DMS-MrHAMER2

In addition to their role as carriers of genetic information, HIV-1 viral RNAs (vRNAs) widely interact with proteins and other RNAs via structured domains to control critical steps in viral pathogenesis, including: splicing, export, stability, translation, and packaging. Despite the wide repertoire of non-coding viral functions that are modulated by RNA structures, an understanding of vRNA structural dynamics beyond the 5' leader remains elusive during early viral transcription due to limitations in current RNA structure probing methods. This has resulted in conflicting evidence on how the temporal dynamics of viral gene expression are regulated: is the equilibrium of spliced vRNA available for translation of viral proteins a function of vRNA structure changes that affect splice junction accessibility, or is it a function of Rev-mediated changes in export of vRNAs containing the Rev-response Element (RRE)? The state-of-the-art in in vivo interrogation of vRNA secondary structure is structure-specific chemical probing with DMS (dimethyl sulfate) or SHAPE (Selective 2´-Hydroxyl Acylation analyzed by Primer Extension) probes, followed by Mutational Profiling (MaP) via short-read next-generation sequencing (NGS). However, a major technological gap exists: the short reads obtained in these structure probing methods cannot deconvolute the prodigious vRNA heterogeneity that manifests as isoform-specific variation during RNA splicing, or as long-range structural variation during viral maturation. In the context of RNA processing/export during early viral transcription, the inability to probe isoform-specific structural variation means that DMS or SHAPE signals cannot be associated with RRE-containing Partially Spliced (Env/Vpu, Vif, Vpr) or with Fully Spliced (Nef, Tat, Rev) vRNAs . To address roadblocks in interrogating vRNA structural heterogeneity, we developed a full-length MaP approach for in vivo single-molecule RNA structure probing using DMS, an approach termed DMS-MrHAMER. This methodology couples our accurate long-read sequencing capabilities (MrHAMER2) with our validated vRNA enrichment and isoform sequencing and analysis strategies to enable DMS chemical probing of both isoform-specific and long-range vRNA structure variation. Compared to the state-of-the-art approaches, our methodology shows 10-fold higher signal-to-noise ratio (SNR) and faithful end-to-end coverage of vRNA, which will enable significant improvements in the accuracy and throughput of structural predictions. We will apply DMS-MrHAMER2 to establish the spatiotemporal dynamics of HIV vRNA architecture in discrete steps of the viral lifecycle with particular focus on the dynamics of vRNA splicing and export during early viral transcription in primary cells.