An HIV-1 Reference Epitranscriptome

Post-transcriptional chemical modifications to RNA, or the epitranscriptome, play important roles in RNA metabolism, gene regulation, and human disease, including viral pathogenesis. Modifications to the RNA viral genome and transcripts of human immunodeficiency virus 1 (HIV-1) have been reported, including methylation of adenosine (m⁶A) and cytosine (m⁵C), acetylation of cytosine, pseudouridylation (psi), and conversion of adenosine to inosine, and their effects on virus and host biology have been investigated. However, diverse experimental approaches have been used, making clear correlations across studies difficult to assess. To address this need, we propose the establishment of a reference HIV-1 epitranscriptome. We sequenced the model NL4-3 HIV-1 genome from infected Jurkat CD4+ T cells cells using the latest nanopore chemistry, custom RNA preparation methods, and commercial base-calling algorithms. This resulted in a reproducible sense and preliminary antisense HIV-1 epitranscriptome where m⁶A, m⁵C, psi, ands inosine could be identified by multiplexed base-calling. Multiplexed base-calling miscalled modifications due to sequence and neighboring modification contexts, which we demonstrate can be corrected with synthetic HIV-1 RNA fragments. We validate m⁶A modification sites with a small molecule inhibitor of methyltransferase-like 3 (METTL3), STM2457. We conclude that modifications do not change substantially under combination antiretroviral therapy (cART) treatment or in primary CD4+ T cells. Samples from patients living with HIV reveal conservation of certain modifications, such as m⁶A. Our approach and reference data offer a straightforward benchmark that can be adopted to help advance rigor, reproducibility, and uniformity across future HIV-1 epitranscriptomics studies.