Hye-Ra Lee

FLIP-mediated autophagy regulation in cell death control.

Autophagy is an active homeostatic degradation process for the removal or turnover of cytoplasmic components wherein the LC3 ubiquitin-like protein undergoes an Atg7 E1-like enzyme/Atg3 E2-like enzyme-mediated conjugation process to induce autophagosome biogenesis. Besides its cytoprotecive role, autophagy acts on cell death when it is abnormally upregulated. Thus, the autophagy pathway requires tight regulation to ensure that this degradative process is well balanced. Two death effector domains (DED1/2) containing cellular FLICE-like inhibitor protein (cFLIP) and viral FLIP (vFLIP) of Kaposis sarcoma-associated herpesvirus (KSHV), Herpesvirus saimiri (HVS), and Molluscum contagiosum virus (MCV) protect cells from apoptosis mediated by death receptors. Here, we report that cellular and viral FLIPs suppress autophagy by preventing Atg3 from binding and processing LC3. Consequently, FLIP expression effectively represses cell death with autophagy, as induced by rapamycin, an mTor inhibitor and an effective anti-tumour drug against KSHV-induced Kaposis sarcoma (KS) and primary effusion lymphoma (PEL). Remarkably, either a DED1 α2-helix ten amino-acid (α2) peptide or a DED2 α4-helix twelve amino-acid (α4) peptide of FLIP is individually sufficient for binding FLIP itself and Atg3, with the peptide interactions effectively suppressing Atg3–FLIP interaction without affecting Atg3-LC3 interaction, resulting in robust cell death with autophagy. Our study thus identifies a checkpoint of the autophagy pathway where cellular and viral FLIPs limit the Atg3-mediated step of LC3 conjugation to regulate autophagosome biogenesis. Furthermore, the FLIP-derived short peptides induce growth suppression and cell death with autophagy, representing biologically active molecules for potential anti-cancer therapies.

Epigenetic Analysis of KSHV Latent and Lytic Genomes

Epigenetic modifications of the herpesviral genome play a key role in the transcriptional control of latent and lytic genes during a productive viral lifecycle. In this study, we describe for the first time a comprehensive genome-wide ChIP-on-Chip analysis of the chromatin associated with the Kaposis sarcoma-associated herpesvirus (KSHV) genome during latency and lytic reactivation. Depending on the gene expression class, different combinations of activating [acetylated H3 (AcH3) and H3K4me3] and repressive [H3K9me3 and H3K27me3] histone modifications are associated with the viral latent genome, which changes upon reactivation in a manner that is correlated with their expression. Specifically, both the activating marks co-localize on the KSHV latent genome, as do the repressive marks. However, the activating and repressive histone modifications are mutually exclusive of each other on the bulk of the latent KSHV genome. The genomic region encoding the IE genes ORF50 and ORF48 possesses the features of a bivalent chromatin structure characterized by the concomitant presence of the activating H3K4me3 and the repressive H3K27me3 marks during latency, which rapidly changes upon reactivation with increasing AcH3 and H3K4me3 marks and decreasing H3K27me3. Furthermore, EZH2, the H3K27me3 histone methyltransferase of the Polycomb group proteins (PcG), colocalizes with the H3K27me3 mark on the entire KSHV genome during latency, whereas RTA-mediated reactivation induces EZH2 dissociation from the genomic regions encoding IE and E genes concurrent with decreasing H3K27me3 level and increasing IE/E lytic gene expression. Moreover, either the inhibition of EZH2 expression by a small molecule inhibitor DZNep and RNAi knockdown, or the expression of H3K27me3-specific histone demethylases apparently induced the KSHV lytic gene expression cascade. These data indicate that histone modifications associated with the KSHV latent genome are involved in the regulation of latency and ultimately in the control of the temporal and sequential expression of the lytic gene cascade. In addition, the PcG proteins play a critical role in the control of KSHV latency by maintaining a reversible heterochromatin on the KSHV lytic genes. Thus, the regulation of the spatial and temporal association of the PcG proteins with the KSHV genome may be crucial for propagating the KSHV lifecycle.

Construction and Manipulation of a New Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Clone

ABSTRACT Efficient genetic modification of herpesviruses such as Kaposis sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (∼5 × 107/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.

MDGAs interact selectively with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development

The MAM domain-containing GPI anchor proteins MDGA1 and MDGA2 are Ig superfamily adhesion molecules composed of six IG domains, a fibronectin III domain, a MAM domain, and a GPI anchor. MDGAs contribute to the radial migration and positioning of a subset of cortical neurons during early neural development. However, MDGAs continue to be expressed in postnatal brain, and their functions during postnatal neural development remain unknown. Here, we demonstrate that MDGAs specifically and with a nanomolar affinity bind to neuroligin-2, a cell-adhesion molecule of inhibitory synapses, but do not bind detectably to neuroligin-1 or neuroligin-3. We observed no cell adhesion between cells expressing neuroligin-2 and MDGA1, suggesting a cis interaction. Importantly, RNAi-mediated knockdown of MDGAs increased the abundance of inhibitory but not excitatory synapses in a neuroligin-2–dependent manner. Conversely, overexpression of MDGA1 decreased the numbers of functional inhibitory synapses. Likewise, coexpression of both MDGA1 and neuroligin-2 reduced the synaptogenic capacity of neuroligin-2 in an artificial synapse-formation assay by abolishing the ability of neuroligin-2 to form an adhesion complex with neurexins. Taken together, our data suggest that MDGAs inhibit the activity of neuroligin-2 in controlling the function of inhibitory synapses and that MDGAs do so by binding to neuroligin-2.

Kaposi's Sarcoma–Associated Herpesvirus Viral IFN Regulatory Factor 3 Stabilizes Hypoxia-Inducible Factor-1α to Induce Vascular Endothelial Growth Factor Expression

Kaposis sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of both developmental and pathologic angiogenesis, composed of an oxygen-sensitive alpha-subunit and a constitutively expressed beta-subunit. HIF-1 activity in tumors depends on the availability of the HIF-1 alpha subunit, the levels of which are increased under hypoxic conditions. Recent studies have shown that HIF-1 plays an important role in KSHV reactivation from latency and pathogenesis. Here, we report a novel mechanism by which KSHV activates HIF-1 activity. Specific interaction between KSHV viral IFN regulatory factor 3 (vIRF3) and the HIF-1 alpha subunit led to the HIF-1 alpha stabilization and transcriptional activation, which induced vascular endothelial growth factor expression and ultimately facilitated endothelial tube formation. Remarkably, the central domain of vIRF3, containing double alpha-helix motifs, was sufficient not only for binding to HIF-1 alpha but also for blocking its degradation in normoxic conditions. This indicates that KSHV has developed a unique mechanism to enhance HIF-1 alpha protein stability and transcriptional activity by incorporating a viral homologue of cellular IRF gene into its genome, which may contribute to viral pathogenesis.

Viral Interferon Regulatory Factors

Upon viral infection, the major defensive strategy employed by the host immune system is the activation of the interferon (IFN)-mediated antiviral pathway, which is overseen by IFN regulatory factors (IRFs). In order to complete their life cycles, viruses must find a way to modulate the host IFN-mediated immune response. Kaposis sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homolog of the cellular IRFs, called vIRFs, into its genome. Here, we summarize the novel evasion mechanisms by which KSHV, through its vIRFs, circumvents IFN-mediated innate immune responses and deregulates the cell growth control mechanism.

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 Targets MDM2 To Deregulate the p53 Tumor Suppressor Pathway

ABSTRACT Cells infected by viruses utilize interferon (IFN)-mediated and p53-mediated irreversible cell cycle arrest and apoptosis as part of the overall host surveillance mechanism to ultimately block viral replication and dissemination. Viruses, in turn, have evolved elaborate mechanisms to subvert IFN- and p53-mediated host innate immune responses. Kaposis sarcoma-associated herpesvirus (KSHV) encodes several viral IFN regulatory factors (vIRF1 to vIRF4) within a cluster of loci, their functions being primarily to inhibit host IFN-mediated innate immunity and deregulate p53-mediated cell growth control. Despite its significant homology and similar genomic location to other vIRFs, vIRF4 is distinctive, as it does not target and antagonize host IFN-mediated signal transduction. Here, we show that KSHV vIRF4 interacts with the murine double minute 2 (MDM2) E3 ubiquitin ligase, leading to the reduction of p53, a tumor suppressor, via proteasome-mediated degradation. The central region of vIRF4 is required for its interaction with MDM2, which led to the suppression of MDM2 autoubiquitination and, thereby, a dramatic increase in MDM2 stability. Consequently, vIRF4 expression markedly enhanced p53 ubiquitination and degradation, effectively suppressing p53-mediated apoptosis. These results indicate that KSHV vIRF4 targets and stabilizes the MDM2 E3 ubiquitin ligase to facilitate the proteasome-mediated degradation of p53, perhaps to circumvent host growth surveillance and facilitate viral replication in infected cells. Taken together, the indications are that the downregulation of p53-mediated cell growth control is a common characteristic of the four KSHV vIRFs and that p53 is indeed a key factor in the hosts immune surveillance program against viral infections.

Non-Human Primate Model of Kaposi's Sarcoma-Associated Herpesvirus Infection

Since Kaposis sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) was first identified in Kaposis sarcoma (KS) lesions of HIV-infected individuals with AIDS, the basic biological understanding of KSHV has progressed remarkably. However, the absence of a proper animal model for KSHV continues to impede direct in vivo studies of viral replication, persistence, and pathogenesis. In response to this need for an animal model of KSHV infection, we have explored whether common marmosets can be experimentally infected with human KSHV. Here, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Marmosets infected with recombinant KSHV rapidly seroconverted and maintained a vigorous anti-KSHV antibody response. KSHV DNA and latent nuclear antigen (LANA) were readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of infected marmosets. Remarkably, one orally infected marmoset developed a KS-like skin lesion with the characteristic infiltration of leukocytes by spindle cells positive for KSHV DNA and proteins. These results demonstrate that human KSHV infects common marmosets, establishes an efficient persistent infection, and occasionally leads to a KS-like skin lesion. This is the first animal model to significantly elaborate the important aspects of KSHV infection in humans and will aid in the future design of vaccines against KSHV and anti-viral therapies targeting KSHV coinfected tumor cells.

Control of B Cell Development by the Histone H2A Deubiquitinase MYSM1

Epigenetic histone modifications play critical roles in the control of gene transcription. Recently, an increasing number of histone H2A deubiquitinases have been identified and characterized. However, the physiological functions for this entire group of histone H2A deubiquitinases remain unknown. In this study, we revealed that the histone H2A deubiquitinase MYSM1 plays an essential and intrinsic role in early B cell development. MYSM1 deficiency results in a block in early B cell commitment and a defect of B cell progenitors in expression of EBF1 and other B lymphoid genes. We further demonstrated that MYSM1 derepresses EBF1 transcription in B cell progenitors by orchestrating histone modifications and transcription factor recruitment to the EBF1 locus. Thus, this study not only uncovers the essential role for MYSM1 in gene transcription during early B cell development but also underscores the biological significance of reversible epigenetic histone H2A ubiquitination.

Modulation of Immune System by Kaposi’s Sarcoma-Associated Herpesvirus: Lessons from Viral Evasion Strategies

Kaposi’s sarcoma-associated herpesvirus (KSHV), a member of the herpesvirus family, has evolved to establish a long-term, latent infection of cells such that while they carry the viral genome gene expression is highly restricted. Latency is a state of cryptic viral infection associated with genomic persistence in their host and this hallmark of KSHV infection leads to several clinical–epidemiological diseases such as KS, a plasmablastic variant of multicentric Castleman’s disease, and primary effusion lymphoma upon immune suppression of infected hosts. In order to sustain efficient life-long persistency as well as their life cycle, KSHV dedicates a large portion of its genome to encode immunomodulatory proteins that antagonize its host’s immune system. In this review, we will describe our current knowledge of the immune evasion strategies employed by KSHV at distinct stages of its viral life cycle to control the host’s immune system.