Chengyu Liang, Ph.D. - US grants

The mission of this Core is to provide state-of-art imaging technology and services to support the study: "Host-pathogen competition in IFN mediated antiviral defense." The proposed study incorporates skilled resources from more than four institutions addressing different but focused research questions involved in understanding the control and regulation of RIG-I- and MDA5-mediated immune surveillance during viral infection, with the overarching goal of developing novel therapeutic strategies for virus-associated disorders. As such, the Core currently features two major imaging systems: a spectral imaging confocal laser scanning microscope system equipped with live-cell imaging observation and a super-resolution molecule imaging system. All five projects will rely heavily on this Core, co-directed by Drs. Liang and Myong. What follows is a description of the confocal live-cell imaging portion of the Core followed by a description of the super-resolution single molecule microscopy services provided by the Core. All data from both systems are accessible to every PI. The data will be uploaded in the Center's website for data sharing and data mining. The amount of time supported varies over the 5-year course. The super-imaging system is more required in Projects 3 and 5, while the confocal system mainly supports Projects 1, 3, 4, and 5. In addition, the Imaging Core will coordinate the scientific and technologic services provided by each project.

DESCRIPTION (provided by applicant): 3-Herpesviruses (3-HVs) have developed a unique mode of interaction with their hosts to establish a life-long persistent infection, which frequently associates with the onset of various malignancies. One critical virulence factor involved in 3-HVs persistence and oncogenicity are the viral homologs of the Bcl-2 protein (referred to as vBcl-2) encoded by all 3-HVs. Alongside its well-characterized anti-apoptotic activity, our preliminary studies have established that the vBcl-2 of the 3-HV family effectively suppresses the anti-viral autophagy pathway ('self-eating', lysosome-dependent degradation and recycling of the intracellular components in response to stress), by directly targeting a key autophagy effector protein, Beclin1. Moreover, vBcl-2 has evolved enhanced anti-autophagic activity when compared to the host counterpart. Based on these findings, we hypothesize that the inhibition of autophagy by vBcl-2 constitutes a novel mechanism by which 3- HVs evade host immunity and confer persistent infection and pathogenesis. To test this hypothesis, we will focus primarily on the vBcl-2 of 3HV68 using well-established in vitro and in vivo systems. 3HV68 shares extensive genetic homology and biological similarity with EBV and KSHV. Infection of mice with 3HV68 provides a genetically tractable in vivo model for characterizing the chronic infection of 3HVs. Notably, the loss of vBcl-2 does not affect the lytic replication of 3HV68. Instead, it severely impairs the ability of 3HV68 to establish chronic infection in mice. In the first aim, we will dissect the molecular mechanism by which vBcl-2 antagonizes Beclin1-dependent autophagy. We have successfully identified the specific mutations that distinguish vBcl-2-mediated inhibition of autophagy from vBcl-2-mediated antagonism of apoptosis. In the second aim, we will investigate the specific roles of vBcl-2-mediated anti-autophagy in viral virulence in vivo. Insights gained from this study will reveal a novel paradigm for the roles of vBcl-2 in 3HVs infection, and establish autophagy as a fundamental host defensive mechanism against viral infections. PUBLIC HEALTH RELEVANCE: Autophagy has been increasingly recognized essential in host anti-viral defense, but its role in 3- herpesviruses infection remains largely unknown. The proposed study is targeted to understand the molecular mechanism of vBcl-2-mediated inhibition of autophagy, and its contribution to the persistent infection and/or pathogenesis of 3-herpesviruses. Insights gained from this study will establish a direct role for autophagy in viral virulence control and suggest strategy for much- needed anti-viral therapeutics.

DESCRIPTION (provided by applicant): Autophagy (Greek, 'self-eating') is an evolutionarily conserved homeostatic process by which cytoplasmic components are sequestered into double-membraned vesicles (autophagosome) and delivered to lysosomes for degradation and recycling. This process has been increasingly recognized as essential for cell survival, differentiation, and development, and is often misregulated in human diseases, including cancer. While it has been speculated that autophagy may both benefit and hinder tumor development/progression, recent data indicates that autophagy principally serves as a tumor suppressor pathway. Yet, despite its importance, the mechanisms by which autophagy functions in tumor suppression remain largely undetermined. The proposed study is directed toward investigating how autophagy contributes to tumor suppression and how its defects contribute to malignancy, with a specific focus on a novel autophagic UVRAG gene that is monoallelically mutated at high frequencies in human cancers. Our preliminary studies have UIKO findings, we hypothesize that UVRAG is a novel autophagic tumor suppressor, which cooperates with Beclin1 and the HOPS complex to activate autophagy and inhibit tumor development. Genetic, biochemical and cell biological studies will primarily focus on defining in mechanistic detail the dual roles of UVRAG in autophagosome formation (Aim 1) and autophagosome maturation (Aim 2), and their functional significance in UVRAG tumor suppressor activity (Aim 3). Insights gained from this study will not only illuminate new views on the autophagy regulatory network, but also suggest new strategy for cancer control.

DESCRIPTION (provided by applicant: Acute lymphoblastic leukemia (ALL), the most common form of malignancies in children, is induced by the transformation of blood (hematopoietic) stem cells and progenitors. The damaged cells produced in the bone marrow crowd out normal cells and also metastasize to other organs. Although the outcome of ALL patients has improved in recent years, significant numbers of patients die from recurrent disease. Better understanding of the molecular basis of this disease and the search for fresh ideas, and new-targeted therapies are thus in high gear. Recent studies have demonstrated that approximately 60% of cases of pediatric T-cell ALL (T- ALL) are marked with aberrant activation of the Notch1 receptor. However, small molecule ?-secretase inhibitors (GSIs), which abrogate oncogenic Notch1 signaling in T-ALL, fail to show objective clinical responses and cause severe toxicity. Therefore, successful targeting of Notch1 in T-ALL demands a considerable refinement of our understanding of Notch1 regulation in T cell leukemia. By using Drosophila as a genetic model, our recent study showed that UVRAG (UV irradiation resistance-associated gene), a highly conserved tumor suppressor, mediates the endocytic degradation of Notch1 and prevents its aberrant activation and cell overgrowth. Additionally, lymphocytes from UVRAG-deficient patient exhibit strongly increased Notch1 activity resulting from an impaired endocytic degradation of Notch1. These observations suggest that Notch1 represents a key target molecule of UVRAG. Given the oncogenic addiction of T-ALL to Notch1, we hypothesize that UVRAG-mediated downregulation of Notch1 plays a critical role in the development and GSIs-resistance of T-ALL. To test this hypothesis, Aim 1 will investigate the molecular mechanism by which UVRAG modulates Notch1 activation and signaling. Aim 2 will investigate the biological significance of UVRAG-mediated downregulation of Notch1 in T cell leukemogenesis by using both cell-based assays and T-ALL xenograft mouse model. With well-established in vitro and in vivo experimental conditions, the proposed study will not only identify UVRAG as a novel and critical player in Notch1-induced T-ALL, but will also illuminate new views on Notch1 regulation for the design of more effective treatment options for patients with leukemia/lymphoma.

Project Summary There is an urgent and unmet need for the development of safe and effective therapeutics against serious human pathogens such as Kaposi's sarcoma herpesvirus (KSHV), which causes significant morbidity and mortality in immune-compromised individuals and remains a clinical challenge. Currently, no FDA approved therapeutics or vaccines are available for KSHV infection. Given that KSHV and other human herpesviruses hijack host proteins and pathways to complete their replication cycles and spread from cell to cell, strategies targeting these pathways and mechanisms will provide broad-spectrum genotype coverage and a high barrier to drug resistance. Herpesviruses including KSHV have long been known to exploit the COPII-mediated secretory pathway for their maturation. However, the mechanisms governing this process remain less understood. This project will fill this gap, capitalizing on our recent discovery that the host nucleoside diphosphates kinase NM23-H2, an important regulator of COPII vesicle transport, is exploited by KSHV for their virion morphogenesis and egress. We found that viral Bcl-2 of KSHV (ks-Bcl-2) directly interacted with, stabilized, and activated NM23-H2 during lytic phase of KSHV. Loss of NM23-H2 or mutations in ks-Bcl-2 that abolished NM23-H2 interaction severely impaired virion production. We thus hypothesize that KSHV activates host protein NM23-H2 to exploit the COPII pathway for efficient virion assembly and release, unraveling a key checkpoint in virus lifecycle that can be targeted for new antiviral therapeutics. We now bring within this proposal a collaboration of experts in KSHV biology and in design of small-molecule inhibitors to identify cellular pathway responsible for virion production of KSHV and develop a new strategy to dampen virus transmission. To achieve this goal, we propose two specific aims, including (1) defining the molecular mechanism by which KSHV ks-Bcl-2 targets NM23-H2 to activate Sar1-mediated COPII transport for efficient virion assembly; and (2) targeting ks-Bcl-2-NM23-H2-mediated COPII mechanism to block KSHV propagation. These aims will be addressed using multidisciplinary approaches that integrate state-of-the-art genetic, biochemistry, live-cell imaging, and physiological assays. Together, we anticipate that our studies will identify host genes/pathways that function in virus assembly and egress, and provide compelling in vivo validation that targeting NM23-H2-dependent host mechanism can abrogate virus transmission within and between individuals.