The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a “submarine-like” budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular “rocket-like” protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses.
The filoviruses, Marburg and Ebola, cause lethal hemorrhagic fever and are highest-priority bioterrorism agents. Filovirus particles contain a rod-like nucleocapsid and are normally filamentous, though other shapes are seen. It is poorly understood how such large filamentous particles are assembled and released from infected cells. Here we have studied Marburg virus production in infected cells using electron tomography. This technique allows virus particles to be visualized in three dimensions at different stages during assembly. We find that in early stages of virus production, highly infectious filamentous viruses are produced, whereas after prolonged infection poorly infectious spherical viruses are released. We also define the sequence of steps in filamentous virus release. The intracellular nucleocapsid first travels to the plasma membrane of the cell, where it binds laterally along its whole length. One end is then wrapped by the plasma membrane and wrapping proceeds rapidly until the virus protrudes vertically from the cell surface. The rear end of the virus particle then pinches off from the cell. We propose that other important filamentous and rod-shaped viruses also follow this series of steps of assembly and budding.
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