DNA methylation may be important in maintaining latency of AIDS virus. It has been demonstrated that virus activation is induced in cells harboring latent virus by treatment with 5-azacytidine, a demethylating agent (Bednarik et al., J. Virol. 61, 1253-1257, 1987). Although it is well established that methylation at regulatory DNA sequences represses transcription of the associated gene (Cedar, Cell 53, 3-4, 1988), the mechanism is not known. DNA methylation may repress transcription by masking binding sites for trans-activating proteins or by binding repressor proteins. The research proposed here will systematically examine the effects on transcription of DNA methylation in the HIV-1 long terminal repeat (LTR), the segment of the AIDS virus genome which regulates transcription. Experimental plasmids will be constructed such that the HIV-1 LTR, methylated at one or more sites, will regulate transcription of a reporter gene. Control plasmids will be constructed in the same manner but without methylated sites. These will be transfected into cultured T helper cells and expression of the reporter gene will be assayed. After determining whether methylated sites block transcription, those sites responsible will be analyzed further. The protein-DNA interactions of these methylated and unmethylated sites will be compared in nuclear extracts. This research will determine which element in the LTR repress transcription when methylated and if repression involves altered binding of transcription factors to methylated sequences. Understanding the role of DNA methylation in regulating HIV-1 transcription may lead to methods to maintain viral latency in infected individuals.
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