Thesis presented November 06, 2024
Abstract:
Advances in genome sequencing have relegated phenotypic evaluation of viruses to mere complements of genotyping. In light of recent advances in native mass spectrometry (MS) approaches for virus characterization, we believe that mass measurements may very well complement genomic-sequence-based phylogenetic approaches at high taxonomic ranks.
To demonstrate the feasibility of such an approach, we computed the total virion mass of human respiratory viruses (HuReVs). We thus observed that they could be grouped into their respective families based solely on mass information. I termed this process of taxonomic separation “phylobaric classification” (from the ancient Greek ‘phûlon’: group, tribe; and ‘baros’: weight). We argue for the need to compile a database of experimental virus masses, which could be helpful in the event of a pandemic, to rapidly determine the class of viral pathogens and help triage patients. Furthermore, measuring virus mass could add a dimension to phylogenetic analysis, in a phenetic (morphological) manner, by allowing evolutionary proximity to be assessed beyond the sequence-based closest relatives.
In continuation of this theoretical framework, additional efforts were devoted to improving the instrumentation of nanoelectromechanical sensors mass spectrometry (NEMS-MS), an experimental approach to perform single-virion MS measurement. This unique method can measure uncharged, individual aerosolized viral particles over a range that perfectly overlaps the estimated mass range of HuReVs (5-6000 MDa). This work led to the development of a newer NEMS-MS prototype, more compact, and user friendly.
Subsequent work focused on characterizing the mass-to-structure relationship of virions by performing structural analyses of a model Virus Like Particle (VLP) which is the phage T5 capsid, a model system well-studied by NEMS-MS. The main comparisons were in investigating the structure differences between T5-VLPs produced by infection or recombination, and checking the presence or absence of the decoration proteins on these capsids. In addition, I also investigated new alternative sources of well-studied viral particles, such as Human Adenoviruses, Murine Noroviruses, and MS2 coliphage.
A methodological approach using a variety of liquid and gas-phase single particle characterization techniques was developed to validate the connection between mass and structure. The main idea was that by performing measurement at every step of the analysis, the fate of the sample is known at each step, from sample preparation to nebulisation, to reaching the mass detector. Nanotracking Analysis (NTA) was used for liquid measurements; Scanning Mobility Particle Sizer (SMPS) for aerosol measurements and Scanning Electron Microscopy (SEM) as a control of deposited particle along the whole pipeline. Additional measurements in liquid by negative staining Transmission Electron Microscopy (TEM), Tunable Resistive Pulse Sensing (TRPS), bottom-up proteomics and Atomic Force Microscopy (AFM) were also performed as controls. This pipeline can be applied to all viruses of interest, as much as safety restrictions allows. This work constitutes a detailed analysis of viruses transfer from the liquid to the gas phase, which could ultimately be compared with the natural aerosolisation of pathogenic respiratory viruses.
In conclusion, this thesis presents evidence that the “molecular mass” of whole virions represents an intrinsic and distinctive viral phenotypic property, which can be used to identify and categorise viruses. In addition, investigations of viruses during transfer from the liquid to the gas-phase transfer provide important information in relating their native structure to their total mass.
Keywords:
Mass Spectrometry (instrumentation), Virus-Like Particles (VLPs), Nano-electromechanical systems (NEMS) detectors, Structural biology, Analytical chemistry, Artificial nanoparticles (NPs)