Comprehensive Transcriptome Annotation of Thousands of HIV-1 Genomes

Alternative splicing in HIV-1 has been a central focus of decades of research, uncovering key mechanisms of viral gene regulation, immune evasion, and therapeutic response - yet, no reference resource has existed to support transcriptome-wide analysis, limiting adoption of modern computational methods. We present HIV Atlas (https://ccb.jhu.edu/HIV_Atlas), the first reference-quality annotation of HIV-1 and SIV transcriptional diversity. We manually curated transcriptomes for HIV-1_HXB2 and SIV_mac239 and developed Vira, an automated annotation-transfer method specifically designed to address unique challenges of viral genome biology, to generate high-quality annotations for 2,077 complete HIV-1 genomes. Using the resources presented in our work, we evaluated conservation of splice sites, revealing near-perfect preservation of major donors and acceptors. Furthermore, using several public datasets, we demonstrate how HIV Atlas enhances methodology, improves the quality and novelty of results, and opens novel avenues for research, supporting more accurate and comprehensive analyses of bulk, single-cell, and spatial RNA-seq in HIV-1 studies.