Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication

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, and this may be important for the initiation of viral DNA replication. Furthermore, interference with RNA4.9 expression drastically reduces the levels of the viral single-stranded DNA-binding protein (ssDBP), and overexpression of ssDBP alleviates the inhibition of viral DNA replication and growth caused by RNA4.9 depletion. We also identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). Knockdown of this lncRNA interferes with MCMV ssDBP expression and viral DNA replication. These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication by coupling oriLyt activity to ssDBP levels, and that this novel activity may be conserved in other betaherpesviruses. Overall design: RNA-sequencing of ribosome protected footprints (FP) of HCMV infected fibroblasts (HFF) that expressed CAS9 and either a control sgRNA (sgUS2) or a sgRNA against the viral lincRNA RNA4.9 (sgRNA4.9) and RNA-seq of according samples; each in replicates. mRNA-seq of HCMV infected fibroblasts (HFF) that expressed CAS9 and either a control sgRNA (sgUS2)or a sgRNA against the viral lincRNA RNA4.9 (sgRNA4.9) at 24 and 48 hpi; each in replicates. Infections were performed in presence of the viral DNA replication inhibitor phosphonoformic acid (PFA). mRNA-seq of fibroblasts (MEF) infected with murid herpesvirus 1.