Charge Detection Mass Spectrometry Reveals Norovirus GII.4 Virus-like Particles Failure to Complete

Virus-like particles (VLPs) are assembled from many identical copies of the virus capsid protein (CP) but lack the genetic material needed for replication. Norovirus is the leading cause of gastroenteritis and norovirus VLPs are vaccine candidates. While the native virus has a T = 3 icosahedral capsid (with 180 CPs), two other structures have been detected for the GII.4 strain VLPs, a T = 4 capsid with 240 CPs and a T = 1 capsid with 60 CPs. Here, charge detection mass spectrometry (CD-MS) was used to measure accurate masses for norovirus GII.4 VLPs. In CD-MS, the masses of individual ions are determined from simultaneous measurement of each ion's m/z ratio and charge. The CD-MS measurements were performed using an electrostatic linear ion trap (ELIT-CD-MS) which allows accurate and precise charge determination for each ion. This in turn enables high resolution and reproducible mass measurements for ions into the gigadalton regime. Peaks corresponding to all three constructs (T = 1, T = 3, and T = 4) were observed in the CD-MS mass distributions for norovirus GII.4 VLPs. However, the peaks were often at a slightly lower mass than expected for the complete icosahedral capsids, suggesting that the icosahedra had missing subunits, i.e., they had failed to complete. Incomplete capsids are expected to be labile and over time the relative abundances of the three particles change dramatically. Structural tools such as cryo-electron microscopy cannot be used to determine whether VLPs are complete because of averaging inherent in the methods obscures missing subunits. This information can only be accessed by accurate mass measurements made using ELIT-CD-MS.