Human Cytomegalovirus long non-coding RNA counteracts nuclear cGAS to facilitate immune evasion

Viruses have evolved diverse immune evasion strategies including targeting host pattern recognition receptors. The role of viral non-coding RNAs in modulating PRR activity is unclear. Here, we show that human cytomegalovirus (HCMV) produces long non-coding RNA4.9 that counteracts nuclear cyclic GMP-AMP synthase (cGAS)-mediated immune response to facilitate viral infection in primary human foreskin fibroblasts (HFFs). RNA4.9 interacts with host cGAS via its 75-nucleotide (nt) RNA region with predicted hairpin loops. Binding of RNA4.9 to cGAS inhibits cGAS enzymatic activity and downstream interferon response, and facilitates productive viral replication. Sterically blocking the folding of 75-nt region with antisense oligonucleotides during HCMV infection restores cGAS activity and impairs viral replication. We also found that the specific localization of RNA4.9, which concentrates near HCMV DNA, is correlated with its efficient binding to cGAS and subsequent immune suppression. Our findings demonstrate viral non-coding RNAs as critical cGAS regulators and potential therapeutic targets. To identify cGAS-bound domain within RNA4.9, HFFs were infected with WT HCMV and cross-linked by UV irradiation. cGAS-RNA complexes were selectively purified by two individual antibodies (D1D3G and E9G9G). The enriched RNAs were fragmented and subjected to construction of cross-linking and immunoprecipitation (CLIP)-libraries. RNAs extracted from non-crosslinked-HCMV infected HFFs were subjected to rRNA depletion, RNA fragmentation, then construction of size-matched input (SMI)-libraries.