Inhibition of AIM2 inflammasome activation by a novel transcript isoform of IFI16.

Mouse p202 is a disease locus for lupus and a dominant-negative inhibitor of AIM2 inflammasome activation. A human homolog of p202 has not been identified so far. Here, we report a novel transcript isoform of human IFI16-designated IFI16-ß, which has a domain architecture similar to that of mouse p202. Like p202, IFI16-ß contains two HIN domains, but lacks the pyrin domain. IFI16-ß is ubiquitously expressed in various human tissues and cells. Its mRNA levels are also elevated in leukocytes of patients with lupus, virus-infected cells, and cells treated with interferon-ß or phorbol ester. IFI16-ß co-localizes with AIM2 in the cytoplasm, whereas IFI16-α is predominantly found in the nucleus. IFI16-ß interacts with AIM2 to impede the formation of a functional AIM2-ASC complex. In addition, IFI16-ß sequesters cytoplasmic dsDNA and renders it unavailable for AIM2 sensing. Enforced expression of IFI16-ß inhibits the activation of AIM2 inflammasome, whereas knockdown of IFI16-ß augments interleukin-1ß secretion triggered by dsDNA but not dsRNA Thus, cytoplasm-localized IFI16-ß is functionally equivalent to mouse p202 that exerts an inhibitory effect on AIM2 inflammasome.

A Screen for Epstein-Barr Virus Proteins That Inhibit the DNA Damage Response Reveals a Novel Histone Binding Protein.

To replicate and persist in human cells, linear double-stranded DNA (dsDNA) viruses, such as Epstein-Barr virus (EBV), must overcome the host DNA damage response (DDR) that is triggered by the viral genomes. Since this response is necessary to maintain cellular genome integrity, its inhibition by EBV is likely an important factor in the development of cancers associated with EBV infection, including gastric carcinoma. Here we present the first extensive screen of EBV proteins that inhibit dsDNA break signaling. We identify the BKRF4 tegument protein as a DDR inhibitor that interferes with histone ubiquitylation at dsDNA breaks and recruitment of the RNF168 histone ubiquitin ligase. We further show that BKRF4 binds directly to histones through an acidic domain that targets BKRF4 to cellular chromatin and is sufficient to inhibit dsDNA break signaling. BKRF4 transcripts were detected in EBV-positive gastric carcinoma cells (AGS-EBV), and these increased in lytic infection. Silencing of BKRF4 in both latent and lytic AGS-EBV cells (but not in EBV-negative AGS cells) resulted in increased dsDNA break signaling, confirming a role for BKRF4 in DDR inhibition in the context of EBV infection and suggesting that BKRF4 is expressed in latent cells. BKRF4 was also found to be consistently expressed in EBV-positive gastric tumors in the absence of a full lytic infection. The results suggest that BKRF4 plays a role in inhibiting the cellular DDR in latent and lytic EBV infection and that the resulting accumulation of DNA damage might contribute to development of gastric carcinoma.IMPORTANCE Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including ∼10% of gastric carcinomas. EBV encodes ∼80 proteins, many of which are believed to manipulate cellular regulatory pathways but are poorly characterized. The DNA damage response (DDR) is one such pathway that is critical for maintaining genome integrity and preventing cancer-associated mutations. In this study, a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and blocks their ubiquitylation at the DNA damage sites. We also present evidence that BKRF4 is expressed in both latent and lytic forms of EBV infection, where it downregulates the DDR, as well as in EBV-positive gastric tumors. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR.

A novel transcript isoform of STING that sequesters cGAMP and dominantly inhibits innate nucleic acid sensing.

STING is a core adaptor in innate nucleic acid sensing in mammalian cells, on which different sensing pathways converge to induce type I interferon (IFN) production. Particularly, STING is activated by 2'3'-cGAMP, a cyclic dinucleotide containing mixed phosphodiester linkages and produced by cytoplasmic DNA sensor cGAS. Here, we reported on a novel transcript isoform of STING designated STING-ß that dominantly inhibits innate nucleic acid sensing. STING-ß without transmembrane domains was widely expressed at low levels in various human tissues and viral induction of STING-ß correlated inversely with IFN-ß production. The expression of STING-ß declined in patients with lupus, in which type I IFNs are commonly overproduced. STING-ß suppressed the induction of IFNs, IFN-stimulated genes and other cytokines by various immunostimulatory agents including cyclic dinucleotides, DNA, RNA and viruses, whereas depletion of STING-ß showed the opposite effect. STING-ß interacted with STING-α and antagonized its antiviral function. STING-ß also interacted with TBK1 and prevented it from binding with STING-α, TRIF or other transducers. In addition, STING-ß bound to 2'3'-cGAMP and impeded its binding with and activation of STING-α, leading to suppression of IFN-ß production. Taken together, STING-ß sequesters 2'3'-cGAMP second messenger and other transducer molecules to inhibit innate nucleic acid sensing dominantly.

PACT Facilitates RNA-Induced Activation of MDA5 by Promoting MDA5 Oligomerization.

MDA5 is a RIG-I-like cytoplasmic sensor of dsRNA and certain RNA viruses, such as encephalomyocarditis virus, for the initiation of the IFN signaling cascade in the innate antiviral response. The affinity of MDA5 toward dsRNA is low, and its activity becomes optimal in the presence of unknown cellular coactivators. In this article, we report an essential coactivator function of dsRNA-binding protein PACT in mediating the MDA5-dependent type I IFN response. Virus-induced and polyinosinic-polycytidylic acid-induced activation of MDA5 were severely impaired in PACT-knockout cells and attenuated in PACT-knockdown cells, but they were potentiated when PACT was overexpressed. PACT augmented IRF3-dependent type I IFN production subsequent to dsRNA-induced activation of MDA5. In contrast, PACT had no influence on MDA5-mediated activation of NF-κB. PACT required dsRNA interaction for its action on MDA5 and promoted dsRNA-induced oligomerization of MDA5. PACT had little stimulatory effect on MDA5 mutants deficient for oligomerization and filament assembly. PACT colocalized with MDA5 in the cytoplasm and potentiated MDA5 recruitment to the dsRNA ligand. Taken together, these findings suggest that PACT functions as an essential cellular coactivator of RIG-I, as well as MDA5, and it facilitates RNA-induced formation of MDA5 oligomers.

PACT- and RIG-I-Dependent Activation of Type I Interferon Production by a Defective Interfering RNA Derived from Measles Virus Vaccine.

UNLABELLED: The live attenuated measles virus vaccine is highly immunostimulatory. Identification and characterization of its components that activate the innate immune response might provide new strategies and agents for the rational design and development of chemically defined adjuvants. In this study, we report on the activation of type I interferon (IFN) production by a defective interfering (DI) RNA isolated from the Hu-191 vaccine strain of measles virus. We found that the Hu-191 virus induced IFN-ß much more potently than the Edmonston strain. In the search for IFN-inducing species in Hu-191, we identified a DI RNA specifically expressed by this strain. This DI RNA, which was of the copy-back type, was predicted to fold into a hairpin structure with a long double-stranded stem region of 206 bp, and it potently induced the expression of IFN-ß. Its IFN-ß-inducing activity was further enhanced when both cytoplasmic RNA sensor RIG-I and its partner, PACT, were overexpressed. On the contrary, this activity was abrogated in cells deficient in PACT or RIG-I. The DI RNA was found to be associated with PACT in infected cells. In addition, both the 5'-di/triphosphate end and the double-stranded stem region on the DI RNA were essential for its activation of PACT and RIG-I. Taken together, our findings support a model in which a viral DI RNA is sensed by PACT and RIG-I to initiate an innate antiviral response. Our work might also provide a foundation for identifying physiological PACT ligands and developing novel adjuvants or antivirals. IMPORTANCE: The live attenuated measles virus vaccine is one of the most successful human vaccines and has largely contained the devastating impact of a highly contagious virus. Identifying the components in this vaccine that stimulate the host immune response and understanding their mechanism of action might help to design and develop better adjuvants, vaccines, antivirals, and immunotherapeutic agents. We identified and characterized a defective interfering RNA from the Hu-191 vaccine strain of measles virus which has safely been used in millions of people for many years. We further demonstrated that this RNA potently induces an antiviral immune response through cellular sensors of viral RNA known as PACT and RIG-I. Similar types of viral RNA that bind with and activate PACT and RIG-I might retain the immunostimulatory property of measles virus vaccines but would not induce adaptive immunity. They are potentially useful as chemically defined vaccine adjuvants, antivirals, and immunostimulatory agents.

CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

MIP-T3 is a negative regulator of innate type I IFN response.

TNFR-associated factor (TRAF) 3 is an important adaptor that transmits upstream activation signals to protein kinases that phosphorylate transcription factors to induce the production of type I IFNs, the important effectors in innate antiviral immune response. MIP-T3 interacts specifically with TRAF3, but its function in innate IFN response remains unclear. In this study, we demonstrated a negative regulatory role of MIP-T3 in type I IFN production. Overexpression of MIP-T3 inhibited RIG-I-, MDA5-, VISA-, TBK1-, and IKKε-induced transcriptional activity mediated by IFN-stimulated response elements and IFN-ß promoter. MIP-T3 interacted with TRAF3 and perturbed in a dose-dependent manner the formation of functional complexes of TRAF3 with VISA, TBK1, IKKε, and IFN regulatory factor 3. Consistent with this finding, retinoic acid-inducible gene I- and TBK1-induced phosphorylation of IFN regulatory factor 3 was significantly diminished when MIP-T3 was overexpressed. Depletion of MIP-T3 facilitated Sendai virus-induced activation of IFN production and attenuated the replication of vesicular stomatitis virus. In addition, MIP-T3 was found to be dissociated from TRAF3 during the course of Sendai virus infection. Our findings suggest that MIP-T3 functions as a negative regulator of innate IFN response by preventing TRAF3 from forming protein complexes with critical downstream transducers and effectors.