African Swine Fever Virus Cysteine Protease pS273R Inhibits Type I Interferon Signaling by Mediating STAT2 Degradation.

African swine fever virus (ASFV) is a large DNA virus that causes African swine fever (ASF), an acute and hemorrhagic disease in pigs with lethality rates of up to 100%. To date, how ASFV efficiently suppress the innate immune response remains enigmatic. In this study, we identified ASFV cysteine protease pS273R as an antagonist of type I interferon (IFN). Overexpression of pS273R inhibited JAK-STAT signaling triggered by type I IFNs. Mechanistically, pS273R interacted with STAT2 and recruited the E3 ubiquitin ligase DCST1, resulting in K48-linked polyubiquitination at K55 of STAT2 and subsequent proteasome-dependent degradation of STAT2. Furthermore, such a function of pS273R in JAK-STAT signaling is not dependent on its protease activity. These findings suggest that ASFV pS273R is important to evade host innate immunity. IMPORTANCE ASF is an acute disease in domestic pigs caused by infection with ASFV. ASF has become a global threat with devastating economic and ecological consequences. To date, there are no commercially available, safe, and efficacious vaccines to prevent ASFV infection. ASFV has evolved a series of strategies to evade host immune responses, facilitating its replication and transmission. Therefore, understanding the immune evasion mechanism of ASFV is helpful for the development of prevention and control measures for ASF. Here, we identified ASFV cysteine protease pS273R as an antagonist of type I IFNs. ASFV pS273R interacted with STAT2 and mediated degradation of STAT2, a transcription factor downstream of type I IFNs that is responsible for induction of various IFN-stimulated genes. pS273R recruited the E3 ubiquitin ligase DCST1 to enhance K48-linked polyubiquitination of STAT2 at K55 in a manner independent of its protease activity. These findings suggest that pS273R is important for ASFV to escape host innate immunity, which sheds new light on the mechanisms of ASFV immune evasion.

African swine fever virus I267L acts as an important virulence factor by inhibiting RNA polymerase III-RIG-I-mediated innate immunity.

ASFV is a large DNA virus that is highly pathogenic in domestic pigs. How this virus is sensed by the innate immune system as well as why it is so virulent remains enigmatic. In this study, we show that the ASFV genome contains AT-rich regions that are recognized by the DNA-directed RNA polymerase III (Pol-III), leading to viral RNA sensor RIG-I-mediated innate immune responses. We further show that ASFV protein I267L inhibits RNA Pol-III-RIG-I-mediated innate antiviral responses. I267L interacts with the E3 ubiquitin ligase Riplet, disrupts Riplet-RIG-I interaction and impairs Riplet-mediated K63-polyubiquitination and activation of RIG-I. I267L-deficient ASFV induces higher levels of interferon-ß, and displays compromised replication both in primary macrophages and pigs compared with wild-type ASFV. Furthermore, I267L-deficiency attenuates the virulence and pathogenesis of ASFV in pigs. These findings suggest that ASFV I267L is an important virulence factor by impairing innate immune responses mediated by the RNA Pol-III-RIG-I axis.

Ubiquitination of TLR3 by TRIM3 signals its ESCRT-mediated trafficking to the endolysosomes for innate antiviral response.

Trafficking of toll-like receptor 3 (TLR3) from the endoplasmic reticulum (ER) to endolysosomes and its subsequent proteolytic cleavage are required for it to sense viral double-stranded RNA (dsRNA) and trigger antiviral response, yet the underlying mechanisms remain enigmatic. We show that the E3 ubiquitin ligase TRIM3 is mainly located in the Golgi apparatus and transported to the early endosomes upon stimulation with the dsRNA analog poly(I:C). TRIM3 mediates K63-linked polyubiquitination of TLR3 at K831, which is enhanced following poly(I:C) stimulation. The polyubiquitinated TLR3 is recognized and sorted by the ESCRT (endosomal sorting complex required for transport) complexes to endolysosomes. Deficiency of TRIM3 impairs TLR3 trafficking from the Golgi apparatus to endosomes and its subsequent activation. Trim3-/- cells and mice express lower levels of antiviral genes and show lower levels of inflammatory response following poly(I:C) but not lipopolysaccharide (LPS) stimulation. These findings suggest that TRIM3-mediated polyubiquitination of TLR3 represents a feedback-positive regulatory mechanism for TLR3-mediated innate immune and inflammatory responses.

RNF152 positively regulates TLR/IL-1R signaling by enhancing MyD88 oligomerization.

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) that are critical for the defense against invading pathogens. IL-1ß is an important pro-inflammatory cytokine that also plays pivotal roles in shaping the adaptive immune response. TLRs and interleukin-1 receptor (IL-1R) share similar cytosolic domains and signaling processes. In this study, we identify the E3 ubiquitin ligase RNF152 as a positive regulator of TLR/IL-1R-mediated signaling. Overexpression of RNF152 potentiates IL-1ß- and LPS-induced NF-κB activation in an ubiquitination-independent manner, whereas knockdown of RNF152 has the opposite effects. RNF152-deficient mice produce less inflammatory cytokines in response to LPS and are more resistant to LPS-induced lethal endotoxemia. Mechanistically, RNF152 interacts with the adaptor protein MyD88 and enhances oligomerization of MyD88, which is essential for the recruitment of downstream signaling components and activation of TLR/IL-1R-mediated signal transduction. Our findings suggest that RNF152-mediated oligomerization of MyD88 is important for TLR/IL-1R-mediated inflammatory response.

YIPF5 Is Essential for Innate Immunity to DNA Virus and Facilitates COPII-Dependent STING Trafficking.

STING plays central roles in the innate immune response to pathogens that contain DNA. Sensing cytoplasmic DNA by cyclic GMP-AMP synthase produces cyclic GMP-AMP, which binds to and activates STING and induces STING translocation from the endoplasmic reticulum to the perinuclear microsome. However, this trafficking process has not been fully elucidated yet. In this study, we identified YIPF5 as a positive regulator of STING trafficking. YIPF5 is essential for DNA virus- or intracellular DNA-triggered production of type I IFNs. Consistently, knockdown of YIPF5 impairs cellular antiviral responses to DNA virus. Mechanistically, YIPF5 interacts with both STING and components of COPII, facilitating STING recruitment to COPII in the presence of cytoplasmic dsDNA. Furthermore, knockdown of components of COPII inhibits DNA virus-triggered production of type I IFNs, suggesting that COPII is involved in innate immune responses to DNA viruses. Collectively, our findings demonstrate that YIPF5 positively regulates STING-mediated innate immune responses by recruiting STING to COPII-coated vesicles and facilitating STING trafficking from the endoplasmic reticulum to Golgi, providing important insights into the molecular mechanisms of intracellular DNA-stimulated STING trafficking and activation.

FAM64A positively regulates STAT3 activity to promote Th17 differentiation and colitis-associated carcinogenesis.

STAT3 is a transcription factor that plays central roles in various physiological processes, including differentiation of Th cells. Its deregulation results in serious diseases, including inflammatory diseases and cancer. The mechanisms related to how STAT3 activity is regulated remain enigmatic. Here we show that overexpression of FAM64A potentiates IL-6-induced activation of STAT3 and expression of downstream target genes, whereas deficiency of FAM64A has the opposite effects. FAM64A interacts with STAT3 in the nucleus and regulates binding of STAT3 to the promoters of its target genes. Deficiency of Fam64a significantly impairs differentiation of Th17 but not Th1 or induced regulatory T cells (iTreg). In addition, Fam64a deficiency attenuates experimental autoimmune encephalomyelitis (EAE) and dextran sulfate sodium (DSS)-induced colitis, which is correlated with decreased differentiation of Th17 cells and production of proinflammatory cytokines. Furthermore, Fam64a deficiency suppresses azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) in mice. These findings suggest that FAM64A regulates Th17 differentiation and colitis and inflammation-associated cancer by modulating transcriptional activity of STAT3.