The Silencing of Human Immunodeficiency Virus

Abstract

A PERSPECTIVE OF RETROVIRAL GENOME METHYLATION
Ten years ago, when I first began to study the methylation of retrovirus genomes, we had quite literally stumbled upon the observation that certain cell lines had the ability to silence viral promoters that were artificially introduced via the introduction of foreign DNA or “transfection.” Most of these experiments were performed using a heterologous promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene. In the case of the HIV-1 long terminal repeat (LTR) linked to the CAT gene, we found that African green monkey cells (VERO) stably transfected with this construct lost the ability to express the CAT mRNA (Bednarik et al. 1987). Rather than discard our stable cell lines that lacked CAT expression, we attempted to stimulate expression by addition of the 5-methylcytosine analog, 5-azacytidine. The significance of this drug is its ability to cause the demethylation or “hypomethylation” of genomic DNA during replication. To our amazement, CAT activity was quickly restored, and even more interesting was the observation that 5-azacytidine worked in a very short period of time, as soon as 8 hours after addition of the drug. This observation launched the next plethora of experiments, which involved testing a spectrum of reagents and physiochemical stimuli, all targeted at LTR reactivation. In this chapter, I present a brief perspective of viral genome methylation and then focus on our most recent observations regarding the methylation of the HIV LTR and how this process might be involved in the establishment of transcriptionally silenced...