Charge Detection Mass Spectrometry Reveals Favored Structures in the Assembly of Virus-Like Particles: Polymorphism in Norovirus GI.1.

The main capsid protein (CP) of norovirus, the leading cause of gastroenteritis, is expected to self-assemble into virus-like particles with the same structure as the wild-type virus, a capsid with 180 CPs in a T = 3 icosahedron. Using charge detection mass spectrometry (CD-MS), we find that the norovirus GI.1 variant is structurally promiscuous, forming a wide variety of well-defined structures, some that are icosahedral capsids and others that are not. The structures that are present evolve with time and vary with solution conditions. The presence of icosahedral T = 3 and T = 4 capsids (240 CPs) under some conditions was confirmed by cryo-electron microscopy (cryo-EM). The cryo-EM studies also confirmed the presence of an unexpected prolate geometry based on an elongated T = 4 capsid with 300 CPs. In addition, CD-MS measurements indicate the presence of well-defined peaks with masses corresponding to 420, 480, 600, and 700 CPs. The peak corresponding to 420 CPs is probably due to an icosahedral T = 7 capsid, but this could not be confirmed by cryo-EM. It is possible that the T = 7 particles are too fragile to survive vitrification. There are no mass peaks associated with the T = 9 and T = 12 icosahedra with 540 and 720 CPs. The larger structures with 480, 600, and 700 CPs are not icosahedral; however, their measured charges suggest that they are hollow shells. The use of CD-MS to monitor virus-like particles assembly may have important applications in vaccine development and quality control.

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation.

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ~1:1 ratio of N to oligonucleotide LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here assembly has a critical concentration of about 1 µM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.