An HIV-1 Reference Epitranscriptome.

Area of Science:

• Molecular Biology
• Virology
• Genomics

Background:

• The epitranscriptome, encompassing RNA modifications, is crucial for RNA metabolism, gene regulation, and diseases like viral pathogenesis.
• Previous studies on human immunodeficiency virus 1 (HIV-1) epitranscriptome modifications (e.g., m6A, m5C, pseudouridylation, inosine) used diverse methods, hindering direct comparisons.

Purpose of the Study:

• To establish a standardized reference HIV-1 epitranscriptome for improved reproducibility and comparability across studies.
• To investigate the impact of combination antiretroviral therapy (cART) and primary CD4+ T cell environments on HIV-1 RNA modifications.

Main Methods:

• Sequencing of the NL4-3 HIV-1 genome in infected Jurkat CD4+ T cells using nanopore technology.
• Development of custom RNA preparation and multiplexed base-calling algorithms for modification detection.
• Validation of m6A sites using a methyltransferase-like 3 (METTL3) inhibitor (STM2457) and synthetic RNA fragments for correction.

Main Results:

• A reproducible sense and preliminary antisense HIV-1 epitranscriptome was generated, identifying m6A, m5C, pseudouridylation, and inosine.
• Correction of miscalled modifications was achieved using synthetic HIV-1 RNA fragments.
• HIV-1 RNA modifications showed minimal changes under cART treatment or in primary CD4+ T cells, with conserved m6A sites observed in patient samples.

Conclusions:

• The developed approach provides a benchmark for HIV-1 epitranscriptomics, enhancing rigor and uniformity.
• The reference data facilitates a deeper understanding of HIV-1 RNA modifications and their role in viral pathogenesis.
• Conserved epitranscriptomic marks like m6A in patient samples suggest their potential significance in HIV-1 biology.