Herpes Simplex Virus 1 UL24 Abrogates the DNA Sensing Signal Pathway by Inhibiting NF-κB Activation.

Cyclic GMP-AMP synthase (cGAS) is a newly identified DNA sensor that recognizes foreign DNA, including the genome of herpes simplex virus 1 (HSV-1). Upon binding of viral DNA, cGAS produces cyclic GMP-AMP, which interacts with and activates stimulator of interferon genes (STING) to trigger the transcription of antiviral genes such as type I interferons (IFNs), and the production of inflammatory cytokines. HSV-1 UL24 is widely conserved among members of the herpesviruses family and is essential for efficient viral replication. In this study, we found that ectopically expressed UL24 could inhibit cGAS-STING-mediated promoter activation of IFN-β and interleukin-6 (IL-6), and UL24 also inhibited interferon-stimulatory DNA-mediated IFN-β and IL-6 production during HSV-1 infection. Furthermore, UL24 selectively blocked nuclear factor κB (NF-κB) but not IFN-regulatory factor 3 promoter activation. Coimmunoprecipitation analysis demonstrated that UL24 bound to the endogenous NF-κB subunits p65 and p50 in HSV-1-infected cells, and UL24 was also found to bind the Rel homology domains (RHDs) of these subunits. Furthermore, UL24 reduced the tumor necrosis factor alpha (TNF-α)-mediated nuclear translocation of p65 and p50. Finally, mutational analysis revealed that the region spanning amino acids (aa) 74 to 134 of UL24 [UL24(74-134)] is responsible for inhibiting cGAS-STING-mediated NF-κB promoter activity. For the first time, UL24 was shown to play an important role in immune evasion during HSV-1 infection.IMPORTANCE NF-κB is a critical component of the innate immune response and is strongly induced downstream of most pattern recognition receptors (PRRs), leading to the production of IFN-β as well as a number of inflammatory chemokines and interleukins. To establish persistent infection, viruses have evolved various mechanisms to counteract the host NF-κB pathway. In the present study, for the first time, HSV-1 UL24 was demonstrated to inhibit the activation of NF-κB in the DNA sensing signal pathway via binding to the RHDs of the NF-κB subunits p65 and p50 and abolishing their nuclear translocation.

Xu H ，Su C ，Pearson A ，Mody CH ，Zheng C ... -  《-》

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

The DNA sensing pathway triggers innate immune responses against DNA virus infection, and NF-κB signaling plays a critical role in establishing innate immunity. We report here that the herpes simplex virus 1 (HSV-1) ubiquitin-specific protease (UL36USP), which is a deubiquitinase (DUB), antagonizes NF-κB activation, depending on its DUB activity. In this study, ectopically expressed UL36USP blocked promoter activation of beta interferon (IFN-β) and NF-κB induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). UL36USP restricted NF-κB activation mediated by overexpression of STING, TANK-binding kinase 1, IκB kinase α (IKKα), and IKKβ, but not p65. UL36USP was also shown to inhibit IFN-stimulatory DNA-induced IFN-β and NF-κB activation under conditions of HSV-1 infection. Furthermore, UL36USP was demonstrated to deubiquitinate IκBα and restrict its degradation and, finally, abrogate NF-κB activation. More importantly, the recombinant HSV-1 lacking UL36USP DUB activity, denoted as C40A mutant HSV-1, failed to cleave polyubiquitin chains on IκBα. For the first time, UL36USP was shown to dampen NF-κB activation in the DNA sensing signal pathway to evade host antiviral innate immunity.IMPORTANCE It has been reported that double-stranded-DNA-mediated NF-κB 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 gene-encoded protein contains the DUB domain and is conserved across all herpesviruses. This study demonstrates that UL36USP abrogates NF-κB activation by cleaving polyubiquitin chains from IκBα and therefore restricts proteasome-dependent degradation of IκBα 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-κB signaling.

Ye R ，Su C ，Xu H ，Zheng C ... -  《-》

Herpes Simplex Virus 1 Serine Protease VP24 Blocks the DNA-Sensing Signal Pathway by Abrogating Activation of Interferon Regulatory Factor 3.

The interferon (IFN)-mediated antiviral response is a central aspect of host defense; however, viruses have evolved multiple strategies to counteract IFN-mediated responses in order to successfully infect the host. Herpes simplex virus 1 (HSV-1), a typical human-restricted DNA virus, is capable of counteracting host immune responses via several distinct viral proteins, thus establishing a lifelong latent infection. In this study, we demonstrate that the VP24 protein, a serine protease of HSV-1 essential for the formation and maturation of capsids, is a novel antagonist of the beta interferon (IFN-β) pathway. Here, VP24 was shown for the first time to dampen interferon stimulatory DNA (ISD)-triggered IFN-β production and inhibit IFN-β promoter activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) and by STING, respectively. Further study demonstrated that ectopic expression of VP24 selectively blocked IFN regulatory factor 3 (IRF3) but not NF-κB promoter activation. In addition, VP24 was demonstrated to downregulate ISD-induced phosphorylation and dimerization of IRF3 during HSV-1 infection with a VP24 stable knockdown human foreskin fibroblast cell line. The underlying molecular mechanism is that VP24 abrogates the interaction between TANK-binding kinase 1 (TBK1) and IRF3, hence impairing IRF3 activation. These results illustrate that VP24 is able to block the production of IFN-β by inhibiting IRF3 activation, which may represent a critical adaptation to enable viral effective replication within the host. This study demonstrated that HSV-1 protein VP24 could inhibit IFN-β production and promoter activation triggered by ISD, cGAS and STING and by STING, respectively. VP24 selectively blocked IRF3 promoter activation and ISD-induced phosphorylation and dimerization of IRF3 without affecting the NF-κB promoter activation during viral infection. VP24 also inhibited IRF3 activation by impeding the interaction between TBK1 and IRF3 during viral infection. This study provides new insights into the immune evasion mediated by HSV-1 and identifies VP24 as a crucial effector for HSV-1 to evade the host DNA-sensing signal pathway.

Zhang D ，Su C ，Zheng C 《-》

Marek's Disease Virus RLORF4 Inhibits Type I Interferon Production by Antagonizing NF-κB Activation.

Marek's disease virus (MDV), which causes T cell lymphomas in chickens, is economically important and has contributed to knowledge of herpesvirus-associated oncogenicity. The DNA-sensing pathway induces innate immune responses against DNA virus infection, and nuclear factor κB (NF-κB) signaling is critical for the establishment of innate immunity. Here, we report that RLORF4, an MDV-specific protein directly involved in viral attenuation, is an inhibitor of the DNA-sensing pathway. The results showed that ectopically expressed RLORF4 blocked beta interferon (IFN-β) promoter activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). RLORF4 selectively inhibited the activation of NF-κB but not IFN-regulatory factor 7. RLORF4 was found to bind the endogenous NF-κB subunits p65 and p50, and it also bound to the Rel homology domains of these subunits. Furthermore, RLORF4 suppressed the nuclear translocation of p65 and p50 mediated by tumor necrosis factor alpha and interferon-stimulatory DNA. Finally, deletion of RLORF4 from the MDV genome promoted IFN-β and interleukin-6 (IL-6) production in vitro and in vivo In the absence of RLORF4, the host cellular immunity was significantly increased, and reduced viral titers were observed during infection of chickens. Our results suggest that the RLORF4-mediated suppression of the host antiviral innate immunity might play an important role in MDV pathogenesis.IMPORTANCE Marek's disease virus (MDV) RLORF4 has been shown to be directly involved in the attenuation of MDV upon serial passages in vitro; however, the exact function of this protein during viral infection was not well characterized. This study demonstrated that RLORF4 significantly inhibits cGAS-STING-mediated NF-κB activation by binding to the Rel homology domains of the NF-κB subunits p65 and p50, interrupting their translocation to the nuclei and thereby inhibiting IFN-β production. Furthermore, RLORF4 deficiency promoted the induction of IFN-β and downstream IFN-stimulated genes during MDV infection in chickens. Our results suggest that the contribution of RLORF4 to MDV virulence may stem from its inhibition of viral DNA-triggered IFN-β responses.

Liu Y ，Gao L ，Xu Z ，Luo D ，Zhang Y ，Gao Y ，Liu C ，Zhang Y ，Qi X ，Cui H ，Li K ，Wang X ... -  《-》

Herpes simplex virus 1-encoded tegument protein VP16 abrogates the production of beta interferon (IFN) by inhibiting NF-κB activation and blocking IFN regulatory factor 3 to recruit its coactivator CBP.

Xing J ，Ni L ，Wang S ，Wang K ，Lin R ，Zheng C ... -  《-》