Binding effects in multivalent Gibbs-Donnan equilibrium

The classical Gibbs-Donnan equilibrium describes excess osmotic pressure associated with confined colloidal charges embedded in an electrolyte solution. In this work, we extend this approach to describe the influence of multivalent ion binding on the equilibrium force acting on a charged rod translocating between two compartments, thereby mimicking ionic effects on force balance during in vitro DNA ejection from bacteriophage. The subtle interplay between Gibbs-Donnan equilibrium and adsorption equilibrium leads to a non-monotonic variation of the ejection force as multivalent salt concentration is increased, in qualitative agreement with experimental observations

Measuring the force ejecting DNA from phage

We discuss how a balance can be established between the force acting to eject DNA from viral capsids and the force resisting its entry into a colloidal suspension which mimics the host cell cytoplasm. The ejection force arises from the energy stored in the capsid as a consequence of the viral genome (double-stranded DNA) being strongly bent and crowded on itself. The resisting force is associated with the osmotic pressure exerted by the colloidal particles in the host solution. Indeed, recent experimental work has demonstrated that the extent of ejection can be progressively limited by increasing the external osmotic pressure; at a sufficiently high pressure the ejection is completely suppressed. We outline here a theoritical analysis that allows a determination of the internal (capsid) pressure by examining the different relations between force and pressure inside and outside the capsid, using the experimentally measured position of the force balance.Comment: 15 pages, 4 figures, accepted for publication in J. Phys. Chem

Viral capsids: Mechanical characteristics, genome packaging and delivery mechanisms

The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its consequent confinement within the capsid. It is proposed that this pressure helps driving the genome into the host, but other mechanisms also seem to play an important role in ejection. DNA packaging and ejection strategies are obviously dependent on the mechanical properties of the capsid. This review focuses on the mechanical properties of viral capsids in general and the elucidation of the biophysical aspects of genome packaging mechanisms and genome delivery processes of double-stranded DNA bacteriophages in particular

Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM

Double-stranded DNA in many bacterial viruses (phage) is strongly confined, which results in internal genome pressures of tens of atmospheres. This pressure is strongly dependent on local ion concentration and distribution within the viral capsid. Here, we have used electron energy loss spectroscopy (EELS), energy-filtered TEM (EFTEM) and X-ray energy dispersive spectroscopy to provide such chemical information from the capsid and the phage tail through which DNA is injected into the cell. To achieve this, we have developed a method to prepare thin monolayers of self-supporting virus/buffer films, suitable for EELS and EFTEM analysis. The method is based on entrapment of virus particles at air–liquid interfaces; thus, the commonly used method of staining by heavy metal salts can be avoided, eliminating the risk for chemical artifacts. We found that Mg(2 + ) concentration was approximately 2–4 times higher in the DNA-filled capsid than in the surrounding TM buffer (containing 10 mM Mg(2 + )). Furthermore, we also analyzed the DNA content inside the phage tail by mapping phosphorus and magnesium

Portal Stability Controls Dynamics of DNA Ejection from Phage

Through a unique combination of time-resolved single-molecule (cryo-TEM) and bulk measurements (light scattering and small-angle X-ray scattering), we provide a detailed study of the dynamics of stochastic DNA ejection events from phage λ. We reveal that both binding with the specific phage receptor, LamB, and thermo-mechanical destabilization of the portal vertex on the capsid are required for initiation of ejection of the pressurized λ-DNA from the phage. Specifically, we found that a measurable activation energy barrier for initiation of DNA ejection with LamB present, Ea = (1.2 ± 0.1) × 10-19 J/phage (corresponding to ∼28 kTbody/phage at Tbody = 37 °C), results in 15 times increased rate of ejection event dynamics when the temperature is raised from 15 to 45 °C (7.5 min versus 30 s average lag time for initiation of ejection). This suggests that phages have a double fail-safe mechanism for ejection - in addition to receptor binding, phage must also overcome (through thermal energy and internal DNA pressure) an energy barrier for DNA ejection. This energy barrier ensures that viral genome ejection into cells occurs with high efficiency only when the temperature conditions are favorable for genome replication. At lower suboptimal temperatures, the infectious phage titer is preserved over much longer times, since DNA ejection dynamics is strongly inhibited even in the presence of solubilized receptor or susceptible cells. This work also establishes a light scattering based approach to investigate the influence of external solution conditions, mimicking those of the bacterial cytoplasm, on the stability of the viral capsid portal, which is directly linked to dynamics of virion deactivation

Influence of Internal Capsid Pressure on Viral Infection by Phage λ

AbstractEjection of the genome from the virus, phage λ, is the initial step in the infection of its host bacterium. In vitro, the ejection depends sensitively on internal pressure within the virus capsid; however, the in vivo effect of internal pressure on infection of bacteria is unknown. Here, we use microfluidics to monitor individual cells and determine the temporal distribution of lysis due to infection as the capsid pressure is varied. The lysis probability decreases markedly with decreased capsid pressure. Of interest, the average lysis times remain the same but the distribution is broadened as the pressure is lowered