Marek's disease virus-encoded microRNA-M6-5p suppresses viral replication by targeting UL42

Marek's disease virus (MDV) is a highly contagious and oncogenic avian alphaherpesvirus that causes severe economic losses on the global poultry industry. MicroRNAs (miRNAs), a class of endogenous noncoding RNAs typically 20-25 nucleotides in length, are crucial regulators of post-transcriptional genes expression. Our previous study found that MDV-encoded miR-M6-5p suppresses viral replication in cells. However, the underlying molecular mechanisms remain incompletely understood. In this study, we identified the viral UL42 transcript, bioinformatically predicted to encode a subunit of the viral DNA polymerase, as a direct target of miR-M6-5p. Through constructing a UL42-null MDV mutant, we demonstrated that the deletion of UL42 completely abolished viral replication in cells by severely impairing MDV DNA polymerase activity. Furthermore, we demonstrated that miR-M6-5p negatively regulates viral DNA polymerase activity, as its overexpression suppressed whereas its knockout enhanced enzymatic activity. Thus, miR-M6-5p-mediated downregulation of UL42 impairs viral DNA polymerase activity, thereby restricting MDV replication. These findings provide novel mechanistic insights into how MDV exploits its own miRNAs to modulate viral replication via the core DNA replication machinery.