Internal DNA pressure modifies stability of WT phage

Abstract

dsDNA in bacteriophages is highly stressed and exerts internal pressures of many atmospheres (1 atm ‫؍‬ 101.3 kPa) on the capsid walls. We investigate the correlation between packaged DNA length in phage (78 -100% of WT DNA) and capsid strength by using an atomic force microscope indentation technique. We show that phages with WT DNA are twice as strong as shorter genome mutants, which behave like empty capsids, regardless of high internal pressure. Our analytical model of DNA-filled capsid deformation shows that, because of DNA-hydrating water molecules, an osmotic pressure exists inside capsids that increases exponentially when the packaged DNA density is close to WT phage. This osmotic pressure raises the WT capsid strength and is approximately equal to the maximum breaking force of empty shells. This result suggests that the strength of the shells limits the maximal packaged genome length. Moreover, it implies an evolutionary optimization of WT phages allowing them to survive greater external mechanical stresses in nature.atomic force microscopy ͉ viral capsids ͉ bacteriophage ͉ DNA hydration forces ͉ osmotic pressure T he majority of viruses have spherical protein shells (capsids) with icosahedral symmetry, with radii varying between 10 and 100 nm, and with thicknesses of few nanometers, corresponding to a single protein layer. Viral capsids protect genomes that can be tens of micrometers in contour length. In prokaryotic viruses (bacteriophages), capsid proteins first assemble in empty capsids before the genome is actively packaged by a molecular motor that is part of the capsid (1, 2). Matching the capsid size and genome length is of great importance for efficient packaging and viral infectivity. For example, WT phage infects Escherichia coli cells and has its DNA (48,502 bp) contained in an icosahedral T ϭ 7 capsid to which a flexible, noncontractile tail is attached. The mature capsid has an outer diameter of 63 nm and a shell thickness of between 1.8 and 4.1 nm (1, 2). phages can be packaged with DNA lengths in the range of 78-106% of WT DNA and remain infectious (3). If the genome is shorter than 78% of the WT-DNA, then the phage fails to infect. When the DNA is longer than 106% of WT length, packaging does not occur. It was recently shown that dsDNA inside many phages is highly stressed because of electrostatic repulsion and the bending energy of the packaged DNA chain, resulting in internal pressures of several tens of atmospheres (1 atm ϭ 101.3 kPa) (4-13). This finding suggests that the infection is in part driven by the internal DNA pressure. Thus, if DNA is significantly shorter than WT, the internal pressure becomes too low and is therefore incapable of injecting enough DNA into the bacteria. On the other hand, if the DNA is longer than WT, the internal pressure might be too high, and the force that builds up in the capsid could exceed either the strength of the packaging motor (4, 14) or the maximum internal force that the capsid can withstand. Thus, the capsid size and strength might limit th...

Modeling and simulation of the mechanical response from nanoindentation test of DNA-filled viral capsids

“…The hydration force is in this case nearly equal to the osmotic pressure of DNA inside the capsid. The strength of the capsid is optimized to the maximum osmotic pressure resulting from packaging the wild-type λ-DNA length [2]. However, the experimental data in ref.…”

“…In case of phage λ, it has been shown that the capsid strength approximately matches the force exerted by the DNA on the capsid walls [2,12]. This suggests that the capsid strength determines the maximum length of DNA that can be packaged, which in turn is also correlated with the strength of the packaging motor complex [2,13]. Therefore, investigating the mechanical properties of DNA-filled versus empty capsids is of great interest.…”

“…Mechanical properties of only a few viral capsids have been investigated experimentally so far. To name a few, bacteriophages λ and φ29 [1,2], plant virus CCMV [3], MVM virus [4], MLV, HIV [5,6], HSV-1 [7] and HK97 [8]. Probing viral shells with an AFM tip generally results in either reversible deformation, often observed when the applied force is below a certain value, or an irreversible capsid rupture when the applied force is above this threshold value.…”

“…In [2] it was experimentally demonstrated that upon AFM cantilever indentation of DNA-filled phage, water hydrating the packaged DNA is being displaced to the bulk through the pores in the capsid. It should be noted that all viral capsids are permeable to water and smaller ions.…”

Probing nanomechanical properties from biomolecules to living cells

“…Like Michel et al [64], Ivanovska et al [38][39][40] have also used AFM to investigate the extent to which the genome of viral capsids contributes to the overall stiffness of the organism. They measured the mechanical properties of λ-phages that were either empty or filled with wild-type DNA to their full or a partial extent.…”