Human immunodeficiency virus-1 (HIV-1) is a retrovirus with a 10-kb single-stranded RNA genome. HIV-1 must express all of its gene products from the same primary transcript, which undergoes alternative splicing to produce diverse protein products, including structural proteins and regulatory factors 1 , 2 . Despite the critical role of alternative splicing, the mechanisms driving splice-site choice are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements 3 . We use DMS-MaPseq to probe the structure of HIV-1 RNA in cells and develop an algorithm called D etection of R NA folding E nsembles using E xpectation- M aximization (DREEM), which reveals alternative conformations assumed by the same RNA sequence. Contrary to previous models, which analyzed population averages 4 , our results reveal the widespread heterogeneous nature of HIV-1 RNA structure. In addition to confirming that in vitro characterized alternative structures for the HIV-1 Rev Responsive Element (RRE) exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis 5 – 7 that RNA conformation heterogeneity regulates splice site usage and viral gene expression.

Increased IFNα production contributes to the pathogenesis of infectious and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) from females produce more IFNα upon TLR7 stimulation than pDCs from males, yet the mechanisms underlying this difference remain unclear. Here, we show that basal levels of interferon regulatory factor 5 (IRF5) in pDCs were significantly higher in females compared to males and positively correlated with the percentage of IFNα-secreting pDCs. Delivery of recombinant IRF5 protein into human primary pDCs increased TLR7-mediated IFNα secretion. In mice, genetic ablation of the estrogen receptor 1 (Esr1) gene in the hematopoietic compartment or DC lineage reduced IRF5 mRNA expression in pDCs and IFNα production. IRF5 mRNA levels furthermore correlated with Esr1 mRNA levels in human pDCs, consistent with IRF5 regulation at the transcriptional level by Esr1. Taken together, these data demonstrate a critical mechanism by which sex differences in basal pDC IRF5 expression lead to higher IFNα production upon TLR7 stimulation in females, and provide novel targets for the modulation of immune responses and inflammation.

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