Biphasic JNK signaling reveals distinct MAP3K complexes licensing inflammasome formation and pyroptosis.

Kinase signaling in the tiered activation of inflammasomes and associated pyroptosis is a prime therapeutic target for inflammatory diseases. While MAPKs subsume pivotal roles during inflammasome priming, specifically the MAP3K7/JNK1/NLRP3 licensing axis, their involvement in successive steps of inflammasome activation is poorly defined. Using live-cell MAPK biosensors to focus on the inflammasome triggering event allowed us to identify a subsequent process of biphasic JNK activation. We find that this biphasic post-trigger JNK signaling initially facilitates the mitochondrial reactive oxygen species generation needed to support core inflammasome formation, then supports the gasdermin-mediated cell permeation required for release of active IL-1ß from human macrophages. We further identify and characterize a xanthine oxidase-ROS activated MAP3K5/JNK2 substrate licensing complex as a novel regulator of the GSDMD mobilization which precedes pyroptosis. We show that inhibitors targeting this MAP3K5 cascade alleviate morbidity in mouse models of colitis and dampen both augmented IL-1ß release and cell permeation in monocytes derived from patients with gain-of-function inflammasomopathies.

Small-molecule screening identifies Syk kinase inhibition and rutaecarpine as modulators of macrophage training and SARS-CoV-2 infection.

Biologically active small molecules can impart modulatory effects, in some cases providing extended long-term memory. In a screen of biologically active small molecules for regulators of tumor necrosis factor (TNF) induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing lipopolysaccharide (LPS)-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages. Rutaecarpine inhibits ß-glucan-induced H3K4Me3 marks at the promoters of several pro-inflammatory cytokines, highlighting the potential of this molecule to modulate chromosomal topology. Syk kinase inhibitor (SYKi IV), another screen hit, promotes an enhanced response to LPS similar to that previously reported for ß-glucan-induced training. Macrophages trained with SYKi IV show a high degree of resistance to influenza A, multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and OC43 coronavirus infection, highlighting a potential application of this molecule and other SYKis as prophylactic treatments for viral susceptibility.

Correction: Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

[This corrects the article DOI: 10.1371/journal.ppat.1009395.].

IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages.

Activation of the TLR4 signaling pathway by lipopolysaccharide (LPS) leads to induction of both inflammatory and interferon-stimulated genes, but the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remain poorly understood. In a genome-wide small interfering RNA (siRNA) screen of the LPS-induced tumor necrosis factor α (TNF-α) response in macrophages, we identify the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identifies a positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of LPS-induced gene classes. We demonstrate that these effects of IFIT1 are mediated through modulation of a Sin3A-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate a function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program.

Inhibition of Tip60 Reduces Lytic and Latent Gene Expression of Kaposi's Sarcoma-Associated Herpes Virus (KSHV) and Proliferation of KSHV-Infected Tumor Cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus responsible for the development of Kaposi's sarcoma, primary effusion lymphoma (PEL), and Multicentric Castleman's disease in immunocompromised individuals. Despite the burden of these diseases there are few treatment options for afflicted individuals, due in part to our limited understanding of virus-host interactions. Tip60, a histone aceytltransferase (HAT) has been previously shown to interact with both the KSHV latency associated nuclear antigen protein (LANA), which is the main factor in maintaining the viral latent state, and ORF36, a viral kinase expressed in the lytic phase. We further investigated Tip60-virus interaction to ascertain Tip60's role in the viral life cycle and its potential as a target for future therapeutics. Through modulation of Tip60 expression in HEK293T cells harboring a plasmid containing the KSHV viral episome, Bac36, we found that Tip60 is vital for both lytic replication as well as efficient expression of latent genes. Interestingly, Tip60 small molecule inhibitors, MG149 and NU9056, similarly inhibited latent and lytic genes, and reduced virion production in wild-type KSHV+/EBV- PEL, BCBL-1 cells. Long-term treatment with these Tip60 inhibitors selectively decreased the viability of KSHV-infected B lymphoma cells compared to uninfected cells. From this study, we conclude that Tip60 is important for KSHV infection and its associated cancer development, and Tip60 is therefore a potential target for future antiviral and anticancer therapeutics.

Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics.

The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.

Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages.

Macrophages play a critical role in the innate immune response to pathogen infection, but few tools exist for systematic dissection of these responses using modern genome-wide perturbation methods. To develop an assay platform for high-throughput analysis of macrophage activation by pathogenic stimuli, we generated reporter systems in human and mouse macrophages with dynamic readouts for NF-κB and/or TNF-α responses. These reporter cells show responsiveness to a broad range of TLR ligands and to gram-negative bacterial infection. There are significant challenges to the use of RNAi in innate immune cells, including efficient small RNA delivery and non-specific immune responses to dsRNA. To permit the interrogation of the macrophage pathogen response pathways with RNAi, we employed the stably expressed reporter genes to develop efficient siRNA delivery protocols for maximal target gene silencing with minimal activation of the innate macrophage response to nucleic acids. We demonstrate the utility of these macrophage cell systems for siRNA screening of pathogen responses by targeting components of the human and mouse TLR pathways, and observe species-specific perturbation of signaling and cytokine responses. Our approach to reporter cell development and siRNA delivery optimization provides an experimental paradigm with significant potential for developing genetic screening platforms in mammalian cells.

Comprehensive identification of host modulators of HIV-1 replication using multiple orthologous RNAi reagents.

RNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene's phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1-host cell interactions.

Amphotericin B increases influenza A virus infection by preventing IFITM3-mediated restriction.

The IFITMs inhibit influenza A virus (IAV) replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB), prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon's protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both a possible mechanism for IFITM-mediated restriction and its negation by AmphoB. Notably, we reveal that mice treated with AmBisome succumbed to a normally mild IAV infection, similar to animals deficient in Ifitm3. Therefore, patients receiving antifungal therapy with clinical preparations of AmphoB may be functionally immunocompromised and thus more vulnerable to influenza, as well as other IFITM3-restricted viral infections.

The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication.

The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3's distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3's restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual's risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction.

A genetic screen identifies interferon-α effector genes required to suppress hepatitis C virus replication.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease. Interferon-α (IFNα) is an important component of anti-HCV therapy; it up-regulates transcription of IFN-stimulated genes, many of which have been investigated for their antiviral effects. However, all of the genes required for the antiviral function of IFNα (IFN effector genes [IEGs]) are not known. IEGs include not only IFN-stimulated genes, but other nontranscriptionally induced genes that are required for the antiviral effect of IFNα. In contrast to candidate approaches based on analyses of messenger RNA (mRNA) expression, identification of IEGs requires a broad functional approach. METHODS: We performed an unbiased genome-wide small interfering RNA screen to identify IEGs that inhibit HCV. Huh7.5.1 hepatoma cells were transfected with small interfering RNAs incubated with IFNα and then infected with JFH1 HCV. Cells were stained using HCV core antibody, imaged, and analyzed to determine the percent infection. Candidate IEGs detected in the screen were validated and analyzed further. RESULTS: The screen identified 120 previously unreported IEGs. From these, we more fully evaluated the following: asparagine-linked glycosylation 10 homolog (yeast, α-1,2-glucosyltransferase); butyrylcholinesterase; dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2); glucokinase (hexokinase 4) regulator; guanylate cyclase 1, soluble, ß 3; MYST histone acetyltransferase 1; protein phosphatase 3 (formerly 2B), catalytic subunit, ß isoform; peroxisomal proliferator-activated receptor-γ-DBD-interacting protein 1; and solute carrier family 27 (fatty acid transporter), member 2; and demonstrated that they enabled IFNα-mediated suppression of HCV at multiple steps of its life cycle. Expression of these genes had more potent effects against flaviviridae because a subset was required for IFNα to suppress dengue virus but not influenza A virus. In addition, many of the host genes detected in this screen (92%) were not transcriptionally stimulated by IFNα; these genes represent a heretofore unknown class of non-IFN-stimulated gene IEGs. CONCLUSIONS: We performed a whole-genome loss-of-function screen to identify genes that mediate the effects of IFNα against human pathogenic viruses. We found that IFNα restricts HCV via actions of general and specific IEGs.

Reactivation of latent HIV-1 by inhibition of BRD4.

HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy. Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell-line models. In multiple instances, JQ1, when used in combination with the NF-κB activators Prostratin or PHA, enhanced the in vitro reactivation of latent HIV-1 in primary T cells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection.

IFITM3 restricts the morbidity and mortality associated with influenza.

The 2009 H1N1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. Recent in vitro studies show that the interferon-inducible transmembrane (IFITM) protein family members potently restrict the replication of multiple pathogenic viruses. Both the magnitude and breadth of the IFITM proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. Using a knockout mouse model, we now test this hypothesis directly and find that IFITM3 is essential for defending the host against influenza A virus in vivo. Mice lacking Ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'Spanish' influenza. Similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of Ifitm3. To test the role of IFITM3 in human influenza virus infection, we assessed the IFITM3 alleles of individuals hospitalized with seasonal or pandemic influenza H1N1/09 viruses. We find that a statistically significant number of hospitalized subjects show enrichment for a minor IFITM3 allele (SNP rs12252-C) that alters a splice acceptor site, and functional assays show the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro. Together these data reveal that the action of a single intrinsic immune effector, IFITM3, profoundly alters the course of influenza virus infection in mouse and humans.

IFITM3 inhibits influenza A virus infection by preventing cytosolic entry.

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.

The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.

Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses antiviral restriction factors to defend against infection. To find host cell modifiers of influenza A H1N1 viral infection, we used a functional genomic screen and identified over 120 influenza A virus-dependency factors with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing. We discovered that the interferon-inducible transmembrane proteins IFITM1, 2, and 3 restrict an early step in influenza A viral replication. The IFITM proteins confer basal resistance to influenza A virus but are also inducible by interferons type I and II and are critical for interferon's virustatic actions. Further characterization revealed that the IFITM proteins inhibit the early replication of flaviviruses, including dengue virus and West Nile virus. Collectively this work identifies a family of antiviral restriction factors that mediate cellular innate immunity to at least three major human pathogens.

Ebola virus VP30 is an RNA binding protein.

The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn(2+), but not Mg(2+), enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

Revealing domain structure through linker-scanning analysis of the murine leukemia virus (MuLV) RNase H and MuLV and human immunodeficiency virus type 1 integrase proteins.

Linker-scanning libraries were generated within the 3' terminus of the Moloney murine leukemia virus (M-MuLV) pol gene encoding the connection-RNase H domains of reverse transcriptase (RT) as well as the structurally related M-MuLV and human immunodeficiency virus type 1 (HIV-1) integrase (IN) proteins. Mutations within the M-MuLV proviral vectors were Tn7 based and resulted in 15-bp insertions. Mutations within an HIV-1 IN bacterial expression vector were based on Tn5 and resulted in 57-bp insertions. The effects of the insertions were examined in vivo (M-MuLV) and in vitro (HIV-1). A total of 178 individual M-MuLV constructs were analyzed; 40 in-frame insertions within RT connection-RNase H, 108 in-frame insertions within IN, 13 insertions encoding stop codons within RNase H, and 17 insertions encoding stop codons within IN. For HIV-1 IN, 56 mutants were analyzed. In both M-MuLV and HIV-1 IN, regions are identified which functionally tolerate multiple-linker insertions. For MuLV, these correspond to the RT-IN proteolytic junction, the junction between the IN core and C terminus, and the C terminus of IN. For HIV-1 IN, in addition to the junction between the IN core and C terminus and the C terminus of IN, insertions between the N terminus and core domains maintained integration and disintegration activity. Of the 40 in-frame insertions within the M-MuLV RT connection-RNase H domains, only the three C-terminal insertions mapping to the RT-IN proteolytic junction were viable. These results correlate with deletion studies mapping the domain and subdomain boundaries of RT and IN. Importantly, these genetic footprints provide a means to identify nonessential regions within RT and IN for targeted gene therapy applications.

Development and application of a high-throughput screening assay for HIV-1 integrase enzyme activities.

Integrase (IN) mediates the covalent insertion of the retroviral genome into its host chromosomal DNA. This enzymatic activity can be reconstituted in vitro with short DNA oligonucleotides, which mimic a single viral DNA end, and purified IN. Herein we report a highly efficient and sensitive high-throughput screen, HIV Integrase Target SRI Assay (HITS), for HIV-1 IN activity using 5' biotin-labeled DNA (5' BIO donor) and 3' digoxygenin-labeled DNA (3' DIG target). Following 3' processing of the 5' BIO donor, strand transfer proceeds with integration of the 5' BIO donor into the 3' DIG target. Products were captured on a streptavidin-coated microplate and the amount of DIG retained in the well was measured. The end point values, measured as absorbance, ranged from 0.9 to 1.5 for IN-mediated reactions as compared with background readings of 0.05 to 0.12. The Z factor for the assay ranged from 0.7 to 0.85. The assay was used to screen drugs in a high-throughput format, and furthermore, we adapted the assay to study mechanistic questions regarding the integration process. For example, using variations of the assay format, we showed high preference of E strand of the long terminal repeat (LTR) viral DNA as a target strand compared with its complementary A strand. The E strand is the strand processed by IN. Furthermore, we explored the reported inhibitory effect of reverse transcriptase on integration.

Rapid, high-throughput purification of HIV-1 integrase using microtiter plate technology.

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) catalyzes the insertion of the retroviral genome into the chromosome of an infected host cell. HIV-1 IN was expressed as a N-terminal hexa-histidine fusion in Escherichia coli. A high-throughput purification strategy was developed using denaturing methods for the initial protein extraction, followed by a one-step nickel-chelating chromatography purification and step-wise refolding. IN was routinely greater than 90% pure with yields exceeding 14 microg of purified IN per ml of E. coli culture. In vitro 3' processing and strand transfer assays showed the enzyme preparations to be highly active. The specific activity of the purified IN was 2.65 pmol/h/microg IN, which is very similar to the activity of IN routinely produced by large-scale column chromatographic methods. This high-throughput platform should be of general utility to those interested in defining the structure-function relationship of proteins and enzymes.