The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• The order *Bunyavirales* comprises numerous lethal negative-sense RNA viruses, including significant human pathogens.
• Approved anti-infective therapies for many bunyaviruses are currently unavailable.
• Bunyavirus ribonucleoproteins (RNPs), formed by nucleocapsid protein (NP) and viral polymerase, are crucial for RNA synthesis and virion assembly but their architecture was poorly understood.

Purpose of the Study:

• To elucidate the structural organization of nucleocapsid protein (NP) within Bunyamwera virus (BUNV) ribonucleoproteins (RNPs).
• To develop a pseudo-atomic model for the NP portion of BUNV RNPs.
• To understand the molecular basis of RNP flexibility and its role in viral replication.

Main Methods:

• Purification of RNPs from Bunyamwera virus (BUNV).
• Negative staining and cryo-electron microscopy (cryo-EM) for imaging RNP structure.
• Molecular dynamics simulations to fit the BUNV NP crystal structure into the cryo-EM average.
• Site-directed mutagenesis of NP using a mini-genome system for functional validation.

Main Results:

• Purified BUNV RNPs exhibited distinct populations, indicating a consistent condensation mechanism.
• Microscopy confirmed a helical arrangement of the nucleocapsid protein (NP) in BUNV RNPs.
• A pseudo-atomic model revealed lateral interactions between NP monomers via flexible N- and C-terminal arms, explaining RNP flexibility.
• RNA was identified as essential for maintaining the native RNP structure.

Conclusions:

• The helical NP structure with flexible terminal arms provides a molecular basis for the inherent flexibility of bunyaviral RNPs.
• This flexibility is critical for essential viral processes like RNA replication and virion assembly.
• The findings offer insights into bunyaviral RNP organization and will inform the development of novel antiviral strategies.