Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages

Abstract

We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phages λ and P22), as well as a eukaryotic virus, human herpes simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the Achilles heel of the virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures, preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation-resistant antivirals.

Importance: A virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage λ) as an experimental model system, we investigated the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome.

FIG 1

DSC scans of bacteriophage λ performed using a capillary-cell geometry at scan rates of 90 (green), 150 (red), and 200 (black) °C/h. The bottom panels show separate enhanced views of the lower- and higher-temperature portions of the scan, focusing on the peaks of DNA release as well as capsid denaturation and DNA melting peaks, respectively. The contribution of the capsid proteins and DNA melting for scans performed at 90°C/h are indicated. The dotted line reveals a capsid protein denaturation event alone scanned at 200°C/h, further revealing the scan rate-dependent shift of capsid denaturation. The central inset illustrates that the amount of DNase-protected viral DNA in phage λ capsids as a function of temperature coincides with the temperature of the exothermic event observed by DSC. The electron microscopy images in the upper panels show virions before (left) and after (right) incubation at 70°C. The heat-treated virions are devoid of their packaged DNA (evidenced by their darker, hollow appearance) and still preserve intact capsid and tail structures.

FIG 2

(a) Results of the DSC scans of phage λ shown in Fig. 1 are plotted as the raw differential power versus time from data collected during the calorimetric scan at 90 (green), 150 (red), and 200 (black) °C/h. For lower scan rates, the duration of the exothermic ejection peak increases with a concomitant decrease in signal intensity. (b) DSC scans of phage λ performed in the presence of 2-fold excess EDTA relative to magnesium concentration (black line). Under these buffer conditions, the ejection peak is well resolved from the larger capsid denaturation (∼72°C) and DNA melting (∼85°C) peaks. The capsid protein peak is revealed by preincubation at 70°C, followed by DNase treatment and subsequent addition of EDTA (red line). (c) Comparison of DSC scans of phage λ performed with calorimeters utilizing either cylindrical-cell (black line) or capillary-cell (green line) geometry (both scans are at 90°C/h in 10 mM MgSO₄-Tris buffer). The cylindrical-cell geometry results in a larger deflection in the apparent signal than that in capillary cells. au, arbitrary units.

FIG 3

(a) DSC scans of phage λ (black), HSV-1 (red), and phage P22 (blue) utilizing cylindrical-cell geometry. (For clarity, the HSV-1 and P22 scans are truncated after the differential power signal drop). (b) DSC scans of DNA-filled HSV-1 C-capsids alone (red) or in the presence of DNase (green), as well as empty HSV-1 A-capsids (purple). au, arbitrary units.

FIG 4

(a) DSC scans of wild-type and 78% genome length λ virions along with wild-type and 91% genome length HSV-1 capsids performed in 10 mM MgCl₂–10 mM Tris buffer. Reduced packaged genome lengths result in ∼2 to 3°C increases in the temperature of genome release. (b) Comparison of DSC ejection temperatures (T_Ejs) for HSV-1 and phages λ and P22 in the presence of 10 mM MgCl₂-Tris and 1 mM MgCl₂-Tris buffers. (c) DNA-DNA interaxial spacings (a_H values) of packaged viral DNA in 10 mM MgCl₂-Tris buffer obtained by SAXS experiments on phage λ (black), phage P22 (blue), and HSV-1 (red) are indicated in parentheses for the 10 mM MgCl₂-Tris buffer DSC scans in panel b. au, arbitrary units.

FIG 5

SAXS experiments demonstrating that heat treatment results in DNA release without loss of the overall capsid structure. The peaks at higher q values (insets) indicate the presence (or absence) of packaged DNA. The broader set of peaks from q values of ∼0.01 to 0.1 Å⁻¹ represents the intact viral capsid structure. Experiments were done on phage λ in 10 mM Mg²⁺ buffer (a) and in buffer plus EDTA (b), as well as on HSV-1 capsids in 10 mM Mg²⁺-Tris buffer (c). In all cases, milder heat treatment (red curves) resulted in loss of the DNA peak without disruption of the overall capsid structure.

FIG 6

(a) Slice through a three-dimensional reconstruction of P22 showing the packaged DNA (green) and the gp26 tail needle protein (yellow) (top). The approximate location of the gp26 tail needle trimer (green) is shown relative to the electron density of gp10 (gray) (bottom). His73 is highlighted in red (structure from reference 58). (b) DSC scans of P22 virions possessing wild-type (solid lines) or mutant (dashed lines) gp26 in 1 mM MgCl₂–10 mM Tris buffer and upon addition of 2 mM EDTA. au, arbitrary units.

FIG 7

Directionality of heat-induced HSV-1 genome ejection. (a) Ethidium bromide-stained gel of partially suppressed DNA remaining within HSV-1 capsids in the presence of 12% PEG 8000. Capsids were heated at 70°C to induce genome release, while the presence of PEG results in ejection of partial genome lengths. The lane on the left represents DNA molecular size markers. (b) The gel lanes from the experiment shown in panel a after Southern blot analysis using probes originating from the unique long (U_L; left two lanes; probe 1) or unique short (U_S; right two lanes; probe 2) genome ends. (c) Linear map of HSV-1 genome indicating the positions of probes 1 (16 to 18 kbp) and 2 (139 to 141 kbp) used in the experiment shown in panel b.