Targeted demethylation and activation of NLRC5 augment cancer immunogenicity through MHC class I.

Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer.

NLRC5/MHC class I transactivator: A key target for immune escape by SARS-CoV-2.

Antigen presentation to CD8+ T cells by MHC class I molecules is essential for host defense against viral infections. Various mechanisms have evolved in multiple viruses to escape immune surveillance and defense to support viral proliferation in host cells. Through in vitro SARS-CoV-2 infection studies and analysis of COVID-19 patient samples, we found that SARS-CoV-2 suppresses the induction of the MHC class I pathway by inhibiting the expression and function of NLRC5, a major transcriptional regulator of MHC class I genes. In this review, we discuss the molecular mechanisms for suppression of the MHC class I pathway and clinical implications for COVID-19.

FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA.

Major histocompatibility complex (MHC) class I and II molecules play critical roles in the activation and regulation of adaptive immunity through antigen presentation to CD8+ and CD4+ T cells, respectively. Strict regulation of MHC expression is critical for proper immune responses. CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, is a master regulator of MHC class II (MHC-II) gene transcription. Although it has been known that CIITA activity is regulated at the transcriptional and protein levels, the mechanism to determine CIITA protein level has not been elucidated. Here, we show that FBXO11 is a bona fide E3 ligase of CIITA and regulates CIITA protein level through ubiquitination-mediated degradation. A nonbiased proteomic approach for CIITA-binding protein identified FBXO11, a member of the Skp1-Cullin-1-F-box E3 ligase complex, as a binding partner of CIITA but not MHC class I transactivator, NLRC5. The cycloheximide chase assay showed that the half-life of CIITA is mainly regulated by FBXO11 via the ubiquitin-proteasome system. The expression of FBXO11 led to the reduced MHC-II at the promoter activity level, transcriptional level, and surface expression level through downregulation of CIITA. Moreover, human and mouse FBXO11-deficient cells display increased levels of MHC-II and related genes. In normal and cancer tissues, FBXO11 expression level is negatively correlated with MHC-II. Interestingly, the expression of FBXO11, along with CIITA, is associated with prognosis of cancer patients. Therefore, FBXO11 is a critical regulator to determine the level of MHC-II, and its expression may serve as a biomarker for cancer.

The balance between nuclear import and export of NLRC5 regulates MHC class I transactivation.

Major histocompatibility complex (MHC) class I molecules play an essential role in regulating the adaptive immune system by presenting antigens to CD8 T cells. CITA (MHC class I transactivator), also known as NLRC5 (NLR family, CARD domain-containing 5), regulates the expression of MHC class I and essential components involved in the MHC class I antigen presentation pathway. While the critical role of the nuclear distribution of NLRC5 in its transactivation activity has been known, the regulatory mechanism to determine the nuclear localization of NLRC5 remains poorly understood. In this study, a comprehensive analysis of all domains in NLRC5 revealed that the regulatory mechanisms for nuclear import and export of NLRC5 coexist and counterbalance each other. Moreover, GCN5 (general control non-repressed 5 protein), a member of HATs (histone acetyltransferases), was found to be a key player to retain NLRC5 in the nucleus, thereby contributing to the expression of MHC class I. Therefore, the balance between import and export of NLRC5 has emerged as an additional regulatory mechanism for MHC class I transactivation, which would be a potential therapeutic target for the treatment of cancer and virus-infected diseases.

MHC class I transactivator NLRC5 in host immunity, cancer and beyond.

The presentation of antigenic peptides by major histocompatibility complex (MHC) class I and class II molecules is crucial for activation of the adaptive immune system. The nucleotide-binding domain and leucine-rich repeat receptor family members CIITA and NLRC5 function as the major transcriptional activators of MHC class II and class I gene expression, respectively. Since the identification of NLRC5 as the master regulator of MHC class I and class-I-related genes, there have been major advances in understanding the function of NLRC5 in infectious diseases and cancer. Here, we discuss the biological significance and mechanism of NLRC5-dependent MHC class I expression.

Interferon stimulation creates chromatin marks and establishes transcriptional memory.

Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNß stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of ∼2,000 IFNß-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.

DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.

RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).

Retinoic acid-inducible gene I-inducible miR-23b inhibits infections by minor group rhinoviruses through down-regulation of the very low density lipoprotein receptor.

In mammals, viral infections are detected by innate immune receptors, including Toll-like receptor and retinoic acid inducible gene I (RIG-I)-like receptor (RLR), which activate the type I interferon (IFN) system. IFN essentially activates genes encoding antiviral proteins that inhibit various steps of viral replication as well as facilitate the subsequent activation of acquired immune responses. In this study, we investigated the expression of non-coding RNA upon viral infection or RLR activation. Using a microarray, we identified several microRNAs (miRNA) specifically induced to express by RLR signaling. As suggested by Bioinformatics (miRBase Target Data base), one of the RLR-inducible miRNAs, miR-23b, actually knocked down the expression of very low density lipoprotein receptor (VLDLR) and LDLR-related protein 5 (LRP5). Transfection of miR-23b specifically inhibited infection of rhinovirus 1B (RV1B), which utilizes the low density lipoprotein receptor (LDLR) family for viral entry. Conversely, introduction of anti-miRNA-23b enhanced the viral yield. Knockdown experiments using small interfering RNA (siRNA) revealed that VLDLR, but not LRP5, is critical for an efficient infection by RV1B. Furthermore, experiments with the transfection of infectious viral RNA revealed that miR-23b did not affect post-entry viral replication. Our results strongly suggest that RIG-I signaling results in the inhibitions of infections of RV1B through the miR-23b-mediated down-regulation of its receptor VLDLR.

SARS-CoV-2 inhibits induction of the MHC class I pathway by targeting the STAT1-IRF1-NLRC5 axis.

The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5.

Production of Antiviral Substance from Sugarcane Bagasse by Chemical Alteration of its Native Lignin Structure through Microwave Solvolysis.

The production of bioactive agents from lignocelluloses has received limited attention because plant cell walls are essentially non-bioactive. In this study, a chemical reaction is reported, which produces a lignin-derived antiviral substance from sugarcane bagasse by microwave heating at 200 °C in aqueous glycerol containing 0.5 % H2 SO4 . The purified fraction, designated as FR200 , strongly inhibited the replication of encephalomyocarditis virus (EMCV) in L929 cells without cytotoxicity. HSQC NMR spectra demonstrated that the principal interunit linkages in the native lignin were cleaved by the reaction. Gel permeation chromatography (GPC) and pyrolysis-GCMS revealed that FR200 is composed of oligomeric lignin with a weight average molecular weight of approximately 2000. When the bagasse was reacted at lower temperatures, 140 °C and 160 °C, the native lignin substructures were partially retained and the antiviral activity significantly decreased. The results thus indicate that the antiviral activity emerged through severe alteration of the native lignin structure. Furthermore, it was revealed that the antiviral lignin inactivated the EMCV virions through direct contact, as the innate immune system of L929 was not activated by FR200 treatment, and no antiviral activity was found when L929 was pre-treated with the lignin before viral infection.

ZFR coordinates crosstalk between RNA decay and transcription in innate immunity.

Control of type I interferon production is crucial to combat infection while preventing deleterious inflammatory responses, but the extent of the contribution of post-transcriptional mechanisms to innate immune regulation is unclear. Here, we show that human zinc finger RNA-binding protein (ZFR) represses the interferon response by regulating alternative pre-mRNA splicing. ZFR expression is tightly controlled during macrophage development; monocytes express truncated ZFR isoforms, while macrophages induce full-length ZFR to modulate macrophage-specific alternative splicing. Interferon-stimulated genes are constitutively activated by ZFR depletion, and immunostimulation results in hyper-induction of interferon ß (IFNß/IFNB1). Through whole-genome analyses, we show that ZFR controls interferon signaling by preventing aberrant splicing and nonsense-mediated decay of histone variant macroH2A1/H2AFY mRNAs. Together, our data suggest that regulation of ZFR in macrophage differentiation guards against aberrant interferon responses and reveal a network of mRNA processing and decay that shapes the transcriptional response to infection.

Structure-dependent antiviral activity of catechol derivatives in pyroligneous acid against the encephalomycarditis virus.

The pyrolysis product, wood vinegar (WV), from Japanese larch exhibited strong antiviral activity against the encephalomycarditis virus (EMCV). Catechol, 3-methyl-, 4-methyl-, 4-ethyl-, and 3-methoxycatechol, and 2-methyl-1,4-benzenediol were identified as the major antiviral compounds. The viral inhibition ability of these compounds was affected by the structure and position of the substituent group attached to the aromatic skeleton. The IC50 of catechol was 0.67 mg mL-1 and those of its derivatives were <0.40 mg mL-1. Methyl and ethyl substitution in the para position relative to a hydroxyl group obviously increased the antiviral activities. The mode of antiviral action was investigated by adding catechol derivatives at different times of the viral life cycle. It was found that direct inactivations of EMCV by these compounds were the major pathway for the antiviral activity. The effect of catechol derivatives on the host immune system was studied by quantification of Il6 and Ifnb1 expression levels. Increased Il6 expression levels indicate NF-κB activation by reactive oxygen species from auto-oxidations of catechol derivatives, which is also a possible antiviral route. The present research provides indices for production of potent antiviral agents form lignocellulose biomass.

SPT6 interacts with NSD2 and facilitates interferon-induced transcription.

The role of the histone chaperone SPT6 in mammalian cells is not fully understood. Here, we investigated the involvement of SPT6 in type I interferon (IFN)-induced transcription in murine fibroblasts. In RNA-seq analysis, Spt6 siRNA attenuates about half of ~ 200 IFN-stimulated genes (ISGs), while not affecting housekeeping genes. ISGs with high mRNA induction are more susceptible to Spt6 siRNA than those with lower levels of induction. ChIP analysis shows that SPT6 is recruited to highly inducible, Spt6 siRNA-sensitive ISGs, but not to other siRNA-insensitive ISGs. Furthermore, SPT6 recruitment is abrogated in cells lacking the histone methyltransferase NSD2. In co-IP experiments, SPT6 interacts with NSD2. In summary, SPT6 facilitates IFN-induced transcription, highlighting its critical role in gene activation.