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. 2020 Aug 6;10:13214. doi: 10.1038/s41598-020-70083-1

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Strain-specific E-box motif binding site for Tal1 and USF1 in the U5 region of CRF22_01A1 HIV-1 strain. (a) E-box canonical and sequence from nucleotide + 107 to + 140 in the U5 region. The CRF22-5′IRDDye-Ebox, which includes the perfect match of the E-box canonical motif (underlined) found in the CRF22_01A1, was used as a probe. Its counterpart from clade B was used as a mutated competitor. (b) The CRF22-5′IRDDye-Ebox oligonucleotide probe was incubated without (lane 1) or with Jurkat nuclear extract showing a DNA–protein complex (lane 2). The specificity of the Ebox-bHLH proteins complex was suggested by the addition of a 100-fold excess of unlabeled CRF22_01A1 sequence (lane 3), a non-specific poly(dI-dC) competitor (lane 4) or a mutated competitor (lane 5). Additionally, we observed a supershift in the E-box-bHLH complex in the presence of antibodies against TAL-1 (lane 8) and USF1 (lane 10). No supershift was seen in the presence of specific antibodies, such as anti-AP4 (lane 6). Also, there was no shift in the presence of the probe and antibodies alone (lanes 7 and 9).