Epigenome-wide characterization reveals aberrant DNA methylation of host genes regulating CD4+ T cell HIV-1 reservoir size in women with HIV

The underlying mechanism of the HIV-1 reservoir, a major barrier to an HIV cure, is largely unknown. The integration of HIV-1 DNA and immune defense mechanisms can disrupt the host epigenetic landscape, potentially silencing HIV-1 replication. Using bisulfite capture DNA methylation sequencing, we profiled approximately 3.2 million CpG sites in CD4 ⁺ T cells isolated from the blood of 427 virally suppressed women with HIV. The average total CD4 ⁺ T cell HIV-1 Reservoir (HR _CD4 ) size was 1,409 copies per million cells. Most proviruses were defective with only a small proportion being intact. We found 245 differentially methylated positions (CpG sites) and 85 methylated regions associated with the total HR _CD4 size. Notably, 52% of significant methylation sites were in intronic regions. HR _CD4 -associated genes were involved in viral replication (e.g., ISG15 ), HIV-1 latency (e.g., MBD2 ), and cell growth and apoptosis (e.g., IRF9 ). A subset of the identified genes with aberrant methylation was an established target of HIV-1 integration (e.g., NFIA, SPPL3, DLEU2, ELMSAN1 ). Overall, HR _CD4 size was inversely associated with DNA methylation of interferon signaling genes and positively associated with methylation at established HIV-1 integration sites. HR _CD4 -associated genes were enriched in pathways including immune defense against the virus (i.e., interferon-α response and interferon-γ response), DNA binding transcription repression, and host-virus interaction such as Tau protein binding. Together, our results show that epigenomic alterations in CD4 ⁺ T cells are associated with total HIV-1 reservoir size, offering new insights into HIV-1 latency and may provide potential molecular targets for future HIV-1 eradication strategies.