An Autographa californica multiple nucleopolyhedrovirus-encoded microRNA, AcMNPV-miR-5, downregulates the expression of viral gene ac66

Four Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-encoded microRNAs (miRNAs) have been characterized previously. Here, we report the fifth AcMNPV-encoded miRNA, AcMNPV-miR-5 (Ac-miR-5), which downregulates the viral gene <i>ac66</i>. Target genes were predicted through sequence analysis and validated using luciferase reporter assays. The regulatory effects of Ac-miR-5 on <i>ac66</i> expression were assessed by reverse transcription quantitative PCR and Western blot. The impact of Ac-miR-5 overexpression on virus infection was analysed by TCID₅₀ assay and quantitative real-time PCR in Sf9 cells. The results showed that Ac-miR-5 downregulates <i>ac66</i> at both the mRNA and protein levels. Meanwhile, the budded virion production and DNA replication were decreased. Furthermore, microscopy revealed a decrease in the number of polyhedra formed. These findings suggest that Ac-miR-5 overexpression restricts viral load, potentially contributing to the establishment of a stable viral infection within the host cell.