Tesis doctoral de Milagros Castellanos Molina
This study is mainly focused on the analysis of the mechanical properties of viral particles, and their possible relationship with the conformational stability or dynamics of such particles. A fundamental goal of this work was to use mutational analysis and atomic force microscopy to provide a first experimental evidence for a biological role of the mechanical stiffness or elasticity of a viral particle. The model system chosen was the minute virus of mice (mvm), one of the structurally simplest viruses known. in the first part of this work we have studied the functional role of evolutionarily conserved glycine residues within a glycine-rich tract located at the amino terminus (nt) of the capsid protein subunits, and their possible implication in capsid conformation dynamics related with translocation processes through capsid pores. The results show that those glycine residues are needed for normal infectivity of the mvm virion, largely because bulkier residues at these positions would impair the externalization of the capsid protein vp2 nt. in the second part of this work we have studied the role of the viral nucleic acid in the mechanical properties of the mvm virion, and a possible relationship between conformational stability and mechanical stiffness of a viral particle. The results show that non-covalent interactions between the viral adn inside the virion and equivalent regions located at the inner capsid wall are responsible for an anisotropic increase in the mechanical stiffness of the viral particle around the 2-fold (s2) and 3-fold (s3) symmetry axes, but not around the 5-fold (s5) axes, where the capsid pores are located. This increase in rigidity of the virion is associated with a higher resistance against thermal inactivation of its infectivity, while at the same time being compatible with the capsid conformational change associated with vp2 nt translocation and required for virus infection. in the third part of this work we have studied the role of residues surrounding capsid pores in the mechanical properties of the mvm capsid, and a possible relationship between conformational dynamics and mechanical flexibility in a viral particle. The results show that the residues located at the base of the pores contribute to preserve a high mechanical flexibility in the s5 regions around the pores. Mutational analysis using as many as 18 mutants revealed a perfect correlation between high mechanical flexibility in the s5 region, the occurrence of the conformational rearrangement of the capsid associated with translocation of the vp2 nt through the pores, and virion infectivity. in the fourth and last part of this work we have studied the role of residues surrounding conserved cavities in the mvm capsid in the mechanical properties of the viral particle. The results indicate that the mvm capsid is kept near a maximum of mechanical flexibility. All of the mutations tested that alter the size and shape of the capsid cavities, as well as several other mutations located in different capsid regions, did not modify the mechanical stiffness of the s5 regions, but increased the stiffness of the s2 and s3 regions. the results of the mechanical analysis of mvm undertaken in this study have allowed us to propose a mechanical model of the mvm particle. This model contemplates that every region in the mvm virion may have acquired during evolution the degree of mechanical stiffness or flexibility required to satisfy an adequate compromise between different structural and functional constraints. On one hand, the interactions between the dna inside the virion and equivalent regions in the inner capsid wall close to the s2 capsid regions increase the mechanical stiffness of the s2 and s3 regions in the virion, possibly impairing a non-productive conformational change and increasing the resistance of the virion against thermal inactivation of its infectivity in the extracellular environment. On the other hand, the absence of dna segments bound close to the s5 capsid regions, and the residues found at the base of the pores located in these regions, keep the s5 regions mechanically flexible enough to allow a productive conformational change in the capsid. This structural rearrangement is associated with translocation processes through the pores and is needed for virus infectivity. in summary, the results obtained support a biological implication for the mechanical stiffness and elasticity of a viral particle, and suggest approaches for the rational manipulation of the mechanical properties of viral particles for nanobiotechnological purposes.
Datos académicos de la tesis doctoral ««análisis mutacional de propiedades estructurales y mecánicas del virus diminuto del ratón y de sus implicaciones biológicas».«
- Título de la tesis: «análisis mutacional de propiedades estructurales y mecánicas del virus diminuto del ratón y de sus implicaciones biológicas».
- Autor: Milagros Castellanos Molina
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 17/06/2011
Dirección y tribunal
- Director de la tesis
- Mauricio Garcia Mateu
- Tribunal
- Presidente del tribunal: jose Lopez carrascosa
- julio Gomez herrero (vocal)
- Jesús Perez gil (vocal)
- nuria Verdaguer massana (vocal)