Papel de interfases y residuos interfásicos en la elasticidad mecánica y dinámica conformacional de partículas del virus diminuto del ratón

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 10-04-2015In this study we have carried out systematic analyses using different techniques, including protein engineering and atomic force microscopy (AFM), to provide new insights into the role of protein‐protein or protein‐nucleic acid interfaces, interfacial residues and interactions into assembly/disassembly, mechanical stability and conformational dynamics of a simple model icosahedral virus, the minute virus of mice (MVM). In the first part of this study we carried out a systematic mutational analysis to investigate the role of electrically charged residues at the inner capsid wall (the interface between capsid and viral nucleic acid) in assembly, dynamics and infectivity of MVM. The results revealed that the presence and distribution of those residues is not generally relevant for assembly or infectivity; however, rings of negatively charged residues around the capsid pores are critical for preserving the pore‐associated capsid dynamics and for virus infectivity. In the second part of this study we subjected single capsids and virions of MVM to mechanical disassembly using AFM, in order to probe the disruption of intersubunit interfaces and identify intermediates in the (dis)assembly pathway. The results allowed us to experimentally confirm the existence of a series of transient intermediaries that had been theoretically predicted using different approaches. These include: i) capsids missing one capsomer (trimer of capsid subunits); ii) capsids missing one pentamer of capsomers; and iii) pentamers of capsomers. In the third part of this study, we carried out a systematic mutational analysis of the localized interfaces between viral DNA segments and DNA‐binding sites in the capsid inner wall, to investigate the relationship between local mechanical elasticity and propensity for thermal inactivation of the MVM virion. The results revealed a quantitative relationship between a decrease in mechanical stiffness of capsid regions where some interactions with bound DNA segments are disrupted by mutation, and an increase in the propensity of the virion to undergo a heat‐mediated structural change that inactivates its infectivity. Application of transition state theory indicated that there exists a linear relationship between mechanical stiffness in a region of a virion, and the energetic barrier of a structural transition in which that region is involved. In the fourth and last part of this study we carried out a systematic mutational analysis of the intersubunit (intertrimer) interfaces in the MVM capsid to investigate by AFM the role of interfacial residues and intersubunit interactions in the mechanical elasticity of the viral particle. The results showed that residues generally located in elements of secondary structure and placed along the major lines that define the intertrimer interfaces exert an important role in maintaining the mechanical elasticity of the capsid. Removal of those interfacial residues almost invariably caused substantial increases in mechanical stiffness. The MVM capsid appears to have evolved towards a maximum of mechanical elasticity. Taken together, the results of this study have provided further insights into the molecular determinants of the properties and functions of simple virus particles. In particular, this work represents a significant advance in the understanding of the molecular determinants of mechanical stiffness in viruses, the relationship between virus elasticity and conformational dynamics, and some biological implications of virus mechanic