Fig. 1

Construction of HTLV-1 proviral clones. Site-directed mutagenesis was utilized to abrogate CTCF binding. a Alignment of the consensus CTCF-binding sequence with HTLV-1, HTLV-1p12Stop, and HTLV-1∆CTCF in the context of accessory gene p12. HTLV-1∆CTCF contains mutations that abolish CTCF binding (blue). These mutations disrupt the p12 reading frame, therefore a stop mutation (red) that truncates p12 by 23 amino acids was introduced immediately upstream. HTLV-1p12Stop serves as a control by containing only the aforementioned stop codon (red). b Abolition of CTCF binding was confirmed via electrophoretic mobility shift assay. EMSA was performed using the Light Chemiluminescent EMSA kit (Thermo Scientific) and following the manufacturer’s protocol with some modification. Briefly, nuclear extract of 293T cells transfected with the plasmid overexpressing human CTCF protein was incubated with biotin labeled target DNA in the presence or absence of the CTCF antibody. Protein bound DNA was separated from unbound DNA in a polyacrylamide gel and transferred to a nylon membrane. DNA was then cross-linked to the membrane. The membrane was incubated with streptavidin–horseradish peroxidase conjugate in blocking buffer and then exposed to the substrate solution. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad)