Autographa californica multiple nucleopolyhedrovirus gene ac81 is required for nucleocapsid envelopment

•ac81 is critical for BV production.•ac81 is essential for nucleocapsid envelopment and subsequent embedding of virions into OBs.•Ac81 predominately localizes to the ODV envelope and contains a functional TM domain.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly pathogenic Baculoviridae that targets insects, whose core gene, ac81, has an unknown function. To determine the role of ac81 in the life cycle of AcMNPV, an ac81-knockout (Ac-81KO-GP) was constructed through homologous recombination in Escherichia coli. We determined that no budded virions were produced in Ac-81KO-GP-transfected Sf9 cells, while there was no effect on viral DNA replication. Electron microscopy (EM) analysis revealed that occlusion-derived virions (ODVs) envelopment and the subsequent embedding of virions into occlusion bodies (OBs) were aborted due to ac81 deletion. Interestingly, confocal microscopy and immunofluorescence analysis revealed that Ac81 was predominantly localized to the ring zone of nuclei during the late phase of infection. In addition, Ac81 was localized to the mature and premature ODVs in virus-infected cells within the ring zone as revealed by immuno-electron microscopy (IEM) analysis. Furthermore, we determined that Ac81 contained a functional hydrophobic transmembrane (TM) domain, whose deletion resulted in a phenotype similar to that of Ac-81KO-GP. These results suggest that Ac81 might be a TM protein that played an important role in nucleocapsid envelopment.