Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

34-POS Board 34 DNA Methylation Analysis to Reveal Latent HIV Reactivation Mechanism in Single Cell Yutaka Negishi 1 , Musa Mhlanga 2 . 1 RIKEN, Yokohama, Japan, 2 Council for Scientific and Industrial Research, Pretoria, South Africa. Despite the suite of effective anti-HIV drugs available, curation of HIV infection is still difficult due to the presence of latent provirus in resting memory T cells. This latent reservoir is established early stage during HIV infection and remains a major barrier to curing HIV infection because latent HIV is unaffected to anti-HIV drugs. One approach to make anti-HIV drugs more effective is reactivating latent HIV artificially. Although some reagents can reactivate latent HIV, it is difficult to reactivate all latent HIV. Therefore, understanding reactivation mechanism of latent HIV in single cell level is important to cure the HIV. Here, we propose to develop a novel technique able to reveal the methylation status of latent HIV in single cell level based on the adaptation of the padlock FISH approach. DNA methylation in the HIV provirus 5′ or 3’ long terminal repeat (LTR) is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and anti- HIV drugs. In our technique, DNA is digested with a DNA methylation sensitive restriction enzyme such as HpaII, and subsequently generated single strand by lambda 5’-3’exonuclease. The padlock probes, which contain the sequence of HIV 5’ and 3’ LTR near the restriction site and detection sequence, are annealed to the target DNA and the ends of the probes are circularized by ligation. Then, phi29 DNA polymerase amplifies circularized DNA by rolling- circle amplification and amplified DNA product is detected by hybridization of fluorescent- labelled oligonucleotides. In this way, we will be able to specifically visualize integrated provirus that is methylated and reveal methylation status in 5’ and 3’ LTR. Our technique will provide a novel approach to develop novel drugs to reactivate latent HIV.

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