Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances

Abstract

Transport of large viral nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, nucleocapsids were transported at ∼200 nm/s; (ii) nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells.dual-color imaging | reverse genetics | viral inclusion bodies T he filoviruses Marburg (MARV) and Ebola (EBOV) cause severe hemorrhagic fever with high-case-fatality rates in humans and nonhuman primates (1-3). Although the interplay of filoviral proteins leading to the transcription and replication of the viral genome and the formation of the viral nucleocapsids (NCs) is rather well understood, we are only just beginning to unravel the complex interactions between the viral and cellular proteins that are necessary to transport the NCs from the sites of their formation to the budding sites. The central protein within the NC is the nucleoprotein NP, which forms complexes with VP35, VP30, and VP24 (4, 5). The helical MARV NC is composed of the RNAdependent RNA polymerase (L), the polymerase cofactor VP35, the viral proteins VP30 and VP24, and NP, which encapsidates the viral genome (5-7). Within the viral particle, the NC is surrounded by a regular lattice of the matrix protein VP40 (5, 8, 9). The outside of the VP40 lattice contacts the viral envelope, in which the glycoprotein GP is inserted (10).MARV virogenesis begins with the formation of perinuclear inclusions which, analogous to the EBOV, are considered to be sites of viral replication and the assembly of new NCs (8, 9, 11). Later in the replication cycle, NCs are detected in the cytosol, at the plasma membrane, and in filopodia, the preferred sites of MARV budding (12, 13). The glycoprotein GP reaches the plasma membrane via vesicular se...

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription

“…Helical nucleocapsids within IBs closely associated with the membranes of the endoplasmic reticulum (ER) have been observed in Marburg virus-infected cells (22). From the replication bodies, Marburg and Ebola virus nucleocapsids were observed to be transported to budding sites, a process dependent on actin polymerization (23,24). For some paramyxoviruses, the formation of IBs in infected cells was demonstrated many years ago by electron microscopy studies (25)(26)(27)(28).…”

Trafficking of Bluetongue Virus Visualized by Recovery of Tetracysteine-Tagged Virion Particles

“…Whereas enveloped viruses have been successfully labeled with genetically encoded fluorescent proteins (21)(22)(23)(24)43), the structural constraints of nonenveloped virus capsid structures have made the insertion of tags difficult. An alternate strategy involving nonspecific labeling of nonenveloped virus capsid structures with fluorescent dyes has helped to elucidate the entry pathway of viruses with naked capsids.…”

Comparing human and algorithmic tracking of subviral particles in fluorescence microscopic image sequences

“…To visualize intracellular movements of subviral particles of Marburg virus, green fluorescent-protein tagged nucleocapsids were used [5]. This method enables the recording of image sequences under biosafety-level 4 conditions.…”