AcMNPV-miR-3 is a miRNA encoded by Autographa californica nucleopolyhedrovirus and regulates the viral infection by targeting ac101

MicroRNAs (miRNAs), which are small noncoding RNAs found in plants, animals, and many viruses, regulate various biological processes. Our group has previously reported the first miRNA encoded by Autographa californica multiple Nucleopolyhedrovirus (AcMNPV), AcMNPV-miR-1, which regulates the expression of three viral genes. This study characterizes another miRNA encoded by AcMNPV, AcMNPV-miR-3. This miRNA is located on the opposite strand of the viral gene ac101 coding sequence in the AcMNPV genome, and it can be detected at 6 h post-infection and accumulated to a peak around 12 h post-infection in AcMNPV infected Sf9 cells. Five viral genes (ac101, ac23, ac25, ac86, and ac98) were verified to be regulated by AcMNPV-miR-3. Ac101 was markedly down-regulated by AcMNPV-miR-3 that may be via a siRNA-like cleavage mode. Administrating excessive AcMNPV-miR-3 resulted in decreased production of infectious budded virions (BV) and accelerated the formation of occlusion-derived virions (ODV). These results suggest that AcMNPV-miR-3 may play a regulatory role in BV and ODV production.