Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication

Author summary Viruses have efficiently organized genomes, mostly consisting of coding genes. Nonetheless, in recent years it became apparent that herpesviruses encode for long non-coding RNAs (lncRNAs). In this study, we show that one of human cytomegalovirus (HCMV) encoded lncRNAs, named RNA4.9, is important for viral DNA replication and viral propagation. RNA4.9 is embedded in the viral origin of replication and its transcription causes the formation of a RNA-DNA hybrid, a structure which is likely important for the viral origin of replication unwinding and initiation of viral DNA replication. Furthermore, interfering with viral origin of replication or with RNA4.9 promoter activities leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the ssDBP levels are coupled to the origin activity. Finally, we discovered a new lncRNA encoded by the murine cytomegalovirus, which seems to have similar features and function as the HCMV encoded RNA4.9. These results suggest a novel mechanism, conserved among betaherpesviruses, by which a viral lncRNA, embedded in the viral origin of replication, regulates viral DNA replication and may play a role in coupling origin activity with the level of ssDBP. Viruses are known for their extremely compact genomes composed almost entirely of protein-coding genes. Nonetheless, four long noncoding RNAs (lncRNAs) are encoded by human cytomegalovirus (HCMV). Although these RNAs accumulate to high levels during lytic infection, their functions remain largely unknown. Here, we show that HCMV-encoded lncRNA4.9 localizes to the viral nuclear replication compartment, and that its depletion restricts viral DNA replication and viral growth. RNA4.9 is transcribed from the HCMV origin of replication (oriLyt) and forms an RNA-DNA hybrid (R-loop) through its G+C-rich 5' end, which may be important for the initiation of viral DNA replication. Furthermore, targeting the RNA4.9 promoter with CRISPR-Cas9 or genetic relocalization of oriLyt leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the levels of ssDBP are coupled to the oriLyt activity. We further identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication, that the levels of ssDBP are coupled to the oriLyt activity, and that these regulatory features may be conserved among