Effects of targeting sumoylation processes during latent and induced Epstein-Barr virus infections using the small molecule inhibitor ML-792.

As the second leading cause of death in the United States, cancer has a considerable impact on society, and one cellular process that is commonly dysregulated in many cancers is the post-translational modification of proteins by the Small Ubiquitin-like Modifier (SUMO; sumoylation). We documented that sumoylation processes are up-regulated in lymphoma tissues in the presence of Latent Membrane Protein-1 (LMP1), the principal oncoprotein of Epstein-Barr virus (EBV). LMP1-mediated dysregulation of cellular sumoylation processes contributes to oncogenesis, modulates innate immune responses, and aids the maintenance of viral latency. Manipulation of protein sumoylation has been proposed for anti-cancer and anti-viral therapies; however, known inhibitors of sumoylation do not only target sumoylation processes. Recently, a specific and selective small-molecule inhibitor of sumoylation (ML-792) was identified; however, nothing is known about the effect of ML-792 on LMP1-mediated dysregulation of cellular sumoylation or the EBV life-cycle. We hypothesized that ML-792 modulates viral replication and the oncogenic potential of EBV LMP1 by inhibiting protein sumoylation. Results showed that ML-792 inhibited sumoylation processes in multiple EBV-positive B cell lines and EBV-positive nasopharyngeal carcinoma cell lines but not in their EBV-negative counterparts. Focusing on its effect on B cells, ML-792 inhibited B-cell growth and promoted cell death at very low doses. ML-792 also modulated LMP1-induced cell migration and cell adhesion, which suggests the abrogation of the oncogenic potential of LMP1. Finally, while higher concentrations of ML-792 were sufficient to induce low levels EBV spontaneous reactivation, they decreased the production of new infectious virus following an induced reactivation and the infection of new cells, suggesting that ML-792 has anti-viral potential. Together, these findings suggest that ML-792 may be a potential therapeutic drug to treat EBV-associated lymphoid malignancies by targeting oncogenesis and the EBV life-cycle.

The Epstein-Barr Virus Oncoprotein, LMP1, Regulates the Function of SENP2, a SUMO-protease.

Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) activates numerous signal transduction pathways using its C-terminal activating regions. We reported that LMP1 increased global levels of sumoylated proteins, which aided the oncogenic nature of LMP1. Because increased protein sumoylation is detected in numerous cancers, we wanted to elucidate additional mechanisms by which LMP1 modulates the sumoylation machinery. Results indicated that SUMO-protease activity decreased in a LMP1-dependent manner, so we hypothesized that LMP1 inhibits SUMO-protease activity, resulting in reduced de-sumoylation of cellular proteins, which contributes to the detected accumulation of sumoylated proteins in EBV-positive lymphomas. Focusing on SENP2, findings revealed that LMP1 expression corresponded with increased sumoylation of SENP2 at K48 and K447 in a CTAR-dependent manner. Interestingly, independent of LMP1-induced sumoylation of SENP2, LMP1 also decreased SENP2 activity, decreased SENP2 turnover, and altered the localization of SENP2, which led us to investigate if LMP1 regulated the biology of SENP2 by a different post-translational modification, specifically ubiquitination. Data showed that expression of LMP1 inhibited the ubiquitination of SENP2, and inhibition of ubiquitination was sufficient to mimic LMP1-induced changes in SENP2 activity and trafficking. Together, these findings suggest that LMP1 modulates different post-translational modifications of SENP2 in order to modulate its biology and identify a third member of the sumoylation machinery that is manipulated by LMP1 during latent EBV infections, which can affect oncogenesis.

Using glycyrrhizic acid to target sumoylation processes during Epstein-Barr virus latency.

Cellular sumoylation processes are proposed targets for anti-viral and anti-cancer therapies. We reported that Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) dysregulates cellular sumoylation processes, contributing to its oncogenic potential in EBV-associated malignancies. Ginkgolic acid and anacardic acid, known inhibitors of sumoylation, inhibit LMP1-induced protein sumoylation; however, both drugs have adverse effects in hosts. Here we test the effects of glycyrrhizic acid, a medicinal botanical extract with anti-inflammatory, anti-carcinogenic, and anti-viral properties, on cellular sumoylation processes. While glycyrrhizic acid is known to inhibit EBV penetration, its affect on cellular sumoylation processes remains to be documented. We hypothesized that glycyrrhizic acid inhibits cellular sumoylation processes and may be a viable treatment for Epstein-Barr virus-associated malignancies. Results showed that glycyrrhizic acid inhibited sumoylation processes (without affecting ubiquitination processes), limited cell growth, and induced apoptosis in multiple cell lines. Similar to ginkgolic acid; glycyrrhizic acid targeted the first step of the sumoylation process and resulted in low levels of spontaneous EBV reactivation. Glycyrrhizic acid did not affect induced reactivation of the virus, but the presence of the extract did reduce the ability of the produced virus to infect additional cells. Therefore, we propose that glycyrrhizic acid may be a potential therapeutic drug to augment the treatment of EBV-associated lymphoid malignancies.

Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells.

Epstein-Barr Virus latent membrane protein-1 (LMP1) interacts with the SUMO-conjugating enzyme Ubc9, which induces protein sumoylation and may contribute to LMP1-mediated oncogenesis. After analyzing human lymphoma tissues and EBV-positive cell lines, we now document a strong correlation between LMP1 and sumo-1/2/3 or SUMO-1/2/3 levels, and show that LMP1-induced sumo expression requires the activation of NF-κB signaling through CTAR1 and CTAR2. Together, these results point to a second mechanism by which LMP1 dysregulates sumoylation processes and adds EBV-associated lymphomas to the list of malignancies associated with increased SUMO expression.

Viral manipulation of the cellular sumoylation machinery.

Viruses exploit various cellular processes for their own benefit, including counteracting anti-viral responses and regulating viral replication and propagation. In the past 20 years, protein sumoylation has emerged as an important post-translational modification that is manipulated by viruses to modulate anti-viral responses, viral replication, and viral pathogenesis. The process of sumoylation is a multi-step cascade where a small ubiquitin-like modifier (SUMO) is covalently attached to a conserved ΨKxD/E motif within a target protein, altering the function of the modified protein. Here we review how viruses manipulate the cellular machinery at each step of the sumoylation process to favor viral survival and pathogenesis.

LMP1-Induced Sumoylation Influences the Maintenance of Epstein-Barr Virus Latency through KAP1.

UNLABELLED: As a herpesvirus, Epstein-Barr virus (EBV) establishes a latent infection that can periodically undergo reactivation, resulting in lytic replication and the production of new infectious virus. Latent membrane protein-1 (LMP1), the principal viral oncoprotein, is a latency-associated protein implicated in regulating viral reactivation and the maintenance of latency. We recently found that LMP1 hijacks the SUMO-conjugating enzyme Ubc9 via its C-terminal activating region-3 (CTAR3) and induces the sumoylation of cellular proteins. Because protein sumoylation can promote transcriptional repression, we hypothesized that LMP1-induced protein sumoylation induces the repression of EBV lytic promoters and helps maintain the viral genome in its latent state. We now show that with inhibition of LMP1-induced protein sumoylation, the latent state becomes less stable or leakier in EBV-transformed lymphoblastoid cell lines. The cells are also more sensitive to viral reactivation induced by irradiation, which results in the increased production and release of infectious virus, as well as increased susceptibility to ganciclovir treatment. We have identified a target of LMP1-mediated sumoylation that contributes to the maintenance of latency in this context: KRAB-associated protein-1 (KAP1). LMP1 CTAR3-mediated sumoylation regulates the function of KAP1. KAP1 also binds to EBV OriLyt and immediate early promoters in a CTAR3-dependent manner, and inhibition of sumoylation processes abrogates the binding of KAP1 to these promoters. These data provide an additional line of evidence that supports our findings that CTAR3 is a distinct functioning regulatory region of LMP1 and confirm that LMP1-induced sumoylation may help stabilize the maintenance of EBV latency. IMPORTANCE: Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) plays an important role in the maintenance of viral latency. Previously, we documented that LMP1 targets cellular proteins to be modified by a ubiquitin-like protein (SUMO). We have now identified a function for this LMP1-induced modification of cellular proteins in the maintenance of EBV latency. Because latently infected cells have to undergo viral reactivation in order to be vulnerable to antiviral drugs, these findings identify a new way to increase the rate of EBV reactivation, which increases cell susceptibility to antiviral therapies.

HCMV reprogramming of infected monocyte survival and differentiation: a Goldilocks phenomenon.

The wide range of disease pathologies seen in multiple organ sites associated with human cytomegalovirus (HCMV) infection results from the systemic hematogenous dissemination of the virus, which is mediated predominately by infected monocytes. In addition to their role in viral spread, infected monocytes are also known to play a key role in viral latency and life-long persistence. However, in order to utilize infected monocytes for viral spread and persistence, HCMV must overcome a number of monocyte biological hurdles, including their naturally short lifespan and their inability to support viral gene expression and replication. Our laboratory has shown that HCMV is able to manipulate the biology of infected monocytes in order to overcome these biological hurdles by inducing the survival and differentiation of infected monocytes into long-lived macrophages capable of supporting viral gene expression and replication. In this current review, we describe the unique aspects of how HCMV promotes monocyte survival and differentiation by inducing a "finely-tuned" macrophage cell type following infection. Specifically, we describe the induction of a uniquely polarized macrophage subset from infected monocytes, which we argue is the ideal cellular environment for the initiation of viral gene expression and replication and, ultimately, viral spread and persistence within the infected host.

KSHV LANA and EBV LMP1 induce the expression of UCH-L1 following viral transformation.

Ubiquitin C-terminal Hydrolase L1 (UCH-L1) has oncogenic properties and is highly expressed during malignancies. We recently documented that Epstein-Barr virus (EBV) infection induces uch-l1 expression. Here we show that Kaposi's Sarcoma-associated herpesvirus (KSHV) infection induced UCH-L1 expression, via cooperation of KSHV Latency-Associated Nuclear Antigen (LANA) and RBP-Jκ and activation of the uch-l1 promoter. UCH-L1 expression was also increased in Primary Effusion Lymphoma (PEL) cells co-infected with KSHV and EBV compared with PEL cells infected only with KSHV, suggesting EBV augments the effect of LANA on uch-l1. EBV latent membrane protein 1 (LMP1) is one of the few EBV products expressed in PEL cells. Results showed that LMP1 was sufficient to induce uch-l1 expression, and co-expression of LMP1 and LANA had an additive effect on uch-l1 expression. These results indicate that viral latency products of both human γ-herpesviruses contribute to uch-l1 expression, which may contribute to the progression of lymphoid malignancies.

Epstein-Barr virus latent membrane protein 1 regulates the function of interferon regulatory factor 7 by inducing its sumoylation.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) induces multiple signal transduction pathways during latent EBV infection via its C-terminal activating region 1 (CTAR1), CTAR2, and the less-studied CTAR3. One mechanism by which LMP1 regulates cellular activation is through the induction of protein posttranslational modifications, including phosphorylation and ubiquitination. We recently documented that LMP1 induces a third major protein modification by physically interacting with the SUMO-conjugating enzyme Ubc9 through CTAR3 and inducing the sumoylation of cellular proteins in latently infected cells. We have now identified a specific target of LMP1-induced sumoylation, interferon regulatory factor 7 (IRF7). We hypothesize that during EBV latency, LMP1 induces the sumoylation of IRF7, limiting its transcriptional activity and modulating the activation of innate immune responses. Our data show that endogenously sumoylated IRF7 is detected in latently infected EBV lymphoblastoid cell lines. LMP1 expression coincided with increased sumoylation of IRF7 in a CTAR3-dependent manner. Additional experiments show that LMP1 CTAR3-induced sumoylation regulates the expression and function of IRF7 by decreasing its turnover, increasing its nuclear retention, decreasing its DNA binding, and limiting its transcriptional activation. Finally, we identified that IRF7 is sumoylated at lysine 452. These data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling, leading to biologic effects. We propose that CTAR3 is an important signaling region of LMP1 that regulates protein function by sumoylation. We have shown specifically that LMP1 CTAR3, in cooperation with CTAR2, can limit the ability of IRF7 to induce innate immune responses by inducing the sumoylation of IRF7.