Viruses have to deliver their genome into the host. Specifically, while eukaryotic viruses are engulfed by the host cell during the endocytosis, bacteriophages eject their nucleic acid in the bacteria from outside upon binding on the outer membrane. In particular, the DNA translocation mechanism of some bacteriophages apparently consists on a free-energy process that injects part of the DNA into the host due to the elastic energy arising from an excess of internal pressure created during the DNA packing process. In contrast, most of eukaryotic viruses just disassemble once they are inside the host to release their fatal load. To understand the above mentioned issues it is crucial not only to know the structure of the viral-nucleic acid complexes, but also the physical properties at the nanometer scale such as the mechanical stability or the elasticity. Thus, although Structural Biology (SB) techniques such as Electron Microscopy or x-ray diffraction provide virus structures successfully, this do not necessarily give information about their physical properties and their averaging character can hide individual details that could be essential. In addition, the static nature of SB data and their intrinsic environment conditions, such as high vacuum or freezing, avoid any real time dynamic characterization. Therefore, in our group we use Atomic Force Microscopy (AFM) in physiological conditions to study both structural and mechanical properties of viral particles.
This line of research was initiated at the Vrije Universiteit (Amsterdam) by Dr. Pedro José de Pablo during his post-doc (Ivanovska, I. L., P. J. de Pablo, B. Ibarra, G. Sgalari, F. C. MacKintosh, J. L. Carrascosa, C. F. Schmidt, and G. J. L. Wuite. 2004. Proc. Natl. Acad. Sci. U. S. A. 101:7600-7605.) and, subsequently, imported to the Nanoforces group at the Universidad Autónoma de Madrid. This technique roughly consist on performing single FZ (force vs. distance) indentations (fig. a), where the AFM tip indents and retracts on a single previously selected virus point while recording the force as a function of tip-sample separation. By considering the virus and the cantilever as two springs in series with constants kv and Kc (fig b), it allows to obtain information about the virus mechanics. The study of the mechanical properties of viruses has become fundamental in physical virology (12) because allows to study not only their physical properties, such as the Young Modulus (20), but also the role of the genome on the viral shell stability and the virus maturation.
Presently, we discuss our research in viruses.
Mechanical properties of the minute virus of mice (MVM) parvovirus
This work is performed with the close collaboration of Dr. Mauricio Garcia Mateu’s group (web the mauricio)
The parvovirus Minute Virus of Mice (MVM) is among the smallest and structurally simplest viruses known. The parvovirus capsid is formed by 60 structurally equivalent subunits arranged in a simple (T=1) icosahedral symmetry. The atomic structures of the protein shell for both the empty capsid and the DNA-containing virion of MVM, as determined by X-ray crystallography, are very similar. In addition, about 25%-30% of the single-stranded viral DNA in the virion was crystallographically visualized as conformationally defined oligonucleotide stretches bound to 60 equivalent, small concavities located at symmetrical positions in the internal surface of the protein shell. Here we show a mechanism to reinforce the strength of an icosahedral virus by using its DNA as a structural element. The mechanical properties of individual empty capsids and DNA-containing virions of the minute virus of mice have been investigated. The stiffness of the empty capsid was found isotropic. Remarkably, the presence of the genomic DNA inside the virion led to an anisotropic reinforcement of the virus stiffness by about 3%, 40% and 140% along the 5-fold, 3-fold and 2-fold symmetry axes, respectively. A finite element model of the virus indicates that this anisotropic mechanical reinforcement is due to DNA stretches bound to 60 concavities of the capsid.
These results, together with evidence of biologically relevant conformational rearrangements of the capsid around pores located at the 5-fold axes, suggest that this virus may have evolved for maximum stiffness without cancelling the conformational changes needed for infectivity. (Carrasco, C., A. Carreira, I. A. T. Schaap, P. A. Serena, J. Gomez-Herrero, M. G. Mateu, and P. J. Pablo. 2006. Proc. Natl. Acad. Sci. U. S. A. 103:13706-13711.) Subsequently, we also showed that Selected amino acid side chains have been truncated to specifically remove major interactions between the capsid and the visible DNA patches, and the effect of the mutations on the stiffness of virus particles has been measured using atomic force microscopy. The mutations do not affect the stiffness of the empty capsid; however, they significantly reduce the difference in stiffness between the DNA-filled virion and the empty capsid. Carrasco, C., M. Castellanos, P. J. de Pablo, and M. G. Mateu. 2008. Proc. Natl. Acad. Sci. U. S. A. 105:4150-4155.
Mechanical properties of bacteriophage phi29
This work is performed with the collaboration of Professor Carrascosa’s group at Centro Nacional de Biotecnología (web de Pepe) and Dr. David Reguera’s group at Universidad Autónoma de Barcelona (web the David).
Here we show that empty prolated phi29 bacteriophage proheads exhibit an intriguing anisotropic stiffness which behaves counter-intuitively different from standard continuum elasticity predictions. By using Atomic Force Microscopy, we find that the phi29 shells are about twice stiffer along the short than along the long axis. This result can be attributed to the existence of a residual stress, a hypothesis that we confirm by coarse-grained simulations. This built-in stress of the virus prohead could be a strategy to provide extra mechanical strength to withstand the
DNA compaction during and after packing and a variety of extracellular conditions, such as osmotic shocks or dehydratation. We show for the first time that the curvature imposses mechanical stress to the proteins of a virus. (C. Carrasco, A. Luque, M.
Hernando-Pérez, R. Miranda, J.L. Carrascosa, P.A. Serena, M. de Ridder, A. Raman, J. Gómez-Herrero, I.A.T. Schaap, D. Reguera and P.J. de Pablo. Biophysical Journal Volume 100, Issue 4, 1100-1108, 16 February 2011.)
Effect of capillary forces in viruses
This work is in collaboration with Dr. Mauricio García Mateu, Dr. Manuel Marqués and Prof. Carrascosa. Here we investigate the water menisci confined in closed geometries by studying the structural effects of their capillary forces on viruses during the final stage of desiccation. We used individual particles of the bacteriophage phi29 and the minute virus of mice. In both cases the genomic DNA was ejected from the capsid. However, while the structural integrity of the minute virus of mice was essentially preserved, the phi29 capsid underwent a wall-to-wall collapse. We provide evidence that the capillary forces of water confined inside the viruses are the main responsible for these effects. Moreover, by performing theoretical simulations with a lattice gas model we found that some structural differences between these two viruses may be crucial to explain the different ways in which they are affected by water menisci forces confined at the nanoscale. Carrasco, C., M. Douas, R. Miranda, M. Castellanos, P. A. Serena, J. L. Carrascosa, M. G. Mateu, M. I. Marques, and P. J. de Pablo. 2009.. Proc. Natl. Acad. Sci. U. S. A. 106:5475-5480.
Measuring internal pressure of phage phi29
This work is a collaboration with Prof. Carrascosa (CNB-CSIC) , Iwan Schaap (Gottingen University) and David Reguera (Universitat de Barcelona). It has been proposed that the phage phi29 dsDNA translocation through the tail to the host is initiated by a push mechanism followed by the bacteria proteins pulling machinery The push mechanism would be triggered by the elastic energy provided by the internal pressure accumulated during the DNA packing process . So far, the demonstration of presence of internal pressure inside phage phi29 has remained elusive. Here we provide evidence of the existence of internal pressure in phi29 virion mainly provoked by the DNA-DNA interaction. By using atomic force microscopy nanoindentation experiments on individual viral particles, we show that the phi29 virion is stiffer than phi29 prohead.
Since there are not major mechanically-relevant structural differences between the prohead and the empty mature head particles, it is likely to consider the packed ds-DNA as the main responsible of such stiffening. In order to distinguish between structural or pressure induced reinforcement, we performed further experiments in the presence spermidin3+, a counter ion that induce DNA condensation even inside viruses. Our real time experiments show that spermidin3+ soften the full virion to prohead values. Back and forth experiments show that this is a reversible process. Theoretical calculations using the model by Purohit et al. (BJ (2005) 88, 851) indicate an internal pressure within the phi29 conditions of about 30 ±10 atm. The evaluation of the internal pressure from the mechanical considerations discovered in the experiments by using Finite Element Analysis, result in a pressure of 40 ±10 atm, in good agreement with the theoretical predictions.
Mercedes Hernando-Pérez, Roberto Miranda, María Aznar, José L. Carrascosa, Iwan. A.T. Schaap, David Reguera and Pedro J. de Pablo. Small (2012).