Chronically persistent viruses are integral components of the organismal ecosystem in humans and animals 1 2 . Many of these viruses replicate and accumulate within the cell nucleus 3 . The nuclear location allows viruses to evade cytoplasmic host viral sensors and promotes viral replication 4 . One of the unexplored and puzzling aspects of the viral nuclear lifecycle involves the virus's ability to deal with the physical constraints of nuclear architecture. To replicate and accumulate within the nucleus in large numbers sufficient for infection spreading, DNA viruses need to overcome the spatial limitations imposed by chromatin and the nuclear matrix. We found that one of the most widespread and potentially lethal human viruses, the JC polyomavirus 5 , interferes with nuclear heterochromatin to create virus-occupied space. The JC virus's impact on heterochromatin is mediated by the viral nonstructural protein, Agnoprotein (Agno). Agno's interference with heterochromatin is governed by structurally diverse mimics of host epigenetic regulators that facilitate virus-induced chromatin reorganization and a dramatic decline in nuclear stiffness in the infected cells. The JCV epigenetic mimicry is critical for the virus infection, as evident from reduced replication of mimic-mutant viruses. Our data suggest that modulation of nuclear mechanical properties is a novel strategy enabling chronicity of the JC and possibly other nuclear virus infections.

The mimicry of host proteins by viruses contributes to their ability to suppress antiviral immunity and hijack host biosynthetic machinery 1 . Host adaptation to evade this exploitation depends on host protein functional redundancy 2 . Non-redundant, essential host proteins have limited potential to adapt without severe consequences 3 . Histones, which are essential for genome architecture and control of gene expression, are among the most evolutionary conserved proteins 4 . Here we show that the capsid protein of the flavivirus yellow fever virus (YFV), mimics histone H4 and interferes with chromatin gene regulation by BRD4, a bromodomain and extraterminal domain (BET) protein. Two acetyl-lysine residues of YFV capsid are embedded in a histone-like motif that interacts with the BRD4 bromodomain, affecting gene expression and influencing YFV replication. These findings reveal histone mimicry as a strategy employed by an RNA virus that replicates in the cytosol 5 and define convergent and distinct molecular determinants for motif recognition of the viral mimic versus histone H4.

Induction of vast transcriptional programs is a central event of innate host responses to viral infections. Here we report a transcriptional program with potent antiviral activity, driven by E74-like ETS transcription factor 1 (ELF1). Using microscopy to quantify viral infection over time, we found that ELF1 inhibits eight diverse RNA and DNA viruses after multi-cycle replication. Elf1 deficiency results in enhanced susceptibility to influenza A virus infections in mice. ELF1 does not feed-forward to induce interferons, and ELF1's antiviral effect is not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNA-seq revealed that the ELF1 transcriptional program is distinct from interferon signatures. Thus, ELF1 provides an additional layer of the innate host response, independent from the action of type I interferons.

The transcription of interferon-stimulated genes (ISGs) is classically triggered via activation of the JAK-STAT pathway, and together, ISGs raise a multifaceted antiviral barrier. An increasing body of evidence reports the existence of additional, non-canonical pathways and transcription factors that coordinate ISG expression. Detailed knowledge of how heterogenous mechanisms regulate ISG expression is crucial for the rational design of drugs targeting the type I interferon response. Here, we characterize the first ETS transcription factor family member as a regulator of non-canonical ISG expression: E74-like ETS transcription factor 1 (ELF1). Using high-content microscopy to quantify viral infection over time, we found that ELF1, itself an ISG, inhibits eight diverse RNA and DNA viruses uniquely at multi-cycle replication. ELF1 did not regulate expression of type I or II interferons, and ELF1’s antiviral effect was not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNAseq revealed that the ELF1 transcriptional program is distinct from, and delayed with respect to, the immediate interferon response. Finally, knockdown experiments demonstrated that ELF1 is a critical component of the antiviral interferon response in vitro and in vivo. Our findings reveal a previously overlooked mechanism of non-canonical ISG regulation that both amplifies and prolongs the initial interferon response by expressing broadly antiviral restriction factors. AUTHOR SUMMARY Over 60 years after their discovery, we still struggle to understand exactly how interferons inhibit viruses. Our gap in knowledge stems, on one hand, from the sheer number of interferon-stimulated effector genes, of which only few have been characterized in mechanistic detail. On the other hand, our knowledge of interferon-regulated gene transcription is constantly evolving. We know that different regulatory mechanisms greatly influence the quality, magnitude, and timing of interferon-stimulated gene expression, all of which may contribute to the antiviral mechanism of interferons. Deciphering these regulatory mechanisms is indispensable for understanding this critical first line of host defense, and for harnessing the power of interferons in novel antiviral therapies. Here, we report a novel mechanism of interferon-induced gene regulation by an interferon-stimulated gene, which, paradoxically, inhibits viruses in the absence of additional interferon signaling: E74-like ETS transcription factor 1 (ELF1) raises an unusually delayed antiviral program that potently restricts propagation of all viruses tested in our study. Reduced levels of ELF1 significantly diminished interferon-mediated host defenses against influenza A virus in vitro and in vivo, suggesting a critical but previously overlooked role in the type I interferon response. The transcriptional program raised by ELF1 is vast and comprises over 400 potentially antiviral genes, which are almost entirely distinct from those known to be induced by interferon. Taken together, our data provide evidence for a critical secondary wave of antiviral protection that adds both “quality” and “time” to the type I interferon response.

Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomain-containing protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire: Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire: Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers.

Studies investigating the causes of autism spectrum disorder (ASD) point to genetic, as well as epigenetic, mechanisms of the disease. Identification of epigenetic processes that contribute to ASD development and progression is of major importance and may lead to the development of novel therapeutic strategies. Here, we identify the bromodomain and extraterminal domain-containing proteins (BETs) as epigenetic regulators of genes involved in ASD-like behaviors in mice. We found that the pharmacological suppression of BET proteins in the brain of young mice, by the novel, highly specific, brain-permeable inhibitor I-BET858 leads to selective suppression of neuronal gene expression followed by the development of an autism-like syndrome. Many of the I-BET858-affected genes have been linked to ASD in humans, thus suggesting the key role of the BET-controlled gene network in the disorder. Our studies suggest that environmental factors controlling BET proteins or their target genes may contribute to the epigenetic mechanism of ASD.

Inflammation is associated with the activation of genes that contribute to immune defense and tissue repair. The bromodomain-containing proteins of the BET family, which recognize histone lysine acetylation, play a key role in the transcriptional control of inflammatory genes. Inhibition of BET proteins by the small-molecule inhibitor I-BET affects the expression of a particular subset of inflammatory genes-namely, ones that follow an "analog-like," but not "digital-like" activation pattern. This ability of I-BET to target genes based on the dynamic pattern of their activation may facilitate the further development of anti-inflammatory treatment protocols that are tuned to the individual or to disease-specific patterns of gene expression.

One of the defining characteristics of human and animal viruses is their ability to suppress host antiviral responses. Viruses express proteins that impair the detection of viral nucleic acids by host pattern-recognition receptors, block signaling pathways that lead to the synthesis of type I interferons and other cytokines, or prevent the activation of virus-induced genes. We have identified a novel mechanism of virus-mediated suppression of antiviral gene expression that relies on the presence of histone-like sequences (histone mimics) in viral proteins. We describe how viral histone mimics can interfere with key regulators of gene expression and contribute to the suppression of antiviral responses. We also describe how viral histone mimics can facilitate the identification of novel mechanisms of antiviral gene regulation and lead to the development of drugs that use histone mimicry for interference with gene expression during diseases.

Latent reservoirs of HIV-1-infected cells are refractory to antiretroviral therapies (ART) and remain the major barrier to curing HIV-1. Because latently infected cells are long-lived, immunologically invisible, and may undergo homeostatic proliferation, a "shock and kill" approach has been proposed to eradicate this reservoir by combining ART with inducers of viral transcription. However, all attempts to alter the HIV-1 reservoir in vivo have failed to date. Using humanized mice, we show that broadly neutralizing antibodies (bNAbs) can interfere with establishment of a silent reservoir by Fc-FcR-mediated mechanisms. In established infection, bNAbs or bNAbs plus single inducers are ineffective in preventing viral rebound. However, bNAbs plus a combination of inducers that act by independent mechanisms synergize to decrease the reservoir as measured by viral rebound. Thus, combinations of inducers and bNAbs constitute a therapeutic strategy that impacts the establishment and maintenance of the HIV-1 reservoir in humanized mice.

Viral infection is commonly associated with virus-driven hijacking of host proteins. Here we describe a novel mechanism by which influenza virus affects host cells through the interaction of influenza non-structural protein 1 (NS1) with the infected cell epigenome. We show that the NS1 protein of influenza A H3N2 subtype possesses a histone-like sequence (histone mimic) that is used by the virus to target the human PAF1 transcription elongation complex (hPAF1C). We demonstrate that binding of NS1 to hPAF1C depends on the NS1 histone mimic and results in suppression of hPAF1C-mediated transcriptional elongation. Furthermore, human PAF1 has a crucial role in the antiviral response. Loss of hPAF1C binding by NS1 attenuates influenza infection, whereas hPAF1C deficiency reduces antiviral gene expression and renders cells more susceptible to viruses. We propose that the histone mimic in NS1 enables the influenza virus to affect inducible gene expression selectively, thus contributing to suppression of the antiviral response.

Effective antiviral immunity depends on the ability of infected cells or cells triggered with virus-derived nucleic acids to produce type I interferon (IFN), which activates transcription of numerous antiviral genes. However, disproportionately strong or chronic IFN expression is a common cause of inflammatory and autoimmune diseases. We describe an epigenetic mechanism that determines cell type-specific differences in IFN and IFN-stimulated gene (ISG) expression in response to exogenous signals. We identify di-methylation of histone H3 at lysine 9 (H3K9me2) as a suppressor of IFN and IFN-inducible antiviral gene expression. We show that levels of H3K9me2 at IFN and ISG correlate inversely with the scope and amplitude of IFN and ISG expression in fibroblasts and dendritic cells. Accordingly, genetic ablation or pharmacological inactivation of lysine methyltransferase G9a, which is essential for the generation of H3K9me2, resulted in phenotypic conversion of fibroblasts into highly potent IFN-producing cells and rendered these cells resistant to pathogenic RNA viruses. In summary, our studies implicate H3K9me2 and enzymes controlling its abundance as key regulators of innate antiviral immunity.

Small molecules that perturb chromatin proteins are an emerging focus of current biomedical research. Two groups reporting in this issue have targeted bromodomain-containing BET proteins that bind acetylated lysine residues during gene activation, arriving at cell-permeable small molecule compounds with similar structures based on fused triazole-diazepine rings. James Bradner and colleagues report the development of a compound named JQ1. The BET protein BRD4, with two bromodomains, is implicated in human squamous cell carcinoma. JQ1 inhibits the growth of BRD4-dependent tumours in mouse models. Alexander Tarakhovsky and colleagues' inhibitor, I-BET, is shown to interfere with the binding of certain BET family members to acetylated histones. It inhibits activation of pro-inflammatory genes in macrophages and has immunomodulatory activity in a mouse model of inflammatory disease. Post-translationally modified histones are recognized by effector proteins which contain specific binding modules; for example, the bromodomain-containing BET proteins bind acetylated lysine residues during gene activation. Here a synthetic small molecule is described that interferes with the binding of certain BET family members to acetylated histones. The compound inhibits activation of pro-inflammatory genes in macrophages and has activity in a mouse model of inflammatory disease. Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression1,2,3,4. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression5,6,7,8,9,10. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by ‘mimicking’ acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.