The JAK-STAT pathway at 30: Much learned, much more to do.

The discovery of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway arose from investigations of how cells respond to interferons (IFNs), revealing a paradigm in cell signaling conserved from slime molds to mammals. These discoveries revealed mechanisms underlying rapid gene expression mediated by a wide variety of extracellular polypeptides including cytokines, interleukins, and related factors. This knowledge has provided numerous insights into human disease, from immune deficiencies to cancer, and was rapidly translated to new drugs for autoimmune, allergic, and infectious diseases, including COVID-19. Despite these advances, major challenges and opportunities remain.

The Human STAT2 Coiled-Coil Domain Contains a Degron for Zika Virus Interferon Evasion.

The ability of viruses to evade the host antiviral immune system determines their level of replication fitness, species specificity, and pathogenic potential. Flaviviruses rely on the subversion of innate immune barriers, including the type I and type III interferon (IFN) antiviral systems. Zika virus infection induces the degradation of STAT2, an essential component of the IFN-stimulated gene transcription factor ISGF3. The mechanisms that lead to STAT2 degradation by Zika virus are poorly understood, but it is known to be mediated by the viral NS5 protein that binds to STAT2 and targets it for proteasome-mediated destruction. To better understand how NS5 engages and degrades STAT2, functional analysis of the protein interactions that lead to Zika virus and NS5-dependent STAT2 proteolysis were investigated. Data implicate the STAT2 coiled-coil domain as necessary and sufficient for NS5 interaction and proteasome degradation after Zika virus infection. Molecular dissection reveals that the first two α-helices of the STAT2 coiled-coil domain contain a specific targeting region for IFN antagonism. These functional interactions provide a more complete understanding of the essential protein-protein interactions needed for Zika virus evasion of the host antiviral response and identify new targets for antiviral therapeutic approaches. IMPORTANCE Zika virus infection can cause mild fever, rash, and muscle pain and in rare cases can lead to brain or nervous system diseases, including Guillain-Barré syndrome. Infections in pregnant women can increase the risk of miscarriage or serious birth defects, including brain anomalies and microcephaly. There are no drugs or vaccines for Zika disease. Zika virus is known to break down the host antiviral immune response, and this research project reveals how the virus suppresses interferon signaling, and may reveal therapeutic vulnerabilities.

The innate immune sensor LGP2 activates antiviral signaling by regulating MDA5-RNA interaction and filament assembly.

Cytoplasmic pattern recognition receptors detect non-self RNAs during virus infections and initiate antiviral signaling. One receptor, MDA5, possesses essential signaling domains, but weak RNA binding. A second receptor, LGP2, rapidly detects diverse dsRNA species, but lacks signaling domains. Accumulating evidence suggests LGP2 and MDA5 work together to detect viral RNA and generate a complete antiviral response, but the basis for their cooperation has been elusive. Experiments presented here address this gap in antiviral signaling, revealing that LGP2 assists MDA5-RNA interactions leading to enhanced MDA5-mediated antiviral signaling. LGP2 increases the initial rate of MDA5-RNA interaction and regulates MDA5 filament assembly, resulting in the formation of more numerous, shorter MDA5 filaments that are shown to generate equivalent or greater signaling activity in vivo than the longer filaments containing only MDA5. These findings provide a mechanism for LGP2 coactivation of MDA5 and a biological context for MDA5-RNA filaments in antiviral responses.

RNA sensor LGP2 inhibits TRAF ubiquitin ligase to negatively regulate innate immune signaling.

The production of type I interferon (IFN) is essential for cellular barrier functions and innate and adaptive antiviral immunity. In response to virus infections, RNA receptors RIG-I and MDA5 stimulate a mitochondria-localized signaling apparatus that uses TRAF family ubiquitin ligase proteins to activate master transcription regulators IRF3 and NFκB, driving IFN and antiviral target gene expression. Data indicate that a third RNA receptor, LGP2, acts as a negative regulator of antiviral signaling by interfering with TRAF family proteins. Disruption of LGP2 expression in cells results in earlier and overactive transcriptional responses to virus or dsRNA LGP2 associates with the C-terminus of TRAF2, TRAF3, TRAF5, and TRAF6 and interferes with TRAF ubiquitin ligase activity. TRAF interference is independent of LGP2 ATP hydrolysis, RNA binding, or its C-terminal domain, and LGP2 can regulate TRAF-mediated signaling pathways in trans, including IL-1ß, TNFα, and cGAMP These findings provide a unique mechanism for LGP2 negative regulation through TRAF suppression and extend the potential impact of LGP2 negative regulation beyond the IFN antiviral response.

Bioinformatic analysis reveals a pattern of STAT3-associated gene expression specific to basal-like breast cancers in human tumors.

Signal transducer and activator of transcription 3 (STAT3), a latent transcription factor associated with inflammatory signaling and innate and adaptive immune responses, is known to be aberrantly activated in a wide variety of cancers. In vitro analysis of STAT3 in human cancer cell lines has elucidated a number of specific targets associated with poor prognosis in breast cancer. However, to date, no comparison of cancer subtype and gene expression associated with STAT3 signaling in human patients has been reported. In silico analysis of human breast cancer microarray and reverse-phase protein array data was performed to identify expression patterns associated with STAT3 in basal-like and luminal breast cancers. Results indicate clearly identifiable STAT3-regulated signatures common to basal-like breast cancers but not to luminal A or luminal B cancers. Furthermore, these differentially expressed genes are associated with immune signaling and inflammation, a known phenotype of basal-like cancers. These findings demonstrate a distinct role for STAT3 signaling in basal breast cancers, and underscore the importance of considering subtype-specific molecular pathways that contribute to tissue-specific cancers.

The TAR-RNA binding protein is required for immunoresponses triggered by Cardiovirus infection.

LGP2 and MDA5 cooperate to detect viral RNA in the cytoplasm of Picornavirus-infected cells and activate innate immune responses. To further define regulatory components of RNA recognition by LGP2/MDA5, a yeast two-hybrid screen was used to identify LGP2-interacting proteins. The screening has identified the TAR-RNA binding protein (TRBP), which is known to be an essential factor for RNA interference (RNAi). Immuno-precipitation experiments demonstrated that TRBP interacted specifically with LGP2 but not with related RIG-I-like receptors, RIG-I or MDA5. siRNA knockdown experiments indicate that TRBP is important for Cardiovirus-triggered interferon responses, but TRBP is not involved in Sendai virus-triggered interferon response that is mediated mainly by RIG-I. To support functional interaction with LGP2, overexpressed TRBP increased Cardiovirus-triggered interferon promoter activity only when LGP2 and MDA5 are co-expressed but not MDA5 alone. Together, our findings illustrate a possible connection between an RNAi-regulatory factor and antiviral RNA recognition that is specifically required for a branch of the virus induced innate immune response.

A conserved role for human Nup98 in altering chromatin structure and promoting epigenetic transcriptional memory.

The interaction of nuclear pore proteins (Nups) with active genes can promote their transcription. In yeast, some inducible genes interact with the nuclear pore complex both when active and for several generations after being repressed, a phenomenon called epigenetic transcriptional memory. This interaction promotes future reactivation and requires Nup100, a homologue of human Nup98. A similar phenomenon occurs in human cells; for at least four generations after treatment with interferon gamma (IFN-γ), many IFN-γ-inducible genes are induced more rapidly and more strongly than in cells that have not previously been exposed to IFN-γ. In both yeast and human cells, the recently expressed promoters of genes with memory exhibit persistent dimethylation of histone H3 lysine 4 (H3K4me2) and physically interact with Nups and a poised form of RNA polymerase II. However, in human cells, unlike yeast, these interactions occur in the nucleoplasm. In human cells transiently depleted of Nup98 or yeast cells lacking Nup100, transcriptional memory is lost; RNA polymerase II does not remain associated with promoters, H3K4me2 is lost, and the rate of transcriptional reactivation is reduced. These results suggest that Nup100/Nup98 binding to recently expressed promoters plays a conserved role in promoting epigenetic transcriptional memory.

Paramyxovirus V protein interaction with the antiviral sensor LGP2 disrupts MDA5 signaling enhancement but is not relevant to LGP2-mediated RLR signaling inhibition.

The interferon antiviral system is a primary barrier to virus replication triggered upon recognition of nonself RNAs by the cytoplasmic sensors encoded by retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology gene 2 (LGP2). Paramyxovirus V proteins are interferon antagonists that can selectively interact with MDA5 and LGP2 through contact with a discrete helicase domain region. Interaction with MDA5, an activator of antiviral signaling, disrupts interferon gene expression and antiviral responses. LGP2 has more diverse reported roles as both a coactivator of MDA5 and a negative regulator of both RIG-I and MDA5. This functional dichotomy, along with the concurrent interference with both cellular targets, has made it difficult to assess the unique consequences of V protein interaction with LGP2. To directly evaluate the impact of LGP2 interference, MDA5 and LGP2 variants unable to be recognized by measles virus and parainfluenza virus 5 (PIV5) V proteins were tested in signaling assays. Results indicate that interaction with LGP2 specifically prevents coactivation of MDA5 signaling and that LGP2's negative regulatory capacity was not affected. V proteins only partially antagonize RIG-I at high concentrations, and their expression had no additive effects on LGP2-mediated negative regulation. However, conversion of RIG-I to a direct V protein target was accomplished by only two amino acid substitutions that allowed both V protein interaction and efficient interference. These results clarify the unique consequences of MDA5 and LGP2 interference by paramyxovirus V proteins and help resolve the distinct roles of LGP2 in both activation and inhibition of antiviral signal transduction. Importance: Paramyxovirus V proteins interact with two innate immune receptors, MDA5 and LGP2, but not RIG-I. V proteins prevent MDA5 from signaling to the beta interferon promoter, but the consequences of LGP2 targeting are poorly understood. As the V protein targets MDA5 and LGP2 simultaneously, and LGP2 is both a positive and negative regulator of both MDA5 and RIG-I, it has been difficult to evaluate the specific advantages conferred by LGP2 targeting. Experiments with V-insensitive proteins revealed that the primary outcome of LGP2 interference is suppression of its ability to synergize with MDA5. LGP2's negative regulation of MDA5 and RIG-I remains intact irrespective of V protein interaction. Complementary experiments demonstrate that RIG-I can be converted to V protein sensitivity by two amino acid substitutions. These findings clarify the functions of LGP2 as a positive regulator of MDA5 signaling, demonstrate the basis for V-mediated LGP2 targeting, and broaden our understanding of paramyxovirus-host interactions.

MDA5 and LGP2: accomplices and antagonists of antiviral signal transduction.

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral transcriptional response that provides an initial barrier to replication and impacts both innate and adaptive immune responses. Retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) proteins mediate intracellular virus recognition and are activated by viral RNA ligands to induce antiviral signal transduction. While the mechanisms of RIG-I regulation are already well understood, less is known about the more enigmatic melanoma differentiation-associated 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). Emerging evidence suggests that these two RLRs are intimately associated as both accomplices and antagonists of antiviral signal transduction.

LGP2 synergy with MDA5 in RLR-mediated RNA recognition and antiviral signaling.

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling. The RIG-I-like receptor (RLR) proteins are expressed in the cytoplasm of nearly all cells and specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RLR family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All RLRs have the ability to detect virus-derived dsRNA and hydrolyze ATP, but display individual differences in enzymatic activity, intrinsic ability to recognize RNA, and mechanisms of activation. Emerging evidence suggests that MDA5 and RIG-I utilize distinct mechanisms to form oligomeric complexes along dsRNA. Aligning of their signaling domains creates a platform capable of propagating and amplifying antiviral signaling responses. LGP2 with intact ATP hydrolysis is critical for the MDA5-mediated antiviral response, but LGP2 lacks the domains essential for activation of antiviral signaling, leaving the role of LGP2 in antiviral signaling unclear. Recent studies revealed a mechanistic basis of synergy between LGP2 and MDA5 leading to enhanced antiviral signaling. This review briefly summarizes the RLR system, and focuses on the relationship between LGP2 and MDA5, describing in detail how these two proteins work together to detect foreign RNA and generate a fully functional antiviral response.

Pan-cancer analysis of TCGA data reveals notable signaling pathways.

BACKGROUND: A signal transduction pathway (STP) is a network of intercellular information flow initiated when extracellular signaling molecules bind to cell-surface receptors. Many aberrant STPs have been associated with various cancers. To develop optimal treatments for cancer patients, it is important to discover which STPs are implicated in a cancer or cancer-subtype. The Cancer Genome Atlas (TCGA) makes available gene expression level data on cases and controls in ten different types of cancer including breast cancer, colon adenocarcinoma, glioblastoma, kidney renal papillary cell carcinoma, low grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian carcinoma, rectum adenocarcinoma, and uterine corpus endometriod carcinoma. Signaling Pathway Impact Analysis (SPIA) is a software package that analyzes gene expression data to identify whether a pathway is relevant in a given condition. METHODS: We present the results of a study that uses SPIA to investigate all 157 signaling pathways in the KEGG PATHWAY database. We analyzed each of the ten cancer types mentioned above separately, and we perform a pan-cancer analysis by grouping the data for all the cancer types. RESULTS: In each analysis several pathways were found to be markedly more significant than all the other pathways. We call them notable. Research has already established a connection between many of these pathways and the corresponding cancer type. However, some of our discovered pathways appear to be new findings. Altogether there were 37 notable findings in the separate analyses, 26 of them occurred in 7 pathways. These 7 pathways included the 4 notable pathways discovered in the pan-cancer analysis. So, our results suggest that these 7 pathways account for much of the mechanisms of cancer. Furthermore, by looking at the overlap among pathways, we identified possible regions on the pathways where the aberrant activity is occurring. CONCLUSIONS: We obtained 37 notable findings concerning 18 pathways. Some of them appear to be new discoveries. Furthermore, we identified regions on pathways where the aberrant activity might be occurring. We conclude that our results will prove to be valuable to cancer researchers because they provide many opportunities for laboratory and clinical follow-up studies.

Activation of RIG-I-like receptor signal transduction.

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling cascades. The RIG-I-like receptors are cytoplasmic DExD/H box proteins that can specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RIG-I-like receptor family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All of these proteins can bind double-stranded RNA species with varying affinities via their conserved DExD/H box RNA helicase domains and C-terminal regulatory domains. The recognition of foreign RNA by the RLRs activates enzymatic functions and initiates signal transduction pathways resulting in the production of antiviral cytokines and the establishment of a broadly effective cellular antiviral state that protects neighboring cells from infection and triggers innate and adaptive immune systems. The propagation of this signal via the interferon antiviral system has been studied extensively, while the precise roles for enzymatic activities of the RNA helicase domain in antiviral responses are only beginning to be elucidated. Here, current models for RLR ligand recognition and signaling are reviewed.

ATP hydrolysis enhances RNA recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology 2 (LGP2).

Laboratory of genetics and physiology 2 (LGP2) is a member of the RIG-I-like receptor family of cytoplasmic pattern recognition receptors that detect molecular signatures of virus infection and initiate antiviral signal transduction cascades. The ATP hydrolysis activity of LGP2 is essential for antiviral signaling, but it has been unclear how the enzymatic properties of LGP2 regulate its biological response. Quantitative analysis of the dsRNA binding and enzymatic activities of LGP2 revealed high dsRNA-independent ATP hydrolysis activity. Biochemical assays and single-molecule analysis of LGP2 and mutant variants that dissociate basal from dsRNA-stimulated ATP hydrolysis demonstrate that LGP2 utilizes basal ATP hydrolysis to enhance and diversify its RNA recognition capacity, enabling the protein to associate with intrinsically poor substrates. This property is required for LGP2 to synergize with another RIG-I-like receptor, MDA5, to potentiate IFNß transcription in vivo during infection with encephalomyocarditis virus or transfection with poly(I:C). These results demonstrate previously unrecognized properties of LGP2 ATP hydrolysis and RNA interaction and provide a mechanistic basis for a positive regulatory role for LGP2 in antiviral signaling.

Amino acid requirements for MDA5 and LGP2 recognition by paramyxovirus V proteins: a single arginine distinguishes MDA5 from RIG-I.

Paramyxovirus V proteins bind to MDA5 (melanoma differentiation-associated gene 5) and LGP2 (laboratory of genetics and physiology gene 2) but not RIG-I (retinoic acid-inducible gene I). The results demonstrate MDA5 R806 is essential for inhibition by diverse V proteins. Complementary substitution for the analogous RIG-I L714 confers V protein recognition. The analogous LGP2 R455 is required for recognition by measles V protein, but not other V proteins. These findings indicate that paramyxoviruses use a single amino acid to distinguish MDA5 from RIG-I and have evolved distinct contact sites for LGP2 interference.

MicroRNA profiling of Sendai virus-infected A549 cells identifies miR-203 as an interferon-inducible regulator of IFIT1/ISG56.

The mammalian type I interferon (IFN) response is a primary barrier for virus infection and is essential for complete innate and adaptive immunity. Both IFN production and IFN-mediated antiviral signaling are the result of differential cellular gene expression, a process that is tightly controlled at transcriptional and translational levels. To determine the potential for microRNA (miRNA)-mediated regulation of the antiviral response, small-RNA profiling was used to analyze the miRNA content of human A549 cells at steady state and following infection with the Cantell strain of Sendai virus, a potent inducer of IFN and cellular antiviral responses. While the miRNA content of the cells was largely unaltered by infection, specific changes in miRNA abundance were identified during Sendai virus infection. One miRNA, miR-203, was found to accumulate in infected cells and in response to IFN treatment. Results indicate that miR-203 is an IFN-inducible miRNA that can negatively regulate a number of cellular mRNAs, including an IFN-stimulated gene target, IFIT1/ISG56, by destabilizing its mRNA transcript.

Influenza A virus infection of human respiratory cells induces primary microRNA expression.

The cellular response to virus infection is initiated by recognition of the invading pathogen and subsequent changes in gene expression mediated by both transcriptional and translational mechanisms. In addition to well established means of regulating antiviral gene expression, it has been demonstrated that RNA interference (RNAi) can play an important role in antiviral responses. Virus-derived small interfering RNA (siRNA) is a primary antiviral response exploited by plants and invertebrate animals, and host-encoded microRNA (miRNA) species have been clearly implicated in the regulation of innate and adaptive immune responses in mammals and other vertebrates. Examination of miRNA abundance in human lung cell lines revealed endogenous miRNAs, including miR-7, miR-132, miR-146a, miR-187, miR-200c, and miR-1275, to specifically accumulate in response to infection with two influenza A virus strains, A/Udorn/72 and A/WSN/33. Known antiviral response pathways, including Toll-like receptor, RIG-I-like receptor, and direct interferon or cytokine stimulation did not alter the abundance of the tested miRNAs to the extent of influenza A virus infection, which initiates primary miRNA transcription via a secondary response pathway. Gene expression profiling identified 26 cellular mRNAs targeted by these miRNAs, including IRAK1, MAPK3, and other components of innate immune signaling systems.

Targeting ULK1 Decreases IFNγ-Mediated Resistance to Immune Checkpoint Inhibitors.

Immune checkpoint inhibitors (ICI) have transformed the treatment of melanoma. However, the majority of patients have primary or acquired resistance to ICIs, limiting durable responses and patient survival. IFNγ signaling and the expression of IFNγ-stimulated genes correlate with either response or resistance to ICIs, in a context-dependent manner. While IFNγ-inducible immunostimulatory genes are required for response to ICIs, chronic IFNγ signaling induces the expression of immunosuppressive genes, promoting resistance to these therapies. Here, we show that high levels of Unc-51 like kinase 1 (ULK1) correlate with poor survival in patients with melanoma and overexpression of ULK1 in melanoma cells enhances IFNγ-induced expression of immunosuppressive genes, with minimal effects on the expression of immunostimulatory genes. In contrast, genetic or pharmacologic inhibition of ULK1 reduces expression of IFNγ-induced immunosuppressive genes. ULK1 binds IRF1 in the nuclear compartment of melanoma cells, controlling its binding to the programmed death-ligand 1 promoter region. In addition, pharmacologic inhibition of ULK1 in combination with anti-programmed cell death protein 1 therapy further reduces melanoma tumor growth in vivo. Our data suggest that targeting ULK1 represses IFNγ-dependent immunosuppression. These findings support the combination of ULK1 drug-targeted inhibition with ICIs for the treatment of patients with melanoma to improve response rates and patient outcomes. IMPLICATIONS: This study identifies ULK1, activated downstream of IFNγ signaling, as a druggable target to overcome resistance mechanisms to ICI therapy in metastatic melanoma.

Dissociation of paramyxovirus interferon evasion activities: universal and virus-specific requirements for conserved V protein amino acids in MDA5 interference.

The V protein of the paramyxovirus subfamily Paramyxovirinae is an important virulence factor that can interfere with host innate immunity by inactivating the cytosolic pathogen recognition receptor MDA5. This interference is a result of a protein-protein interaction between the highly conserved carboxyl-terminal domain of the V protein and the helicase domain of MDA5. The V protein C-terminal domain (CTD) is an evolutionarily conserved 49- to 68-amino-acid region that coordinates two zinc atoms per protein chain. Site-directed mutagenesis of conserved residues in the V protein CTD has revealed both universal and virus-specific requirements for zinc coordination in MDA5 engagement and has also identified other conserved residues as critical for MDA5 interaction and interference. Mutation of these residues produces V proteins that are specifically defective for MDA5 interference and not impaired in targeting STAT1 for proteasomal degradation via the VDC ubiquitin ligase complex. Results demonstrate that mutation of conserved charged residues in the V proteins of Nipah virus, measles virus, and mumps virus also abolishes MDA5 interaction. These findings clearly define molecular determinants for MDA5 inhibition by the paramyxovirus V proteins.

Immune regulator LGP2 targets Ubc13/UBE2N to mediate widespread interference with K63 polyubiquitination and NF-κB activation.

Lysine 63-linked polyubiquitin (K63-Ub) chains activate a range of cellular immune and inflammatory signaling pathways, including the mammalian antiviral response. Interferon and antiviral genes are triggered by TRAF family ubiquitin ligases that form K63-Ub chains. LGP2 is a feedback inhibitor of TRAF-mediated K63-Ub that can interfere with diverse immune signaling pathways. Our results demonstrate that LGP2 inhibits K63-Ub by association with and sequestration of the K63-Ub-conjugating enzyme, Ubc13/UBE2N. The LGP2 helicase subdomain, Hel2i, mediates protein interaction that engages and inhibits Ubc13/UBE2N, affecting control over a range of K63-Ub ligase proteins, including TRAF6, TRIM25, and RNF125, all of which are inactivated by LGP2. These findings establish a unifying mechanism for LGP2-mediated negative regulation that can modulate a variety of K63-Ub signaling pathways.

A point mutation, E95D, in the mumps virus V protein disengages STAT3 targeting from STAT1 targeting.

Mumps virus, like other paramyxoviruses in the Rubulavirus genus, encodes a V protein that can assemble a ubiquitin ligase complex from cellular components, leading to the destruction of cellular signal transducer and activator of transcription (STAT) proteins. While many V proteins target the interferon-activated STAT1 or STAT2 protein, mumps virus V protein is unique in its ability to also target STAT3 for ubiquitin modification and proteasome-mediated degradation. Here we report that a single amino acid substitution in the mumps virus V protein, E95D, results in defective STAT3 targeting while maintaining the ability to target STAT1. Results indicate that the E95D mutation disrupts the ability of the V protein to associate with STAT3. A recombinant mumps virus carrying the E95D mutation in its P and V proteins replicates normally in cultured cells but fails to induce targeting of STAT3. Infection with the recombinant virus results in the differential regulation of a number of cellular genes compared to wild-type mumps virus and increases cell death in infected cells, producing a large-plaque phenotype.