Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As

Alice Baek^1,2,3#, Ga-Eun Lee^1,2,3,4#, Sarah Golconda^1,2,3, Asif Rayhan⁵, Anastasios A. Manganaris^4,6, Shuliang Chen^1,2, Nagaraja Tirumuru^1,2, Hannah Yu^1,2,3, Shihyoung Kim^1,2,3, Christopher Kimmel^2,4, Olivier Zablocki^7,8, Matthew B Sullivan^7,8,9, Balasubrahmanyam Addepalli⁵, Li Wu¹⁰, and Sanggu Kim^1,2,3,4,11*

¹Center for Retrovirus Research, The Ohio State University, Columbus, OH, USA

²Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, USA

³Infectious Diseases Institute, The Ohio State University, Columbus, OH, USA

⁴Translational Data Analytics Institute, The Ohio State University, Columbus, OH, USA

⁵Rieveschl laboratories for mass spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA

⁶Department of Computer Science and Engineering, The Ohio State University, Columbus, OH, USA

⁷Center of Microbiome Science, Ohio State University, Columbus, OH, USA

⁸Department of Microbiology, Ohio State University, Columbus, OH, USA

⁹Department of Civil, Environmental and Geodetic Engineering, Ohio State University, Columbus, OH, USA

¹⁰Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.

¹¹Center for RNA Biology, The Ohio State University, Columbus, OH, USA.

^#These authors contributed equally: Alice Baek, Ga-Eun Lee

^*Corresponding author: Sanggu Kim,✉e-mail:kim.6477@osu.edu

SUMMARY

Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in 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 analyzed chemical modifications on HIV-1 RNAs at the full-length, single-RNA level, and nucleotide resolution using novel direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N6-methyladenosine (m6A) modifications near the 3' end. More densely installed in spliced viral mRNAs than in genomic RNAs, these m6As play a crucial role in maintaining normal levels of RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m6A ensembles, and maintaining multiple of these m6As on its RNAs provides additional stability and resilience to HIV-1 replication—an unexplored RNA-level evolutionary strategy.

USAGE

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