Lights, Camera, Actin: Divergent roles of beta- and gamma-cytoplasmic actin in vaccinia virus infection

Abstract

Intracellular pathogens require access to host cells for their replication and spread. The host actin cytoskeleton represents a physical barrier to them, although many have evolved ways to circumvent, or hijack, this system to their advantage. Vaccinia virus (VACV) can manipulate the host actin cytoskeleton to facilitate dissemination. It expedites its cellular egress by nucleating actin beneath its particles, creating filamentous actin (F-actin) comets that propel virions across the cell surface. Tagging VACV proteins with fluorescent markers is used to study virus-host interactions, and define host molecular mechanisms, particularly within dynamic actin pathways. To this end, we developed a novel, optimised protocol for generating recombinant VACV. We then used this to create a recombinant VACV expressing Lifeact-GFP, a fluorescent marker that can highlight F-actin on infection, enabling live tracking of VACV comets via real-time fluorescence microscopy. F-actin comprises two cytoplasmic isoforms: β- and γ-actin. Despite differing only by four N-terminal amino acids, recent studies outlined their distinct localisations and functions in cell lines and whole organisms. We performed a detailed study of their roles in VACV actin-based motility. Initiation of comet formation appears to have an essential requirement for β-actin. Conversely, speed of virus movement was enhanced when γ-actin was silenced, indicating a moderating effect on the rate of actin polymerisation by this isoform. We aimed to define the site of β-actin dependency for VACV actin-based motility by biochemical pull-down assays. This represents the first investigation of the role of actin isoforms in pathogen motility, implicating the importance of their relative distribution in initiating VACV-induced actin comets. Further studies may underpin the importance of β- over γ-actin in other organisms using actin-based motility, providing a route to curb actin-assisted spread of intracellular pathogens.