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. 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 family.

To TRIM or not to TRIM: the balance of host–virus interactions mediated by the ubiquitin system

“…To date, most studies have implicated TRIM25 as a critical mediator of innate immune signalling through ubiquitination of RIG-I [2,11,[101][102][103][104][105][106][107][108][109][110][111][112][113][114][115][116][117][118], while only a handful of studies have proposed RIG-I-independent mechanisms [46,119]. Recent years have brought both exciting discoveries and rousing controversies regarding how ubiquitin and E3 ligases precisely function to activate RIG-I for antiviral signalling, with some groups suggesting a single factor operates as the critical component while others have put forth a cooperative model based on trends gleaned from numerous studies.…”

“…HCV-PAMP), or upon live virus infection (e.g. SeV, HCV, IAV, HPV) [101,102,105,113,114,125]. Additional evidence showing regulation of RIG-I by TRIM25 comes from studies that have uncovered several host proteins that regulate the E3 ligase activity of TRIM25 (e.g.…”

“…For example, a full in vitro reconstitution of the RIG-I pathway utilizing TRIM25 as the E3 ligase is known to potently activate RIG-I signalling through unanchored K63-linked polyubiquitin chains [21]. In addition, viral products have been found to directly antagonize TRIM25, consequently reducing RIG-I ubiquitination and downstream signalling, including paramyxoviruses V protein, the papillomavirus E6 oncoprotein, the severe acute respiratory syndrome-related coronavirus (SARS-CoV) N protein, the NSs protein of severe fever with thrombocytopenia syndrome virus (SFTSV), the nucleocapsid protein (N) of porcine reproductive and respiratory syndrome virus (PRRSV) and potentially DENV subgenomic RNA [106,107,111,113,115,135]. Specifically, the IAV NS1 protein inhibits both TRIM25 and Riplet, and this occurs in a species-specific manner [104].…”

“…As mentioned above, since its identification as a critical component for induction of RIG-I-mediated innate immunity, TRIM25 has received much attention not only for its importance in antiviral defence, but also for how a diverse range of pathogens have zeroed in and neutralized its function [101,102,104,106,107,109,[111][112][113]115]. Besides TRIM25, other TRIMs have been found to be targeted by viruses.…”

Insertional oncogenesis by HPV70 revealed by multiple genomic analyses in a clinically HPV‐negative cervical cancer

“…These findings suggest that only low levels of E6 might be translated in this tumor. In addition to binding to TP53, E6 also interacts with a number of antiapoptotic proteins and PDZ domain‐containing proteins . It also activates the TERT promoter .…”