Influence of Internal DNA Pressure on Stability and Infectivity of Phage λ

Abstract

Viruses must remain infectious while in harsh extracellular environments. An important aspect of viral particle stability for double-stranded DNA viruses is the energetically unfavorable state of the tightly confined DNA chain within the virus capsid creating pressures of tens of atmospheres. Here, we study the influence of internal genome pressure on the thermal stability of viral particles. Using differential scanning calorimetry to monitor genome loss upon heating, we find that internal pressure destabilizes the virion, resulting in a smaller activation energy barrier to trigger DNA release. These experiments are complemented by plaque assay and electron microscopy measurements to determine the influence of intra-capsid DNA pressure on the rates of viral infectivity loss. At higher temperatures (65-75°C), failure to retain the packaged genome is the dominant mechanism of viral inactivation. Conversely, at lower temperatures (40-55°C), a separate inactivation mechanism dominates, which results in non-infectious particles that still retain their packaged DNA. Most significantly, both mechanisms of infectivity loss are directly influenced by internal DNA pressure, with higher pressure resulting in a more rapid rate of inactivation at all temperatures.

Keywords: DNA pressure; capsid stability; differential scanning calorimetry; infectivity; phage lambda.

Figure 1

DSC scans of phage λ reveal the exothermic peak of heat-induced DNA release. DSC scan of phage λ performed in 10 mM MgCl₂ Tris-buffer at 200 °C/hour. The exothermic peak near 75 °C (inset) represents DNA release from virions. (Upper panel) Negative stain electron microscopy (EM) images showing DNA-filled virions that retain their packaged genome after brief incubation below the DNA release temperature at 50 °C (left), whereas virions incubated at the temperature of the exothermic peak (75 °C) no longer contain packaged genomes, but rather still show intact capsid and tail structures (right). (Lower panel) Gel electrophoresis bands showing that the decreasing amount of protected DNA within capsids corresponds with the temperature of the exothermic peak in DSC scans (bands correspond to incubations at 55, 65, 70, and 75 °C).

Figure 2

(A) DSC scans at 90 °C/hour showing that the reduced packaging density of 78% DNA λ (black) results in a higher temperature to trigger heat-induced DNA release compared to wt DNA λ (red) in 5 mM MgCl₂. (B) Increasing the Mg²⁺ concentration from 5 mM (black) to 20 mM (blue) further stabilizes the 78% DNA length λ virion, resulting in a higher temperature of DNA release. Influence of temperature scanning rate on wt (C) and 78% (D) DNA length λ virions performed in 10 mM MgCl₂ buffer. DSC scans were performed at 60 (blue), 90 (green), 150 (red), and 200 °C/hour (black). For both viral strains, faster scanning rates result in shifting the DNA release peak toward higher temperature (insets). The minimum for the peak of DNA release was reproducible to within 0.1 °C.

Figure 3

(A) The dependence of the DNA release peak position (plotted as inverse temperature) on the temperature scanning rate (60, 90, 150, and 200 °C/hour). Analysis was performed on wt (solid lines) and 78% (dotted lines) DNA length λ virions in 5 (red), 10 (green), and 20 (blue) mM MgCl₂ buffers. (B) Activation energy for heat-induced DNA release determined from the linear fits in (A) (see text for further description) for each buffer and DNA length condition. (C) Schematic illustrating that decreased internal pressure (from either reduced DNA packaging or ionic conditions) results in higher activation energies for DNA release from λ virions. This is due to higher internal pressures resulting in an increased internal energy, which effectively lowers the overall energy barrier height.

Figure 4

(A) Logarithm of the remaining proportion of viral infectivity over time for wt DNA λ in 10 mM MgCl₂. Experiments were performed for various temperatures between 40 and 75 °C. For each temperature the rate constant for loss of viral infectivity (k) was determined from the exponential fits (solid lines). (B) Viral inactivation rate constants (determined from plaque assay experiments) are plotted as a function of inverse temperature (Arrhenius plot). (The corresponding values for the inverse Celsius temperatures are shown in 5 °C increments on the upper horizontal axis). Rates of viral infectivity loss are shown for wt DNA length λ in 10 mM MgCl₂ (red squares), along with 78% DNA length λ in 5 mM (green triangles) and 10 mM (blue diamonds) MgCl₂. The inset shows the influence of DNA packaging density for the lower temperature region.

Figure 5

(A) The proportion of remaining DNA filled virions observed over time by EM analysis at 70 °C (black circles) closely agrees with the proportion of infectious particles over time determined by plaque assay (red squares). (B) The same comparison for incubation at 55 °C shows significantly reduced kinetics for DNA release compared to infectivity loss.

Figure 6

Rate constants describing heat-induced genome release (black circles) for wt DNA length λ plotted against inverse temperature along with the rates of viral inactivation (red squares) from Figure 4B. At the higher temperature range, inactivation is dominated by genome release (black line), whereas an alternative mechanism dominates inactivation at lower temperatures (red line). Reaction rates for both mechanisms follow an anticipated Arrhenius dependence on temperature, as revealed by the linearity of the separate higher and lower (inset) temperature regions. Summing the extrapolated rate values for two separate mechanisms (shaded region) accounts for a significant degree of the observed variation in the measured rate constants.