Poster abstracts
Poster number 40 submitted by Michael Martinez
Role of Chromatin Insulator CTCF in HTLV-1 Retroviral Pathogenesis
Michael P. Martinez (Department of Veterinary Biosciences, The Ohio State University, Columbus, OH), Jacob J. Al-Saleem (Department of Veterinary Biosciences, The Ohio State University, Columbus, OH), Amanda R. Panfil (Department of Veterinary Biosciences, The Ohio State University, Columbus, OH), Lee Ratner (Department of Medicine, Washington University, St. Louis, MO), Patrick L. Green (Department of Veterinary Biosciences, The Ohio State University, Columbus, OH)
Abstract:
Human T-cell leukemia virus (HTLV-1) is a deltaretrovirus endemic to the Caribbean and Japan. HTLV-1 is the etiologic agent of adult T-cell leukemia and the neurological disorder HAM/TSP. Approximately 5-10% of infected individuals will develop disease after a prolonged latency. The exact mechanisms through which this variable latency is regulated remains nebulous. CCCTC-binding factor (CTCF) is an 11-zinc finger transcriptional repressor that acts through the induction of conformational changes in chromatin structure and subsequent enhancer-blocking activity. A CTCF-binding site was recently identified within the HTLV-1 provirus and shown to affect the anti-sense derived hbz transcript. HBZ provides tumor maintenance function and supports viral persistence. Therefore, we propose to study the epigenetic effects of CTCF on HTLV-1-induced in vitro immortalization using short-term viral co-culture assays. Using an HTLV-1 proviral molecular clone, a ∆CTCF mutant was generated using site-directed mutagenesis. The ∆CTCF mutation prevents CTCF-binding via EMSA, but does not disrupt overlapping reading frames or splice sites. The ∆CTCF mutant is transcriptionally similar to WT, as demonstrated by LTR-based reporter gene assays and viral gag release. Since efficient in vitro infection of naïve T-cells by HTLV-1 requires co-cultivation with infected cells, we next used stable transfection of the WT and ∆CTCF proviral clones into a susceptible human B-cell line to produce consistent and quantifiable amounts of virus. Briefly, irradiated viral producer cells are co-cultured with freshly isolated peripheral blood lymphocytes (PBL). The initiation of transformation is apparent within 5-6 weeks following co-culture as detected by expansion of cells from the PBL cell population. Preliminary results indicate the ∆CTCF mutant has similar immortalization potential compared to WT. Future experiments will examine the effect of the CTCF mutation on viral persistence using a rabbit model of infection. Ultimately, understanding epigenetic regulation of HTLV-1 latency could provide meaningful insights into mechanisms of immune evasion.
Keywords: CTCF, HTLV-1, ATL