CTCF is a key insulator sequence that plays an important role in the 3D structure of chromatin, and also serves as a transcriptional repressor and a regulator of splicing, that has been shown to regulate latency of ?- herpesviruses. A single CTCF binding site was identified in HTLV-1, at the boundary within integrated proviruses between methylated and unmethylated DNA, and between inactive and active chromatin marks. The current project is among the first studies to examine the role of insulators in retrovirus biology and pathogenesis. For this purpose, we constructed a mutant virus lacking the CTCF binding site. We will compare the wild type and mutant virus in vitro (Aim1) and in vivo (Aim 2), with regards to regulation of viral RNA synthesis and splicing, viral protein and particle production, latency, immortalization, and in vivo, stimulation of immune responses and induction of leukemia.
In Aim 1, we will determine if loss of the CTCF binding site disrupts proviral DNA methylation and marks of chromatin silencing in the integrated provirus. Moreover, we will determine if this mutation affects viral transcription and splicing. In addition, we will use a novel model of viral latency in order to assess the effect of the mutation on establishment and/or reactivation from latency, and efficiency of lymphocyte immortalization.
In Aim 2, we will use humanized mouse and rabbit models of HTLV-1 infection. The rabbit model is an established system that will be used to assess the effects of the mutation on virus replication and antibody responses. Humanized mice model is a newly described model, in which we can analyze HTLV-1 replication, humoral and cellular immunity, and development of leukemia. In summary, this project will determine if CTCF is an important regulator of HTLV-1 replication and pathogenesis in vitro and in vivo. Data and reagents from this study will provide a new avenue of investigation of epigenomic regulation of HTLV-1 and potential novel therapy approaches.
The current application is for novel studies of the 3-dimensional structure of DNA with associated proteins (chromatin) in human T-cell leukemia virus (HTLV) infected cells. A specific protein, known as CTCF, binds to the HTLV genome when integrated in the cell, dividing the genome into inactive and active domains. The current study will examine an HTLV mutant that no longer binds CTCF. We will determine if this mutation affects the structure of the chromatin and modifications of the DNA of the integrated virus genome. We will determine, with studies in tissue culture, if this CTCF binding mutant has alterations in virus RNA and protein synthesis, virus particle production and infection of adjacent cells, as well as its ability to remain latent in or immortalize lymphocytes. With studies in infected rabbits, we will examine the rate of virus replication and the immune response to the virus. With infection studies in immunodeficient mice transplanted with a human lymphoid system, we will examine rates of virus replication, immune responses, and development of leukemia. This project opens a new field of investigation of control of retroviruses by proteins that regulate DNA and chromatin structure and activity. This could provide an avenue to novel therapeutics against oncogenic viruses, such as HTLV-1.
