Resolving sequencing-based HIV-1 epitranscriptomics

The collection of HIV-1 RNA chemical modifications and their functional consequences in viral gene expression, host interactions, and the viral life cycle, referred to as HIV-1 epitranscriptomics, remain incompletely understood. While the field is evolving, diverse modification discovery methods, cell lines, HIV-1 sequences, and bioinformatics methods make a consensus view of the HIV-1 epitranscriptome difficult to resolve. Here, we review methods for identifying and interpreting N⁶-methyladenosine (m⁶A), 5-methylcytosine (m⁵C), pseudouridine (Ψ), 2´-O-methylation (N_m), and N⁴-acetylcytidine (ac⁴C) modifications in HIV-1, including antibody-based selection methods, chemical-treatment-based selection methods, and detection by nanopore direct RNA sequencing. We recommend the adoption of the latter as a standardized sequencing strategy to enable better benchmarking across diverse studies and help resolve HIV-1 epitranscriptomics.