Enhancer activation requires trans-recruitment of a mega transcription factor complex.

Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.

Enhancer release and retargeting activates disease-susceptibility genes.

The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).

Dismissal of RNA Polymerase II Underlies a Large Ligand-Induced Enhancer Decommissioning Program.

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.

Long-distance association of topological boundaries through nuclear condensates.

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.

The transcription factor KLF2 restrains CD4⁺ T follicular helper cell differentiation.

T follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4(+) T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4(+) T cells led to increased Tfh cell generation and B cell priming, whereas KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4(+) T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization and by regulation of lineage-defining transcription factors.

Condensin I and II Complexes License Full Estrogen Receptor α-Dependent Enhancer Activation.

Enhancers instruct spatio-temporally specific gene expression in a manner tightly linked to higher-order chromatin architecture. Critical chromatin architectural regulators condensin I and condensin II play non-redundant roles controlling mitotic chromosomes. But the chromosomal locations of condensins and their functional roles in interphase are poorly understood. Here we report that both condensin complexes exhibit an unexpected, dramatic estrogen-induced recruitment to estrogen receptor α (ER-α)-bound eRNA(+) active enhancers in interphase breast cancer cells, exhibiting non-canonical interaction with ER-α via its DNA-binding domain (DBD). Condensins positively regulate ligand-dependent enhancer activation at least in part by recruiting an E3 ubiquitin ligase, HECTD1, to modulate the binding of enhancer-associated coactivators/corepressors, including p300 and RIP140, permitting full eRNA transcription, formation of enhancer:promoter looping, and the resultant coding gene activation. Collectively, our results reveal an important, unanticipated transcriptional role of interphase condensins in modulating estrogen-regulated enhancer activation and coding gene transcriptional program.

Global epigenomic analysis of KSHV-infected primary effusion lymphoma identifies functional MYC superenhancers and enhancer RNAs.

Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function.

Effects of equine-assisted activities on attention and quality of life in children with cerebral palsy in a randomized trial: examining the comorbidity with attention-deficit/hyperactivity disorder.

BACKGROUND: Attention problems and decreased quality of life are frequently accompanied in Cerebral Palsy (CP), which can negatively affect rehabilitation of physical disability. However, the majority of affected children remain untreated in the aspects of attention or psychosocial factors. Equine-Assisted Activities and Therapies (EAAT) use horse as a therapeutic modality including grooming as well as mounted riding activities in which patients exercise and experience mounted stimulation. It is known to help improve attention in children with ADHD, so that it can be an exercise therapy that is expected to improvement of attention as well as rehabilitating effects in CP patients. EAA may be a promising strategy to address the unmet need for CP patients. This study aims to investigate the efficacy of EAA for children with CP, those with both CP and ADHD and confirm the comorbidity between CP and ADHD. METHODS: Forty-six children with cerebral palsy participated in this study. For the exercise group, they participated in a 40-min session twice a week for a 16-week period, while the control group engaged in daily life without any special treatments. Each children individually were assessed on attention and psychological wellbeing at baseline and post-treatment. Comorbidity were identified based on the Diagnostic and Statistical Manual of Mental Disorder 5th edition (DSM-5) and confirmed by Korean Kiddie-Schedule for Affective Disorders and Schizophrenia Present and Lifetime Version (K-SADS-PL). RESULTS: Perseveration rated using the Conner's Performance Test (CPT) showed a significant decrease only in the exercise group (p < .024). However, no significant improvement in children's quality of life was observed after EAA program compared with control group. Among the total participants, fifteen children (31.91%) were diagnosed with ADHD. When conducting an additional analysis with the subsample of CP patients diagnosed with ADHD, the d', commission error and perseveration showed a significant decrease only in the exercise group. Children with CP and ADHD reported an improvement in quality of life both in exercise and control group, but only in the exercise group social functioning exhibited a significant difference. CONCLUSION: The positive effects of the EAA on attention and quality of life were confirmed. Children with CP in the exercise group were more capable to sustain their attention longer. Those with CP and ADHD showed an increase in attention and perceived to have better social skills after receiving 16 weeks of EAA compared to those in the control group. Considering high comorbidity of CP and ADHD, it seems that the EAA program could be the better alternative treatment for CP with attentional problem. The results of this study will contribute to growing evidence for the efficacy of EAA in children especially with CP and ADHD. TRIAL REGISTRATION: This trial was registered on ClinicalTrials.gov ( NCT03870893 ). Registered 26 July 2017.

Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation.

The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs). However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17ß-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer-promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer-promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.

Enhanced production of enveloped viruses in BST-2-deficient cell lines.

Despite all the advantages that cell-cultured influenza vaccines have over egg-based influenza vaccines, the inferior productivity of cell-culture systems is a major drawback that must be addressed. BST-2 (tetherin) is a host restriction factor which inhibits budding-out of various enveloped viruses from infected host cells. We developed BST-2-deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST-2 gene knock-out resulted in increased release of viral particles into the culture medium, by at least 2-fold and up to 50-fold compared to release from wild-type counterpart cells depending on cell line and virus type. The effect was not influenza virus/MDCK/Vero-specific, but was also present in a broad range of host cells and virus families; we observed similar results in murine, human, canine, and monkey cell lines with viruses including MHV-68 (Herpesviridae), influenza A virus (Orthomyxoviridae), porcine epidemic diarrhea virus (Coronaviridae), and vaccinia virus (Poxviridae). Our results suggest that the elimination of BST-2 expression in virus-producing cell lines can enhance the production of viral vaccines. Biotechnol. Bioeng.2017;114: 2289-2297. © 2017 Wiley Periodicals, Inc.

UVRAG is required for virus entry through combinatorial interaction with the class C-Vps complex and SNAREs.

Enveloped viruses exploit the endomembrane system to enter host cells. Through a cascade of membrane-trafficking events, virus-bearing vesicles fuse with acidic endosomes and/or lysosomes mediated by SNAREs triggering viral fusion. However, the molecular mechanisms underlying this process remain elusive. Here, we found that UV-radiation resistance-associated gene (UVRAG), an autophagic tumor suppressor, is required for the entry of the prototypic negative-strand RNA virus, including influenza A virus and vesicular stomatitis virus, by a mechanism independent of IFN and autophagy. UVRAG mediates viral endocytic transport and membrane penetration through interactions with the class C vacuolar protein sorting (C-Vps) tethering complex and endosomal glutamine-containing SNAREs [syntaxin 7 (STX7), STX8, and vesicle transport through t-SNARE homolog 1B (Vti1b)], leading to the assembly of a fusogenic trans-SNARE complex involving vesicle-associated membrane protein (VAMP8), but not VAMP7. Indeed, UVRAG stimulates VAMP8 translocation to virus-bearing endosomes. Inhibition of VAMP8, but not VAMP7, significantly reduces viral entry. Our data indicate that UVRAG, in concert with C-Vps, regulates viral entry by assembling a specific fusogenic SNARE complex. Thus, UVRAG governs downstream viral entry, highlighting an important pathway capable of potential antiviral therapeutics.

Chem-seq permits identification of genomic targets of drugs against androgen receptor regulation selected by functional phenotypic screens.

Understanding the mechanisms by which compounds discovered using cell-based phenotypic screening strategies might exert their effects would be highly augmented by new approaches exploring their potential interactions with the genome. For example, altered androgen receptor (AR) transcriptional programs, including castration resistance and subsequent chromosomal translocations, play key roles in prostate cancer pathological progression, making the quest for identification of new therapeutic agents and an understanding of their actions a continued priority. Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as "SD70," initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-sequencing-like approach, referred to as "Chem-seq," we were next able to efficiently map the genome-wide binding locations of this small molecule, revealing that it largely colocalized with AR on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C. Global location of candidate drugs represents a powerful strategy for new drug development by mapping genome-wide location of small molecules, a powerful adjunct to contemporary drug development strategies.

Enhancer-promoter specificity in gene transcription: molecular mechanisms and disease associations.

Although often located at a distance from their target gene promoters, enhancers are the primary genomic determinants of temporal and spatial transcriptional specificity in metazoans. Since the discovery of the first enhancer element in simian virus 40, there has been substantial interest in unraveling the mechanism(s) by which enhancers communicate with their partner promoters to ensure proper gene expression. These research efforts have benefited considerably from the application of increasingly sophisticated sequencing- and imaging-based approaches in conjunction with innovative (epi)genome-editing technologies; however, despite various proposed models, the principles of enhancer-promoter interaction have still not been fully elucidated. In this review, we provide an overview of recent progress in the eukaryotic gene transcription field pertaining to enhancer-promoter specificity. A better understanding of the mechanistic basis of lineage- and context-dependent enhancer-promoter engagement, along with the continued identification of functional enhancers, will provide key insights into the spatiotemporal control of gene expression that can reveal therapeutic opportunities for a range of enhancer-related diseases.

Diagnosis of ADHD using virtual reality and artificial intelligence: an exploratory study of clinical applications.

Introduction: Diagnosis of Attention Deficit/Hyperactivity Disorder (ADHD) is based on clinical evaluation of symptoms by a psychiatrist, referencing results of psychological tests. When diagnosing ADHD, the child's behavior and functionality in real-life situations are critical components. However, direct observation by a clinician is often not feasible in practice. Therefore, such information is typically gathered from primary caregivers or teachers, which can introduce subjective elements. To overcome these limitations, we developed AttnKare-D, an innovative digital diagnostic tool that could analyze children's behavioral data in Virtual Reality using Artificial Intelligence. The purpose of this study was to explore the utility and safety of AttnKare-D for clinical application. Method: A total of 21 children aged between 6 and 12 years were recruited for this study. Among them, 15 were children diagnosed with ADHD, 5 were part of a normal control group, and 1 child was excluded due to withdrawal of consent. Psychological assessments, including K-WISC, Conners CPT, K-ARS, and K-CBCL, were conducted for participants and their primary caregivers. Diagnoses of ADHD were confirmed by child and adolescent psychiatrists based on comprehensive face-to-face evaluations and results of psychological assessments. Participants underwent VR diagnostic assessment by performing various cognitive and behavioral tasks in a VR environment. Collected data were analyzed using an AI model to assess ADHD diagnosis and the severity of symptoms. Results: AttnKare-D demonstrated diagnostic performance with an AUC of 0.893 when compared to diagnoses made by child and adolescent psychiatrist, showing a sensitivity of 0.8 and a specificity of 1.0 at a cut-off score of 18.44. AttnKare-D scores showed a high correlation with K-ARS scores rated by parents and experts, although the correlation was relatively low for inattention scores. Conclusion: Results of this study suggest that AttnKare-D can be a useful tool for diagnosing ADHD in children. This approach has potential to overcome limitations of current diagnostic methods, enhancing the accuracy and objectivity of ADHD diagnoses. This study lays the groundwork for further improvement and research on diagnostic tools integrating VR and AI technologies. For future clinical applications, it is necessary to conduct clinical trials involving a sufficient number of participants to ensure reliable use.

Identification of Interleukin (IL)-33 Inhibitory Constituents from Canavalia gladiata Pods.

Interleukin (IL)-33, a member of the IL-1 cytokine family, plays a vital role in immune system regulation and inflammation, with oxidative stress being implicated in its expression. During the search for compounds from natural sources with potential as therapeutic agents for allergic diseases via IL-33 signal modulation, we discovered significant IL-33 inhibitory activity in the methanol extract of Canavalia gladiata (sword bean) pods. Through chromatographic separation and liquid chromatography-mass spectrometry, we isolated 11 compounds (1-11) from the methanol extract. Furthermore, we assessed the inhibitory effects of these substances on IL-33/ST2 signaling in processes related to inflammatory and autoimmune diseases using an enzyme-linked immunosorbent assay. Among them, compounds 7, 10, and 11 exhibited substantial IL-33 inhibitory efficacy, with values reaching 78%, 86%, and 79% at 100 µM, respectively. Remarkably, compounds 7, 10, and 11 demonstrated significant and dose-dependent inhibition of IL-33 signaling at concentrations of 10, 50, and 100 µM. Computational molecular docking and dynamic simulations further elucidated the underlying mechanisms. These findings have promising pharmacological implications for allergy prevention and treatment associated with flavonoid glycosides derived from C. gladiata.

Recruitment of CTCF to the SIRT1 promoter after Oxidative Stress mediates Cardioprotective Transcription.

Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.

Transcriptional and Epigenetic Regulation of Context-Dependent Plasticity in T-Helper Lineages.

Th cell lineage determination and functional specialization are tightly linked to the activation of lineage-determining transcription factors (TFs) that bind cis-regulatory elements. These lineage-determining TFs act in concert with multiple layers of transcriptional regulators to alter the epigenetic landscape, including DNA methylation, histone modification and three-dimensional chromosome architecture, in order to facilitate the specific Th gene expression programs that allow for phenotypic diversification. Accumulating evidence indicates that Th cell differentiation is not as rigid as classically held; rather, extensive phenotypic plasticity is an inherent feature of T cell lineages. Recent studies have begun to uncover the epigenetic programs that mechanistically govern T cell subset specification and immunological memory. Advances in next generation sequencing technologies have allowed global transcriptomic and epigenomic interrogation of CD4+ Th cells that extends previous findings focusing on individual loci. In this review, we provide an overview of recent genome-wide insights into the transcriptional and epigenetic regulation of CD4+ T cell-mediated adaptive immunity and discuss the implications for disease as well as immunotherapies.

Direct observation of surface charge and stiffness of human metaphase chromosomes.

Metaphase chromosomes in which both polynucleotides and proteins are condensed with hierarchies are closely related to life phenomena such as cell division, cancer development, and cellular senescence. Nevertheless, their nature is rarely revealed, owing to their structural complexity and technical limitations in analytical methods. In this study, we used surface potential and nanomechanics mapping technology based on atomic force microscopy to measure the surface charge and intrinsic stiffness of metaphase chromosomes. We found that extra materials covering the chromosomes after the extraction process were positively charged. With the covering materials, the chromosomes were positively charged (ca. 44.9 ± 16.48 mV) and showed uniform stiffness (ca. 6.23 ± 1.98 MPa). In contrast, after getting rid of the extra materials through treatment with RNase and protease, the chromosomes were strongly negatively charged (ca. -197.4 ± 77.87 mV) and showed relatively non-uniform and augmented stiffness (ca. 36.87 ± 17.56 MPa). The results suggested undulating but compact coordination of condensed chromosomes. Additionally, excessive treatment with RNase and protease could destroy the chromosomal structure, providing an exceptional opportunity for multiscale stiffness mapping of polynucleotides, nucleosomes, chromatin fibers, and chromosomes in a single image. Our approach offers a new horizon in terms of an analytical technique for studying chromosome-related diseases.

Dynamics of Viral and Host 3D Genome Structure upon Infection.

Eukaryotic chromatin is highly organized in the 3D nuclear space and dynamically regulated in response to environmental stimuli. This genomic organization is arranged in a hierarchical fashion to support various cellular functions, including transcriptional regulation of gene expression. Like other host cellular mechanisms, viral pathogens utilize and modulate host chromatin architecture and its regulatory machinery to control features of their life cycle, such as lytic versus latent status. Combined with previous research focusing on individual loci, recent global genomic studies employing conformational assays coupled with high-throughput sequencing technology have informed models for host and, in some cases, viral 3D chromosomal structure re-organization during infection and the contribution of these alterations to virus-mediated diseases. Here, we review recent discoveries and progress in host and viral chromatin structural dynamics during infection, focusing on a subset of DNA (human herpesviruses and HPV) as well as RNA (HIV, influenza virus and SARS-CoV-2) viruses. An understanding of how host and viral genomic structure affect gene expression in both contexts and ultimately viral pathogenesis can facilitate the development of novel therapeutic strategies.

Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68.

Gamma-herpesviruses (gammaHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine gamma-herpesvirus 68 (gammaHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant gammaHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which gammaHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of gamma-herpesviruses.