Latent HIV-1 reactivation in transgenic mice requires cell cycle -dependent demethylation of CREB/ATF sites in the LTR

Abstract

We previously produced a line of transgenic mice that carried the HIV-1 genome deficient in the gene. Although the HIV-1 genome in the lymphocytes was dormant under normal physiological conditions, it could be reactivated by lipopolysaccharide (LPS) administration via induction of interleukin-1alpha/beta and tumour necrosis factor-alpha. In this report, we analysed further the reactivation mechanism of the latent HIV-1 using this transgenic mouse model. and methods: Possible involvement of CpG methylation in HIV-1 latency was examined by treating transgenic lymphocytes with a demethylating agent, 5'-azacytidine. CpG methylation in the HIV-1 long terminal repeat (LTR) was analysed using the bisulfite genomic sequencing method. As previous studies suggested that CpG demethylation depended on the cell cycle progression, we analysed the relation between cell cycle progression and LPS-induced reactivation of HIV-1 by labelling lymphocytes with an intracellular fluorescein, carboxyfluorescein diacetate succinimidyl ester. We found that 5'-azacytidine enhanced HIV-1 expression ninefold compared to treatment with LPS alone. Furthermore, HIV-1 p24 induction by LPS was observed only in cells that had undergone cell division, while induction was prevented in cells in which cell cycle progression was blocked either by mimosine, aphidicolin, or nocodazole. LPS-induced HIV-1 reactivation was associated with demethylation of two CpG sites located in the CREB/ATF binding sites in the HIV-1 LTR in a cell cycle-dependent manner. These observations indicate that cell cycle progression-dependent demethylation of the CREB/ATF sites in the LTR is crucial for the reactivation of latent HIV-1 genome in transgenic mice.