Actomyosin‐driven motility and coalescence of phase‐separated viral inclusion bodies are required for efficient replication of a plant rhabdovirus

Abstract

Summary Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood. Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle‐associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live‐cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells. We show that TYMaV IBs are phase‐separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation in vivo and in vitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication. Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation.

Plant viruses and biomolecular condensates: novel perspectives in virus replication strategies

“…Interestingly, the host casein kinase 1 (CK1) phosphorylates P protein, hindering both its phase separation and BYSMV replication (Fang et al ., 2022). In another example of where host machinery influences viral condensates, it was found that microfilaments are necessary for condensate maturation during Tomato yellow mottle‐associated virus (TYMaV, Rhabdoviridae family) infection (Liang et al ., 2023). The viral nucleoprotein (N) and phosphoprotein (P) drive condensate formation and move along microfilaments when anchored to myosin XI motor proteins via the P protein.…”

“…The viral nucleoprotein (N) and phosphoprotein (P) drive condensate formation and move along microfilaments when anchored to myosin XI motor proteins via the P protein. Interestingly, actin‐depolymerization with Latrunculin B significantly reduced both the number and motility of N‐P condensates, as demonstrated by live‐cell imaging of N‐P condensates using a recombinant TYMaV virus expressing a split green fluorescent protein (split‐GFP; Liang et al ., 2023).…”