Ultrastructural Organization of Recombinant Marburg Virus Nucleoprotein: Comparison with Marburg Virus Inclusions

Abstract

HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP)have been studied by immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral genome and other nucleocapsid-associated proteins.Marburg virus (MBGV) and Ebola virus (EBOV) make up the family of Filoviridae, which belongs to the order Mononegavirales. MBGV causes a severe hemorrhagic disease in humans and nonhuman primates (22). The recent outbreak of MBGV hemorrhagic fever in the Democratic Republic of the Congo underlines the emerging potential of this pathogen (25). MBGV is an enveloped virus with a nonsegmented negativestrand RNA genome 19.1 kb in length, which encodes seven structural proteins (5, 8). The nucleocapsid of the virion is composed of the viral RNA and four proteins, the nucleoprotein (NP), P (formerly called VP35), the viral protein VP30, and the catalytic subunit of the polymerase (L). Two putative matrix proteins, VP24 and VP40, are located between the nucleocapsid and the envelope, which is decorated with the surface protein (GP) (2,3,7,13,15,20).Viral reproduction takes place in the cytoplasm, and the FIG. 1. IEM analysis of ultrathin section of MVA-T7-infected HeLa cells expressing recombinant NP. Immunogold labeling was carried out using a monoclonal antibody directed against MBGV NP (dilution, 1:10) and a goat anti-mouse antibody conjugated with colloidal gold (bead diameter, 5 nm) (dilution, 1:50). NP aggregates appeared as two long sheets of a reticular network of electron-dense material heavily labeled with gold particles. The NP aggregates are in close association with the membrane of the rER. Bar, 150 nm.

Filovirus Replication and Transcription

“…MARV NP forms large helical structures resembling the nucleocapsids found in mature virions [22,34]. For EBOV, the minimal protein requirements for the formation of nucleocapsid-like structures are NP, VP35 and VP24 [17,35].…”

“…These are the NP, the polymerase cofactor VP35, the transcription activator VP30, and the major component of the RNA-dependent RNA polymerase, L ( Figure 1C). The nucleocapsid proteins play a dual role in the viral replication cycle; they are structural components of the nucleocapsid complex and are, therefore, involved in viral morphogenesis and catalyze the replication and transcription of the RNA genome [22][23][24][25].…”

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

“…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).…”

“…Although exchanging the gene encoding the EBOV polymerase with a gene encoding a fluorescently labeled version of the enzyme does not impair viral growth and enables the visualization of viral inclusions, its fluorescent intensity is not sufficient to observe individual EBOV NCs, most likely due to the few L particles associated with NCs (11). The filamentous MARV NCs of 892 times 91 nm could be large enough for visualization by fluorescence microscopy if the number of fluorescent molecules per NC is enhanced (6,9). We hypothesized that labeling VP30, one of the abundant structural NC components, would allow us to visualize the NCs in living cells.…”

“…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).…”