Electron Tomography Reveals the Steps in Filovirus Budding

Welsch, Sonja; Kolesnikova, Larissa; Krähling, Verena; Riches, James D.; Becker, Stephan; Briggs, John

doi:10.1371/journal.ppat.1000875

Cited by 70 publications

(69 citation statements)

References 53 publications

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“…MARV NCs are considered to be formed in the virus-induced inclusions and then transported to the plasma membrane, where budding takes place (6,9,26). However, so far it has not been possible to trace individual NCs as they leave the inclusions on their way to the cell periphery.…”

Section: Resultsmentioning

confidence: 99%

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

Schudt

Kolesnikova

Dolnik

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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122

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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...

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Section: Resultsmentioning

confidence: 99%

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

Schudt

Kolesnikova

Dolnik

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

122

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“…Arm-like structures protrude from alternate interfaces between NPs, and immuno-electron microscopy analysis locates VP24 and VP35 to these protrusions. The NC is incorporated into virions by envelopment at the plasma membrane initiated at one end of the NC (16,17).In the present study, EBOV virions were imaged using cryoEM and cryo-electron tomography (cryoET) to describe their structure in a near-native state. Image-processing techniques were applied to define the 3D structure of the NC within the virion.…”

mentioning

confidence: 99%

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confidence: 99%

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography

Bharat

Noda

Riches

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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222

221

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Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.

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“…In the budding process, membrane-associated VP40 extruded from the cells accompanying surface GP and the nucleocapsid, and infectious virions are released by pinching off the plasma membrane [87] (Fig. 2b).…”

Section: Assembly/egressmentioning

confidence: 99%

Host Cell Factors Involved in Filovirus Infection

Kajihara

Takada

2015

Curr Trop Med Rep

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Filoviruses (ebolaviruses and marburgviruses) cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates of up to 90 %. The latest epidemic of Ebola virus disease in Western African countries has underscored the urgent need for effective prophylactic and therapeutic interventions for this deadly infectious disease. However, neither approved prophylactics nor therapeutics are currently available for filovirus diseases. Recent studies have been unveiling the molecular mechanisms underlying the filovirus lifecycle, including cellular entry, egress, and the evasion from host immunity, suggesting possibilities to develop effective pan-filovirus drugs.

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Electron Tomography Reveals the Steps in Filovirus Budding

Cited by 70 publications

References 53 publications

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

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

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography

Host Cell Factors Involved in Filovirus Infection

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