Functional and molecular dissection of viral lncRNAs throughout HCMV life cycle

Small, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs) RNA1.2, RNA2.7, RNA4.9, and RNA5.0. These lncRNAs account for majority of the viral transcriptome, but their functions remain largely unknown. Here, we showed that HCMV lncRNAs, except for RNA5.0, are required throughout the entire viral life cycle. Deletion of each lncRNA resulted in a decrease in viral progeny during lytic replication and failing to efficiently establish latent reservoirs and reactivate. Nanopore direct RNA sequencing of native lncRNA molecules revealed that each lncRNA exhibited a dynamic modification landscape, depending on the state of infection. Global analysis of the lncRNA interactome identified 32, 11, and 89 host factors that specifically bind to RNA1.2, RNA2.7, and RNA4.9, respectively. Moreover, 52 proteins commonly bound to the three lncRNAs were identified, including 11 antiviral immunity-related proteins. Our molecular analyses found that three lncRNAs are modified with N⁶-methyladenosine (m6A) and interact with m6A readers in all infection states. In-depth functional analysis revealed that m6A–mediated lncRNA stabilization as the key mechanism by which lncRNAs are maintained at high levels. Our study lays the groundwork for understanding viral lncRNA–mediated regulation of host-virus interaction throughout the HCMV life cycle.