Decoding the spliced HIV-1 transcriptome with accurate long-read RNA sequencing

Abstract Following integration into the host genome, HIV co-opts host-cell transcriptional machinery to regulate its activation and proliferation. The resulting viral RNAs (vRNAs) are subject to extensive alternative splicing, generating highly heterogenous isoform mixtures that are critical for the regulation of HIV gene expression. In this context, a major technological gap exists — short-read sequencing is unable to deconvolute alternatively-spliced HIV vRNAs, preventing effective interrogation of viral gene expression dynamics. To fully decode vRNA heterogeneity and glean insights into the processes that drive HIV persistence, we developed a highly accurate long-read RNA sequencing methodology to quantitatively assess viral transcriptional dynamics in contexts where HIV-infected cells transition to quiescence to evade immune surveillance. This nanopore sequencing-based approach, termed MrHAMER 2.0, couples a novel transcript enrichment strategy with dual UMIs for the accurate and unbiased PCR amplification of rare singlemolecule viral transcripts, resulting in unambiguous end-to-end isoform assignment, along with per nucleotide accuracy levels exceeding those in Illumina sequencing. We have applied MrHAMER 2.0 using the new Oxford Nanopore Q20+ chemistries to understand the transcriptional program in HIV-infected cells that drives viral persistence and is a barrier to cure strategies. We are exploring the viral transcriptional signatures gleaned from these studies as possible targets for new therapeutic approaches or to improve ‘functional cure’ strategies that rely on modulation of viral transcription and activation. Biography Christian Gallardo is an RNA genomics expert who develops long-read RNA sequencing methods to decode RNA interactions and dynamics during RNA processing, viral infections, and gene delivery. Christian has been sequencing long RNAs using Oxford Nanopore technology for over six years and is currently leading programs on RNA structure/function and mutational profiling of viral RNAs in the context of host-pathogen interactions. Christian is a Senior Scientist at the Center of Immunity and Immunotherapies at Seattle Children’s Research Institute. He completed his PhD in Genomics at Scripps Research and his Master’s in Drug Discovery and Development at the Keck Graduate Institute