Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As
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Abstract
Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here we analysed chemical modifications on HIV-1 RNAs at the full-length, single RNA level and nucleotide resolution using direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N 6 -methyladenosine (m 6 A) modifications near the 3′ end. More densely installed in spliced viral messenger RNAs than in genomic RNAs, these m 6 As play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m 6 A ensembles, and maintaining multiple of these m 6 As on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy.
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Demethylated RNA is more efficiently packaged, while the methylated form (m6A-marked RNA) supports translation.
“demethylated RNA is more efficiently packaged, while the methylated form (m6A-marked RNA) supports translation”
From Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication (2026) · cites this paper as Baek et al. 2024
The Results (‘Higher m6A stoichiometry on HIV-1 mRNAs than genomic RNA’; ‘Triple m6A mutations reduced HIV-1 protein translation’) report more m6A on translating HIV-1 mRNAs and reduced translation after mutation of three m6A sites. But the packaging claim is stronger than the paper’s finding: the Results say fewer m6As on genomic RNA *may* be favoured during packaging, and the Discussion says the mechanism needs further investigation. The paper does not establish that demethylated RNA is packaged more efficiently. (LLM, conf=0.87)
Experimental — accuracy not yet measuredRNA modifications, particularly N6-methyladenosine (m6A), impact gRNA fate.
“RNA modifications, particularly N6-methyladenosine (m6A), impact gRNA fate.”
From Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication (2026) · cites this paper as Baek et al. 2024
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RNA modifications detection by comparative Nanopore direct RNA sequencingPreprint
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Epitranscriptomic addition of m 6 A regulates HIV-1 RNA stability and alternative splicingPreprint
Tsai, K., Bogerd, H. P., Kennedy, E. M., Emery, A., Swanstrom, R. I., & Cullen, B. R. (2021) · Genes & Development · doi:10.1101/gad.348508.121 · Cited by 68 · see citing works
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A call for direct sequencing of full-length RNAs to identify all modifications
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From A to m6A: The Emerging Viral Epitranscriptome
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Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases
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Self-citationVisualizing the translation and packaging of HIV-1 full-length RNA
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METTL3 regulates breast cancer-associated alternative splicing switches
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Epitranscriptomic regulation of HIV-1 full-length RNA packaging
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Short- and long-range interactions in the HIV-1 5′ UTR regulate genome dimerization and packaging
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Nanopore-Based Detection of Viral RNA Modifications
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Identification of host proteins differentially associated with HIV-1 RNA splice variants
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Self-citationFunctional Impacts of Epitranscriptomic m6A Modification on HIV-1 Infection
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Self-citationQuantitative Evaluation of HIV and SIV Co-Receptor Use With GHOST(3) Cell Assay
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Anomalous HIV-1 RNA, How Cap-Methylation Segregates Viral Transcripts by Form and Function
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Sequestering the 5′‐cap for viral RNA packaging
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Alternative splicing modulation by G-quadruplexesPreprint
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Functional and structural segregation of overlapping helices in HIV-1
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Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS
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Self-citationProtocol for the generation of HIV-1 genomic RNA with altered levels of N6-methyladenosine
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Self-citation
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Functional Impacts of Epitranscriptomic m6A Modification on HIV-1 Infection
Mishra, T., Phillips, S. L., Huang, S., & Wu, L. J. (2024) · Viruses · doi:10.3390/v16010127 · Cited by 24 · see citing works
Self-citationMolina, E. M., Bech‐Serra, J. J., Franco‐Trepat, E., Jarne-Sanz, I., Perez‐Zsolt, D., Badía, R., Riveira‐Muñoz, E., García-Vidal, E., Sancho, L. R., Franco, S., et al. (2025) · Nature Communications · doi:10.1038/s41467-025-56151-y · Cited by 20 · see citing works
Epitranscriptomic m 6 A modifications during reactivation of HIV-1 latency in CD4 + T cells
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Self-citationRecent insights into N6-methyladenosine during viral infection
Horner, S. M., & Reaves, J. V. (2024) · Current Opinion in Genetics & Development · doi:10.1016/j.gde.2024.102213 · Cited by 18 · see citing works
Yi, Q., Liao, Y., Sun, W., Li, J., Yang, D., Shang, H., & Sun, W. (2025) · International Journal of Molecular Medicine · doi:10.3892/ijmm.2025.5605 · Cited by 17 · see citing works
Advances in nanopore direct RNA sequencing and its impact on biological research
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Nanopore direct RNA sequencing and the epitranscriptome: Advances in mapping native RNA landscapes
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Quantitative and Multiplexing Analysis of MicroRNAs by Direct Full-Length Sequencing in Nanopores
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HIV-1 Infection Reduces NAD Capping of Host Cell snRNA and snoRNA
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Inhibition of ALKBH5 demethylase of m6A pathway potentiates HIV-1 reactivation from latency
Ali, H., Wadas, J., Bendoumou, M., Chen, H., Maiuri, P., Dutilleul, A., Selberg, S., Nestola, L., Lalik, K., Avettand-Fènoël, V., et al. (2025) · Virology Journal · doi:10.1186/s12985-025-02744-4 · Cited by 9 · see citing works
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Nanopore sequencing in veterinary medicine: from concepts to clinical applications
Chaves, M., Hashish, A., Goraichuk, I. V., Caserta, L. C., Mears, M. C., Gadu, E., Bakre, A. A., Morris, E. R. A., Shelkamy, M. M. S., Nadendla, S., et al. (2025) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2025.1701570 · Cited by 8 · see citing works
YTHDF1 and YTHDC1 m 6 A reader proteins regulate HTLV-1 tax and hbz activity
King, E. M., Midkiff, A., McClain, K., Kim, S., & Panfil, A. R. (2025) · Journal of Virology · doi:10.1128/jvi.02063-24 · Cited by 8 · see citing works
Self-citationHuang, S., Zhao, Y., Phillips, S. L., Warrick, J. E., Kearse, M. G., He, C., & Wu, L. J. (2025) · Journal of Virology · doi:10.1128/jvi.01536-25 · Cited by 7 · see citing works
Self-citationAdvancing DNA and RNA Modification Detection via Nanopore Sequencing
He, B., Fan, Y., Liu, J., Chen, Y., Yang, J., Xiang, H., Peng, J., & Yi, C. (2025) · ACS Nano · doi:10.1021/acsnano.5c11784 · Cited by 7 · see citing works
Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS
Baek, A., Rayhan, A., Lee, G.-E., Golconda, S., Yu, H. J., Kim, S., Limbach, P. A., Addepalli, B., & Kim, S. (2024) · Methods and Protocols · doi:10.3390/mps7010007 · Cited by 7 · see citing works
Self-citationMishra, T. K., Phillips, S. L., Maldonado, C., Stapleton, J. T., & Wu, L. J. (2024) · AIDS Research and Human Retroviruses · doi:10.1089/aid.2024.0003 · Cited by 7 · see citing works
Self-citationRetroviral PBS-segment sequence and structure: Orchestrating early and late replication events
Heng, X., Herrera, A. P., Song, Z., & Boris‐Lawrie, K. (2024) · Retrovirology · doi:10.1186/s12977-024-00646-x · Cited by 6 · see citing works
Mears, M. C., Read, Q. D., & Bakre, A. A. (2024) · Journal of Virological Methods · doi:10.1016/j.jviromet.2024.115103 · Cited by 6 · see citing works
Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication
Jehad, M. A., Ali, L. M., Pillai, V. N., Prabhu, S. G., Mustafa, F., & Rizvi, T. A. (2026) · FEMS Microbiology Reviews · doi:10.1093/femsre/fuaf066 · Cited by 5 · see citing works
“It has been shown that demethylated RNA is more efficiently packaged, while the methylated form (m 6 A-marked RNA) supports translation (Pereira-Montecinos et al. 2022 , Singh et al. 2022b , Baek et al. 2024 ).”
When animal viruses meet N6-methyladenosine (m6A) modifications: for better or worse?
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Self-citationRNA modification is the mark and strategy for host-microbe interactions
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Emerging role of SETD2 in the development and function of immune cells
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Past, Present, and Future of RNA Modifications in Infectious Disease Research
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A dual context-aware basecaller for nanopore direct RNA sequencing
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RNA ‐centric world of retroviruses: unravelling the molecular strategies of genomic RNA packaging
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Pharmacological METTL3 inhibition attenuates HIV-1 latency reversal in CD4 + T cells
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Self-citationDetection of Base Modifications Using Long-Read Sequencing: Methods, Applications, and Challenges
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Resolving sequencing-based HIV-1 epitranscriptomics
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APHIX: Analysis Pipeline for HIV-1 Isoform eXploration Using Long-read RNA Sequencing DataPreprint
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Transcriptomic and Epitranscriptomic Landscape of Integrated HTLV-1 in MT2 Cells
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Advanced deep learning strategies in nanopore RNA sequencing
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Advantages and Challenges of G-Quadruplexes in Regulating Alternative Splicing
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Self-citationSUMOylated viral polymerase reprograms host epitranscriptome to promote infection in plantsPreprint
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- Authors
- Baek, A., Lee, G.-E., Golconda, S., Rayhan, A., Manganaris, A. A., Chen, S., Tirumuru, N., Yu, H., Kim, S., Kimmel, C., Zablocki, O., Sullivan, M. B., Addepalli, B., Wu, L., & Kim, S.
- Affiliations
- The Ohio State University (US)
- University of Cincinnati (US)
- University of Iowa (US)
- Corresponding author
- Kim, Shihyoung
- Kim, Sanggu
- ORCID
- Venue
- Nature Microbiology
- Published
- 2024-04-11
- Volume
- 9
- Issue
- 5
- Pages
- 1340-1355
- ISSN
- 2058-5276 (ISSN-L)
- PMC
- PMC11087264
- OpenAlex
- W4394718034
- Cited by
- 52
- References
- 112
- Citations by year
- 2026: 13 · 2025: 26 · 2024: 13
- FWCI
- 7.08
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Nature Microbiology (publishedVersion, cc-by)
- Research Square (acceptedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- Funders
- U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (R01HG010318, HG010108)
- U.S. Department of Defense (HT9425-23-1-0582)
- University of Cincinnati
- U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (R61AI169659, R21AI170070)
- U.S. Department of Energy (248445, DE-SC0023307)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- HIV-1, N6-methyladenosine, epitranscriptomics, RNA modification, single-molecule RNA sequencing, viral RNA splicing, viral RNA translation, RNA stability, 3'-end modification, HIV-1 replication
- MeSH terms
- Adenosine, Humans, RNA Splicing, RNA, Messenger, RNA, Viral, Virus Replication, HIV-1, HIV Infections, Sequence Analysis, RNA, Transcriptome
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