Herpes Simplex Virus 1 Ubiquitin-Specific Protease UL36 Abrogates NF-κB Activation in DNA Sensing Signal Pathway

Abstract

The DNA sensing pathway triggers innate immune responses against DNA virus infection, and NF-B signaling plays a critical role in establishing innate immunity. We report here that the herpes simplex virus 1 (HSV-1) ubiquitinspecific protease (UL36USP), which is a deubiquitinase (DUB), antagonizes NF-B activation, depending on its DUB activity. In this study, ectopically expressed UL36USP blocked promoter activation of beta interferon (IFN-␤) and NF-B induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). UL36USP restricted NF-B activation mediated by overexpression of STING, TANKbinding kinase 1, IB kinase ␣ (IKK␣), and IKK␤, but not p65. UL36USP was also shown to inhibit IFN-stimulatory DNA-induced IFN-␤ and NF-B activation under conditions of HSV-1 infection. Furthermore, UL36USP was demonstrated to deubiquitinate IB␣ and restrict its degradation and, finally, abrogate NF-B activation. More importantly, the recombinant HSV-1 lacking UL36USP DUB activity, denoted as C40A mutant HSV-1, failed to cleave polyubiquitin chains on IB␣. For the first time, UL36USP was shown to dampen NF-B activation in the DNA sensing signal pathway to evade host antiviral innate immunity.IMPORTANCE It has been reported that double-stranded-DNA-mediated NF-B activation is critical for host antiviral responses. Viruses have established various strategies to evade the innate immune system. The N terminus of the HSV-1 UL36 geneencoded protein contains the DUB domain and is conserved across all herpesviruses. This study demonstrates that UL36USP abrogates NF-B activation by cleaving polyubiquitin chains from IB␣ and therefore restricts proteasome-dependent degradation of IB␣ and that DUB activity is indispensable for this process. This study expands our understanding of the mechanisms utilized by HSV-1 to evade the host antiviral innate immune defense induced by NF-B signaling.KEYWORDS HSV-1, DNA sensor, UL36, NF-B, IB␣ C ells have developed plenty of ways to govern their homeostasis, and the recognition of double-stranded DNA (dsDNA) by DNA sensors is a central host cellular defense against DNA virus infection. Among these DNA sensors, cyclic GMP-AMP synthase (cGAS), which is a nucleotidyltransferase, has been demonstrated to be the predominant cytosolic DNA sensor. Upon binding to DNA fragments, cGAS utilizes GTP and ATP to produce cyclic-GMP-AMP (cGAMP) through its enzymatic activity, and cGAMP activates an endoplasmic reticulum (ER)-resident receptor, stimulator of interferon genes (STING) (1). Activated STING then recruits TANK-binding kinase 1 (TBK1) and traffics from the ER to a perinuclear endosomal compartment, leading to the activation of transcription factors NF-B and interferon (IFN) regulatory factor 3 (IRF3), which induce IFN-␤ production (1-4).

Regulatory interplay between deubiquitinating enzymes and cytokines

“…In a different study regarding the function of UL36USP, it was found that UL36USP inhibits cGAS and STING dependent IFN-β production [49]. NF-κB activation from overexpressing STING, TBK1, IKKα and IKKβ was also inhibited, but not from overexpressing p65 [49]. In this study, human foreskin fibroblast (HFF) cells were infected with either HSV-1 or HSV-1 C40A mutant and stimulated with IFN stimulatory DNA [49].…”

“…NF-κB activation from overexpressing STING, TBK1, IKKα and IKKβ was also inhibited, but not from overexpressing p65 [49]. In this study, human foreskin fibroblast (HFF) cells were infected with either HSV-1 or HSV-1 C40A mutant and stimulated with IFN stimulatory DNA [49]. As a result, the level of endogenous IκBα in HFF cells with mutant HSV-1 significantly decreased compared to HFF cells with WT HSV-1, supporting that UL36USP decreases the degradation of IκBα in a DUB activity-dependent manner [49].…”

“…In a different study regarding the function of UL36USP, it was found that UL36USP inhibits cGAS and STING dependent IFN-β production [49]. NF-κB activation from overexpressing STING, TBK1, IKKα and IKKβ was also inhibited, but not from overexpressing p65 [49].…”

Emerging drug development technologies targeting ubiquitination for cancer therapeutics

“…In order to promote infection and replication in host cells, while evading the immune response, many human pathogenic viruses have evolved the ability to hijack and manipulate the UPS (Luo, 2016). For example, the Ub-specific protease of the herpes simplex virus 1 antagonizes NF-κB activation, whereas the E6 oncoprotein of the human papillomavirus recruits host E3 ligases to promote Ub-dependent degradation of p53 and consequent carcinogenesis (Hoppe-Seyler, Bossler, Braun, Herrmann, & Hoppe-Seyler, 2018;Ye, Su, Xu, & Zheng, 2017). We developed highly selective and potent UbVs targeting the DUBs of the Middle East respiratory syndrome coronavirus (MERS-CoV) and the Crimean-Congo hemorrhagic fever virus (CCHFV), two emergent and globally important viruses (W. Zhang, Bailey-Elkin, et al, 2017).…”

The Role of Nucleic Acid Sensing in Controlling Microbial and Autoimmune Disorders

“…Through a variety of mechanisms, several DNA viruses such as adenovirus, herpes simplex virus (HSV)-1, and human papilloma virus (HPV) have been shown to induce or actively inhibit a STING-dependent type I IFN response (Anghelina et al, 2016; Ishikawa et al, 2009; Lam and Falck-Pedersen, 2014; Lam et al, 2014; Liang et al, 2015; Sunthamala et al, 2014). For example, over a dozen proteins of HSV-1 have been found to actively suppress cytosolic-DNA recognition by the cGAS/STING pathway (Christensen et al, 2016; Horan et al, 2013; Ishikawa et al, 2009; Kalamvoki and Roizman, 2014; Su and Zheng, 2017; Xu et al, 2017; Ye et al, 2017; Zhang et al, 2016; Zheng, 2018). Similarly, the E2 proteins of HPV16 inhibit the transcription of different ISGs by targeting STING (Sunthamala et al, 2014).…”