Evidence of Viral Capsid Dynamics Using Limited Proteolysis and Mass Spectrometry

Abstract

Virus particles are stable yet exhibit highly dynamic character given the events that shape their life cycle. Isolated from their hosts, the nucleoprotein particles are macromolecules that can be crystallized and studied by x-ray diffraction. During assembly, maturation and entry, however, they are highly dynamic and display remarkable plasticity. These dynamic properties can only be inferred from the x-ray structure and must be studied by methods that are sensitive to mobility. We have used matrix-assisted laser desorption/ionization mass spectrometry combined with time resolved, limited proteolysis (Cohen, S. L., Ferre-D'Amare, A. R., Burley, S. K., and Chait, B. T. (1995) Protein Sci. 4, 1088 -1099; Kriwacki, R. W., Wu, J., Tennant, T., Wright, P. E., and Siuzdak, G. (1997) J. Chromatogr. 777, 23-30; Kriwacki, R. W., Wu, J., Siuzdak, G., and Wright, P. E. (1996) J. Am. Chem. Soc. 118, 5320 -5321) to examine the viral capsid of flock house virus. Employing less than 10 g of virus, time course digestion products were assigned to polypeptides of the subunit. Although surface regions in the three-dimensional structure were susceptible to cleavage on extended exposure to the protease, the first digestion products were invariably from parts of the subunit that are internal to the x-ray structure. Regions in the N-and C-terminal portions of the subunit, located within the shell in the x-ray structure, but implicated in RNA neutralization and RNA release and delivery, respectively, were the most susceptible to cleavage demonstrating transient exposure of these polypeptides to the viral surface.The protein capsid of virions is a noncovalent association of protein subunits that is responsible for an array of functions, including cell attachment, cell entry, and RNA release. Mobile regions associated with these events can only be inferred from inherently static methods such as x-ray crystallography (4) and cryo-electron microscopy ( 5). To better understand the dynamic nature of the viral capsid it is necessary to develop methods that are sensitive to functional mobility. One recently developed method has combined limited proteolysis with mass spectrometry (1-3) to explore protein/DNA (1) and protein/ protein interactions (2, 3). While previous applications of mass spectrometry to viruses have either focused on characterizing protein and DNA structure ( 6 -11) or the intact virus (12), here we have applied the proteolysis method with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) 1 to study the surface-accessible regions of the viral particle.MALDI-MS typically provides picomole sensitivity and accuracy on the order of 0.05% (i.e. Ϯ 0.5 Da on a 1000-Da peptide) and therefore offers a useful method for identifying the proteolysis products of the virus. The limited proteolysis/MALDI-MS experiments were performed on flock house virus (FHV), a non-enveloped, icosahedral, RNA animal virus ( Fig. 1) with dimensions similar to the rhino and polio viruses (ϳ300 Å). Its protein coat or capsid is compose...

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

“…In those previous reports, single proteins with nucleic acids or ligands [24,26] or virus complexes [28] were investigated by limited proteolysis with mass spectrometry. Most limited proteolysis studies previously reported have been carried out for observing the conformational change of a protein with and without bound nucleic acid or ligands, and for probing the structural and dynamic differences between the holo and apo form of a protein [25].…”

“…Mass spectrometry has been used as the readout step in a limited proteolysis approach for characterizing macromolecular structure [24][25][26][27][28][29]. In those previous reports, single proteins with nucleic acids or ligands [24,26] or virus complexes [28] were investigated by limited proteolysis with mass spectrometry.…”

The structure of a thermophilic archaeal virus shows a double-stranded DNA viral capsid type that spans all domains of life

“…It is possible that these structures play a role in host recognition and͞or attachment. We speculate that the central channel in each turret might act as a portal for translocation of the viral nucleic acid, as is typical of the 5-fold vertices in many viruses (25,26). Based on these unusual structural features, we propose that this virus be named STIV for Sulfolobus turreted icosahedral virus.…”

Viral capsid mobility: A dynamic conduit for inactivation

“…One mechanism involves the modification of proteins, which would cause inhibition of viral cell entry or the release of the genome. The second mechanism allows the alkylating reagents direct access to the viral genome through a mobile protein capsid (8)(9)(10)(11)(12). The recent findings (8)(9)(10)(11) that the protein capsids of viruses in solution have a much higher degree of dynamics than their crystallized counterparts suggested that the second mechanism might be the means of inactivation.…”

“…The recent findings (8)(9)(10)(11) that the protein capsids of viruses in solution have a much higher degree of dynamics than their crystallized counterparts suggested that the second mechanism might be the means of inactivation. Focusing on this latter idea, the ability of small alkylating agents to react with either the capsid or encapsidated nucleic acid was investigated initially by using flock house virus (FHV), an RNA-containing model virus used in previous studies (8,9).…”