Abstract 3791: Understanding the function of LMP1 CTAR3 in EBV-associated cancers
Authors: McGreal Analise , Minko Aimee , Visalli Melissa A. , Visalli Robert J. , Bentz Gretchen L. 
Published: August 2019

Abstract
Background: Over 90% of the human population is infected with Epstein-Barr virus (EBV), which has been linked to cancers such as Hodgkin’s lymphoma and nasopharyngeal carcinoma. The principal oncoprotein responsible for cellular proliferation, increased cell invasion, evasion of apoptosis, and metastasis observed in EBV-associated cancers is latent membrane protein-1 (LMP-1). LMP-1 constitutively activates multiple signal transduction pathways via its cytoplasmic C-terminal activating regions (CTARs). Although, the functions of CTAR1 and CTAR2 regions have been extensively defined, the functions of a third regulatory region, CTAR3, are less understood. CTAR3 participates in protein sumoylation, which regulates evasion of immune responses and oncogenesis. The CTAR3 region is composed of 11-amino acid repeats and JAK binding motifs that contribute to the sumoylating activity of LMP-1. Variation in the number of repetitive elements has been noted in clinical isolates, but the exact function of these elements in oncogenesis is unknown. Therefore, the present study aims to generate EBV LMP-1 CTAR3 mutations within the repetitive elements to study their role in oncogenesis.
Methods: Recombineering using an EBV-containing bacterial artificial chromosomes (BACs) was utilized to prepare mutant CTAR3 constructs with a N-terminal hemagglutinin (HA) epitope tag. Mutant BACs were transfected into HEK 293 cells and clones selected using hygromycin. Expression of mutant CTAR3 proteins was detected by immunoblot using HA antibody.
Results: Preliminary results indicate the successful generation of infectious EBV clones containing the following LMP-1 CTAR3 mutations: (1) HA - Wild Type, (2) HA - JAK Only, (3) HA - No JAK, and (4) HA - ΔCTAR3. Clones for each mutant were selected after transfection into HEK 293 cells. Expression of mutant CTAR3 proteins was confirmed via immunoblot. The ability of the mutants to produce infectious virus was determined after induction of HEK CTAR3 clones and infection of B cells (RAJI) with culture supernatant. All CTAR3 mutants produced infectious virus and expressed CTAR3 mutant proteins of the expected molecular weight.
Conclusions: An EBV-BAC was recombineered to create EBV strains with targeted mutations in CTAR3. The novel BAC mutants will be used to assess the function of CTAR3 domains in EBV replication and oncogenesis. The role that CTAR3 plays in envelopment and egress during lytic replication will be determined. Additionally, the role of CTAR3 during EBV-mediated immortalization of naïve B-cells will be examined. Ultimately, mutant EBV strains will be studied in a SCID mouse model to determine the role of CTAR3 sumoylation in cell migration and metastasis in vivo. Overall, further appreciation of the role of LMP-1 in tumorigenesis will identify potential targets for therapeutic intervention in patients with EBV-related cancers.
*Analise McGreal and Aimee Minko contributed equally to this work.
Citation Format: Analise McGreal, Aimee Minko, Melissa A. Visalli, Robert J. Visalli, Gretchen L. Bentz. Understanding the function of LMP1 CTAR3 in EBV-associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3791.