Epstein-Barr Nuclear Antigen 1 Contributes to Nasopharyngeal Carcinoma through Disruption of PML Nuclear Bodies

Latent Epstein-Barr virus (EBV) infection is strongly associated with several cancers, including nasopharyngeal carcinoma (NPC), a tumor that is endemic in several parts of the world. We have investigated the molecular basis for how EBV latent infection promotes the development of NPC. We show that the viral EBNA1 protein, previously known to be required to maintain the EBV episomes, also causes the disruption of the cellular PML (promyelocytic leukemia) nuclear bodies (or ND10s). This disruption occurs both in the context of a native latent infection and when exogenously expressed in EBV-negative NPC cells and involves loss of the PML proteins. We also show that EBNA1 is partially localized to PML nuclear bodies in NPC cells and interacts with a specific PML isoform. PML disruption by EBNA1 requires binding to the cellular ubiquitin specific protease, USP7 or HAUSP, but is independent of p53. We further observed that p53 activation, DNA repair and apoptosis, all of which depend on PML nuclear bodies, were impaired by EBNA1 expression and that cells expressing EBNA1 were more likely to survive after induction of DNA damage. The results point to an important role for EBNA1 in the development of NPC, in which EBNA1-mediated disruption of PML nuclear bodies promotes the survival of cells with DNA damage

Biphasic euchromatin-to-heterochromatin transition on the KSHV genome following de novo infection.

The establishment of latency is an essential step for the life-long persistent infection and pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). While the KSHV genome is chromatin-free in the virions, the viral DNA in latently infected cells has a chromatin structure with activating and repressive histone modifications that promote latent gene expression but suppress lytic gene expression. Here, we report a comprehensive epigenetic study of the recruitment of chromatin regulatory factors onto the KSHV genome during the pre-latency phase of KSHV infection. This demonstrates that the KSHV genome undergoes a biphasic chromatinization following de novo infection. Initially, a transcriptionally active chromatin (euchromatin), characterized by high levels of the H3K4me3 and acetylated H3K27 (H3K27ac) activating histone marks, was deposited on the viral episome and accompanied by the transient induction of a limited number of lytic genes. Interestingly, temporary expression of the RTA protein facilitated the increase of H3K4me3 and H3K27ac occupancy on the KSHV episome during de novo infection. Between 24-72 hours post-infection, as the levels of these activating histone marks declined on the KSHV genome, the levels of the repressive H3K27me3 and H2AK119ub histone marks increased concomitantly with the decline of lytic gene expression. Importantly, this transition to heterochromatin was dependent on both Polycomb Repressive Complex 1 and 2. In contrast, upon infection of human gingiva-derived epithelial cells, the KSHV genome underwent a transcription-active euchromatinization, resulting in efficient lytic gene expression. Our data demonstrate that the KSHV genome undergoes a temporally-ordered biphasic euchromatin-to-heterochromatin transition in endothelial cells, leading to latent infection, whereas KSHV preferentially adopts a transcriptionally active euchromatin in oral epithelial cells, resulting in lytic gene expression. Our results suggest that the differential epigenetic modification of the KSHV genome in distinct cell types is a potential determining factor for latent infection versus lytic replication of KSHV

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

Tap: A Novel Cellular Protein That Interacts with Tip of Herpesvirus Saimiri and Induces Lymphocyte Aggregation

AbstractTip of herpesvirus saimiri associates with Lck and down-regulates Lck-mediated activation. We identified a novel cellular Tip-associated protein (Tap) by a yeast two-hybrid screen. Tap associated with Tip following transient expression in COS-1 cells and stable expression in human Jurkat-T cells. Expression of Tip and Tap in Jurkat-T cells induced dramatic cell aggregation. Aggregation was likely caused by the up-regulated surface expression of adhesion molecules including integrin α, L-selectin, ICAM-3, and H-CAM. Furthermore, NF-κB transcriptional factor of aggregated cells had approximately 40-fold higher activity than that of parental cells. Thus, Tap is likely to be an important cellular mediator of Tip function in T cell transformation by herpesvirus saimiri

The Antiviral Effector IFITM3 Disrupts Intracellular Cholesterol Homeostasis to Block Viral Entry

SummaryVesicle-membrane-protein-associated protein A (VAPA) and oxysterol-binding protein (OSBP) regulate intracellular cholesterol homeostasis, which is required for many virus infections. During entry, viruses or virus-containing vesicles can fuse with endosomal membranes to mediate the cytosolic release of virions, and alterations in endosomal cholesterol can inhibit this invasion step. We show that the antiviral effector protein interferon-inducible transmembrane protein 3 (IFITM3) interacts with VAPA and prevents its association with OSBP, thereby disrupting intracellular cholesterol homeostasis and inhibiting viral entry. By altering VAPA-OSBP function, IFITM3 induces a marked accumulation of cholesterol in multivesicular bodies and late endosomes, which inhibits the fusion of intraluminal virion-containing vesicles with endosomal membranes and thereby blocks virus release into the cytosol. Consequently, ectopic expression or depletion of the VAPA gene profoundly affects IFITM3-mediated inhibition of viral entry. Thus, IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry, further underscoring the importance of cholesterol in virus infection

Deregulation of HDAC5 by Viral Interferon Regulatory Factor 3 Plays an Essential Role in Kaposi's Sarcoma-Associated Herpesvirus-Induced Lymphangiogenesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS), which is one of the most common HIV-associated neoplasms. The endothelium is the thin layer of squamous cells where vascular blood endothelial cells (BECs) line the interior surface of blood vessels and lymphatic endothelial cells (LECs) are in direct contact with lymphatic vessels. The KS lesions contain a prominent compartment of neoplastic spindle morphology cells that are closely related to LECs. Furthermore, while KSHV can infect both LECs and BECs in vitro, its infection activates genetic programming related to lymphatic endothelial cell fate, suggesting that lymphangiogenic pathways are involved in KSHV infection and malignancy. Here, we report for the first time that viral interferon regulatory factor 3 (vIRF3) is readily detected in over 40% of KS lesions and that vIRF3 functions as a proangiogenic factor, inducing hypersprouting formation and abnormal growth in a LEC-specific manner. Mass spectrometry analysis revealed that vIRF3 interacted with histone deacetylase 5 (HDAC5), which is a signal-responsive regulator for vascular homeostasis. This interaction blocked the phosphorylation-dependent cytosolic translocation of HDAC5 and ultimately altered global gene expression in LECs but not in BECs. Consequently, vIRF3 robustly induced spindle morphology and hypersprouting formation of LECs but not BECs. Finally, KSHV infection led to the hypersprouting formation of LECs, whereas infection with a ΔvIRF3 mutant did not do so. Collectively, our data indicate that vIRF3 alters global gene expression and induces a hypersprouting formation in an HDAC5-binding-dependent and LEC-specific manner, ultimately contributing to KSHV-associated pathogenesis.IMPORTANCE Several lines of evidences indicate that KSHV infection of LECs induces pathological lymphangiogenesis and that the results resemble KS-like spindle morphology. However, the underlying molecular mechanism remains unclear. Here, we demonstrated that KSHV vIRF3 is readily detected in over 40% of various KS lesions and functions as a potent prolymphangiogenic factor by blocking the phosphorylation-dependent cytosolic translocation of HDAC5, which in turn modulates global gene expression in LECs. Consequently, vIRF3-HDAC5 interaction contributes to virus-induced lymphangiogenesis. The results of this study suggest that KSHV vIRF3 plays a crucial role in KSHV-induced malignancy

Structural and Biochemical Bases for the Inhibition of Autophagy and Apoptosis by Viral BCL-2 of Murine γ-Herpesvirus 68

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine γ-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus

The cap-snatching SFTSV endonuclease domain is an antiviral target

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%-30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (I

Inhibition of T Cell Receptor Signal Transduction by Tyrosine Kinase–interacting Protein of Herpesvirus saimiri

T cells play a central role in orchestrating immunity against pathogens, particularly viruses. Thus, impairing T cell activation is an important strategy employed by viruses to escape host immune control. The tyrosine kinase–interacting protein (Tip) of the T lymphotropic Herpesvirus saimiri (HVS) is constitutively present in lipid rafts and interacts with cellular Lck tyrosine kinase and p80 endosomal protein. Here we demonstrate that, due to the sequestration of Lck by HVS Tip, T cell receptor (TCR) stimulation fails to activate ZAP70 tyrosine kinase and to initiate downstream signaling events. TCR ζ chains in Tip-expressing T cells were initially phosphorylated to recruit ZAP70 molecule upon TCR stimulation, but the recruited ZAP70 kinase was not subsequently phosphorylated, resulting in TCR complexes that were stably associated with inactive ZAP70 kinase. Consequently, Tip expression not only markedly inhibited TCR-mediated intracellular signal transduction but also blocked TCR engagement with major histocompatibility complexes on the antigen-presenting cells and immunological synapse formation. These results demonstrate that a lymphotropic herpesvirus has evolved a novel mechanism to deregulate T cell activation to disarm host immune surveillance. This process contributes to the establishment and maintenance of viral latency