# Role of the CTCF binding site in Human T-Cell Leukemia Virus-1 pathogenesis

## TL;DR

This study shows that a specific DNA site in the HTLV-1 virus affects how the virus spreads and causes disease in mice.

## Contribution

The study demonstrates the role of the CTCF binding site in HTLV-1 pathogenesis using a humanized mouse model.

## Key findings

- Mutation of the CTCF binding site in HTLV-1 delayed disease progression and reduced proviral load in mice.
- The CTCF binding site regulates Tax expression and T cell activation in vivo.
- Proviral load in peripheral blood correlated with mortality in mice infected with the mutated virus.

## Abstract

During HTLV-1 infection, the virus integrates into the host cell genome as a provirus with a single CCCTC binding protein (CTCF) binding site (vCTCF-BS), which acts as an insulator between transcriptionally active and inactive regions. Previous studies have shown that the vCTCF-BS is important for maintenance of chromatin structure, regulation of viral expression, and DNA and histone methylation. Here, we show that the vCTCF-BS also regulates viral infection and pathogenesis in vivo in a humanized (Hu) mouse model of adult T-cell leukemia/lymphoma. Three cell lines were used to initiate infection of the Hu-mice, i) HTLV-1-WT which carries an intact HTLV-1 provirus genome, ii) HTLV-1-CTCF, which contains a provirus with a mutated vCTCF-BS which abolishes CTCF binding, and a stop codon immediately upstream of the mutated vCTCF-BS which deletes the last 23 amino acids of the p12 gene, and iii) HTLV-1-p12stop that contains the intact vCTCF-BS, but retains the same stop codon in p12 as in the HTLV-1-CTCF cell line. Hu-mice were infected with mitomycin-treated or irradiated HTLV-1 producing cell lines. There was a delay in pathogenicity when Hu-mice were infected with the HTLV-1-CTCF virus compared to mice infected with either HTLV-1-p12 stop or HTLV-1-WT virus. Proviral load (PVL), spleen weights, and CD4 T cell counts were significantly lower in HTLV-1-CTCF infected mice compared to HTLV-1-p12stop infected mice. Furthermore, we found a direct correlation between the PVL in peripheral blood and death of HTLV-1-CTCF infected mice. In cell lines, we found that the vCTCF-BS regulates Tax expression in a time-dependent manner. The scRNAseq analysis of splenocytes from infected mice suggests that the vCTCF-BS plays an important role in activation and expansion of T lymphocytes in vivo. Overall, these findings indicate that the vCTCF-BS regulates Tax expression, proviral load, and HTLV pathogenicity in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) is a cause of leukemia and lymphoma, and several inflammatory medical disorders. The virus integrates in the host cell DNA, and it includes a single binding site for a cellular protein designated CTCF. This protein is important in regulation of many viruses, as well as properties of normal and malignant cells. In order to define the role of CTCF in HTLV-1 pathogenesis in vivo, we analyzed a mutant virus lacking the binding site in humanized mice. We found that this mutation slowed virus spread and attenuated the development of disease. Gene expression studies demonstrated a dynamic role of CTCF in regulating viral gene expression and T lymphocyte activation.