The latent HIV-1 reservoir is populated with clones of cells infected with stably integrated, intact, but transcriptionally-silent proviruses. Previously, we described the integration site of one such clone harboring a replication-competent provirus called AMBI-1. We hypothesized that the silencing of HIV-1 gene expression from this and other clones was due to DNA methylation of the 5’LTR promoter. To address this question, we investigated methylation at the single-proviral level in known CpG islands in the HIV-1 proviral genome, including one in the 5′ LTR promotor region.
Using a bisulfite-based, methylation-specific single-genome-sequencing (SGS) assay, we measured the levels of methylation in CpG islands from ART-suppressed, chronically-infected individuals with samples from PBMC and lymph node mononuclear cells (LNMC) (including the donor with the AMBI-1 clone).
From 4 individuals an average of 30 (range: 13 to 91) bisulfite-treated SGS were obtained from each PBMC and LNMC sample. We found no significant difference in any provirus between the level of methylation of the CpG island in the 5′ LTR promoter and the assay background of cytosines not in CpG sites (averaging 3.8% and 3.3% respectively p=0.9). Furthermore, the presumed AMBI-1 provirus (matching LTR sequence) was not found to be methylated above assay background. Interestingly, we did find a significantly higher level of methylation in the CpG island in the env-tat-rev overlapping reading frame in multiple proviruses in each of the samples [averaging 21% of all CpG sites methylated vs. an average of 6% assay background (p=0.03)]. In each PBMC and LNMC sample, 78% of genomes were methylated at >1 CpG site in the env-tat-rev island and 46% were methylated at ≥3 CpG sites.
Surprisingly, we did not find evidence that methylation of the 5’LTR promotor maintains HIV-1 latency in vivo, including LTRs of proviruses that are known to be intact and latent. Significant levels of methylation were found in a CpG island in env but its role, if any, in transcriptional silencing is unknown. Since it is well known that methylation of transcriptional enhancers located many kilobases from mRNA start sites can result in gene silencing, it is important to determine if the identified methylated CpG island in the env gene has any function in HIV-1 latency in vivo.