CHARACTERIZING VIRUSES WITH MASS SPECTROMETRY

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DESCRIPTION: (applicant's abstract) The study of virus structure is both scientifically interesting and medically relevant. We seek to gain information on viruses by taking advantage of the inherent speed and accuracy of mass spectrometry and by using the techniques we have developed to study viral structure. While viruses can be analyzed by other spectroscopic techniques, those methods are by no means routine or complete. Since viruses are composed of multiple protein-protein subunits, viral proteolysis followed by MALDI-MS will be used to map the amino acids that are exposed on the viral surface and to provide information about the protein dynamics through comparisons with crystallographic data. In order to perform these analyses we have initiated studies on a model system, the flock house virus (FHV), which is a small RNA icosahedral animal virus that is well-characterized crystallographically. Our MALDI-MS/proteolysis approach has already been shown to be useful on less than 10 ug of FHV, whereas crystallographic data typically requires 100 mg of material. Once the approach is established we will move to viruses that have not been crystallized or fully characterized such as cucumber mosaic virus. Viral autocatalytic proteolysis events will also be examined and, through identification of protein fragments, we will investigate their role in initiating viral infectivity. The FHV also exhibits a structural feature termed the "pocket factor" that is analogous to a feature observed in human rhinoviruses. Interestingly, crystallographic studies do not reveal the structure of the organic molecule, most likely due to structural disorder or low occupancy. The existence of the "pocket factor", however, is closely associated with viral infectivity in FHV and rhinoviruses and has not yet been identified. Our goal is to investigate/characterize this factor using methods that we previously developed. In addition to characterizing viral substructure, we are also designing a mass spectrometer to perform mass analysis on whole viruses, preliminary research has allowed us to demonstrate the retention of virus ultrastructure and infectivity throughout the ESI mass analysis process. The ability to mass measure whole viruses and gain structural information on viruses could provide virologists with an accurate means of viral identification and characterization. The aim of our work is to develop new methods for understanding local and global viral structure.