Filoviruses are filamentous enveloped viruses belonging to the family Filoviridae, in the order Mononegavirales. Some filovirus members, such as Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and non-human primates. The filovirus ribonucleoprotein complex, called the nucleocapsid, forms a double-layered helical structure in which a non-segmented, single-stranded negative-sense RNA genome is encapsidated by nucleoprotein (NP), viral protein 35 (VP35), VP24, VP30, and RNA-dependent RNA polymerase (L). The inner layer consists of the helical NP–RNA complex, acting as a scaffold for the binding of VP35 and VP24 that constitute the outer layer. Recent structural studies using cryo-electron microscopy have advanced our understanding of the molecular mechanism of filovirus nucleocapsid formation. Here, we review the key characteristics of the EBOV and MARV nucleocapsid structures, highlighting the similarities and differences between the two viruses. In particular, we focus on the structure of the helical NP–RNA complex, the RNA binding mechanism, and the NP–NP interactions in the helix. The structural analyses reveal a possible mechanism of nucleocapsid assembly and provide potential targets for the anti-filovirus drug design. Mini-abstract Filovirus nucleocapsid is responsible for viral genome transcription and replication. Here, we summarize the helical nucleocapsid structure of Ebola and Marburg viruses, focusing on the nucleoprotein-RNA helix of the nucleocapsid core structure, which has been recently determined by cryo-electron microscopy.

Lloviu virus (LLOV) is a novel filovirus detected in Schreiber’s bats in Europe. The isolation of the infectious LLOV from bats has raised public health concerns. However, the virological and molecular characteristics of LLOV remain largely unknown. The nucleoprotein (NP) of LLOV encapsidates the viral genomic RNA to form a helical NP-RNA complex, which acts as a scaffold for nucleocapsid formation and de novo viral RNA synthesis. In this study, using single-particle cryo-electron microscopy, we determined two structures of the LLOV NP-RNA helical complex, comprising a full-length and a C-terminally truncated NP. The two helical structures were identical, demonstrating that the N-terminal region determines the helical arrangement of the NP. The LLOV NP-RNA protomers displayed a structure similar to that in the Ebola and Marburg virus, but the spatial arrangements in the helix differed. Structure-based mutational analysis identified amino acids involved in the helical assembly and viral RNA synthesis. These structures advance our understanding of the filovirus nucleocapsid formation, and provide a structural basis for the development of anti-filoviral therapeutics.