of K63-linked ubiquitination of endogenous RIG-I was determined by IP with anti-RIG-I antibody, followed by IB with anti-HA and anti-RIG-I antibodies. WCLs were further immunoblotted with anti-HA, anti-FLAG, and anti-actin antibodies. (C) HEK 293T cells were transfected with GST or GST-RIG-I-2CARD together with MAVS-CARD-PRD-FLAG and either empty vector or increasing amounts of 16E6-FLAG. Forty-eight hours later, cells were treated with 50 μM MG132 for 5 h. WCLs were prepared and subjected to PD with anti-GST, followed by IB with anti-FLAG and anti-GST antibodies. WCLs were further immunoblotted with anti-FLAG to confirm the expression of MAVS-CARD-PRD-FLAG and 16E6-FLAG.

FIG 4

HPV16 E6 inhibits USP15- and TRIM25-induced RIG-I signaling. (A) HEK 293T cells were transfected with GST-RIG-I-2CARD together with empty vector, V5-TRIM25, or V5-TRIM25 and USP15-Myc and increasing amounts of 16E6-FLAG as well as IFN-β luciferase reporter construct and a plasmid expressing constitutive β-galactosidase (pGK-β-gal). Forty-eight hours later, luciferase and β-galactosidase values were determined. Results are expressed as means ± standard deviations (SD) (n = 3). (B) HEK 293T cells were transfected with empty vector or increasing amounts of 16E6-FLAG. Twenty-four hours later, cells were infected with SeV (50 HA units/ml) or left uninfected. Sixteen hours later, cells were treated with 50 μM MG132 for 5 h. WCLs were prepared and subjected to IB with the indicated antibodies. (C) HEK 293T cells were transfected with empty vector, 16E6-FLAG, or 16E7-FLAG. Twenty-four hours later, cells were either infected with SeV (50 HA units/ml) or left uninfected. Sixteen hours later, cells were treated with 50 μM MG132 for 5 h. WCLs were prepared and subjected to IB with the indicated antibodies. (D) C33A cells were transfected with 1 μg of empty vector or with plasmids encoding 16E6-FLAG or 16E7-FLAG. Twenty-four hours posttransfection, cells were infected with SeV (10 HA units/ml) for 16 h or left uninfected (Mock). Transcript levels of the indicated genes were assessed by qPCR and the values were normalized to those for GAPDH, with each mock-infected value set to 1. Results are expressed as means ± SD (n = 3). **, P < 0.005; ***, P < 0.001 (unpaired t test).

FIG 5

The TRIM25-RIG-I-MAVS signaling pathway is important for an antiviral response to HPV16 infection. (A) HaCaT cells were mock-treated or infected with HPV16 (MOI of 50) for 48 h. Transcript levels of the indicated genes were assessed by qPCR, and values were normalized to those for human GAPDH. Results are expressed as means ± SD (n = 3). **, P < 0.005; ***, P < 0.001 (unpaired t test). (B) Validation of generated HaCaT-CRISPR-KO cell lines. WCLs from HaCaT cells expressing nontargeting control guide RNA (NT) or transduced with guide RNA targeting TRIM25 (TRIM25 KO), RIG-I (RIG-I KO), or MAVS (MAVS KO) were subjected to IB analysis with anti-TRIM25, anti-RIG-I, or anti-MAVS antibody to confirm the absence of protein expression for the individual genes. IB with anti-actin served as a loading control. (C) HaCaT NT, TRIM25 KO, RIG-I KO, and MAVS KO cells were mock treated or infected with HPV16 (MOI of 50) for 48 h. Transcript levels of the indicated genes were assessed by qPCR, and values were normalized to those for human GAPDH. Results are expressed as means ± SD (n = 3). *, P < 0.05; **, P < 0.005, ***, P < 0.001 (unpaired t test).