Molecular insights into nucleocapsid assembly and transport in Marburg and Ebola viruses

Abstract

Live-cell imaging enables visualization of the spatiotemporal dynamics of signals in cells. Intracytoplasmic movement of nucleocapsids is crucial during the life cycle of enveloped viruses; however, the molecular mechanisms governing their assembly and transport are not fully understood. Using a Marburg virus (MARV) live-cell imaging system, we identified three nucleocapsid proteins—nucleoprotein (NP), VP35, and VP24—that are necessary and sufficient to form transport-competent nucleocapsid-like structures (NCLSs). These findings are consistent with observations in Ebola virus (EBOV). Interestingly, despite incompatibility among these proteins, VP30 interacts with nucleocapsid proteins from both MARV and EBOV, supporting viral transcription and replication in heterologous systems. Furthermore, we show that the conserved PPxPxY motif at the C-terminus of NP regulates NP-VP30 interactions in both homologous and heterologous contexts and is crucial for VP30 association with NCLSs. Because this motif is conserved across filoviruses, it represents a promising target for antiviral development. Our findings advance the understanding of nucleocapsid formation and offer new avenues for therapeutic intervention against MARV and EBOV. IMPORTANCE: This study provides crucial insights into the molecular mechanisms of nucleocapsid assembly and transport in filoviruses, specifically Marburg virus (MARV) and Ebola virus (EBOV). Using advanced live-cell imaging, we uncovered how the conserved PPxPxY motif in nucleoprotein (NP) mediates its interactions with VP30, thereby regulating nucleocapsid formation and viral replication. Notably, while VP30’s role differs between MARV and EBOV, the underlying mechanism of NP-VP30 interaction via this motif appears conserved across filoviruses, making it a promising target for broad-spectrum antiviral strategies. These findings deepen our understanding of nucleocapsid protein compatibility and virus-host interactions, offering new avenues for therapeutic intervention against these deadly pathogens.