Ionic effects on viral DNA packaging and portal motor function in bacteriophage phi 29

Abstract

In many viruses, DNA is confined at such high density that its bending rigidity and electrostatic self-repulsion present a strong energy barrier in viral assembly. Therefore, a powerful molecular motor is needed to package the DNA into the viral capsid. Here, we investigate the role of electrostatic repulsion on single DNA packaging dynamics in bacteriophage phi 29 via optical tweezers measurements. We show that ionic screening strongly affects the packing forces, confirming the importance of electrostatic repulsion. Separately, we find that ions affect the motor function. We separate these effects through constant force measurements and velocity versus load measurements at both low and high capsid filling. Regarding motor function, we find that eliminating free Mg(2+) blocks initiation of packaging. In contrast, Na(+) is not required, but it increases the motor velocity by up to 50% at low load. Regarding internal resistance, we find that the internal force was lowest when Mg(2+) was the dominant ion or with the addition of 1 mM Co(3+). Forces resisting DNA confinement were up to approximately 80% higher with Na(+) as the dominant counterion, and only approximately 90% of the genome length could be packaged in this condition. The observed trend of the packing forces is in accord with that predicted by DNA charge-screening theory. However, the forces are up to six times higher than predicted by models that assume coaxial spooling of the DNA and interaction potentials derived from DNA condensation experiments. The forces are also severalfold higher than ejection forces measured with bacteriophage lambda.

Fig. 1.

Schematic illustration of the experiment. (Bottom Left) Prohead–motor complexes were attached to antibody-coated microspheres and captured in one optical trap. (Top Left) Biotinylated DNA molecules were tethered to streptavidin-coated microspheres and captured in a second optical trap. The bottom trap was moved with respect to the top one while measuring the DNA tension. To initiate packaging, the microspheres were brought into near contact for ≈1 s (Middle) and then quickly separated to probe for DNA binding and translocation (Right).

Fig. 2.

Dependence of the average motor velocity on force was determined from measurements on n = 25–58 complexes (mean n = 38) for selected ionic conditions. Error bars report standard errors. The different colors and symbols indicate the different ionic conditions studied, as described in Table 1. The lines are fits of the data to a theoretical model (10), as explained in Methods.

Fig. 3.

Velocity decrease due to filling. (A) Dependence of the average rate of packaging on capsid filling (expressed as percent of the native φ29 genome packaged) for selected ionic conditions. The measurements were made using the force-clamp method with F = 5 pN. The different colors and symbols indicate the different ionic conditions studied, which are described in Table 1. Points are averages in bins over n = 26–59 individual data sets (mean n = 36). The solid lines were obtained by filtering of the raw velocity data for all complexes (see Methods). (B) Velocity normalized by the maximum velocity. For clarity, only the smoothed lines from A are plotted.

Fig. 4.

Force resisting DNA packaging. (A) Internal force versus capsid filling for selected ionic conditions. The different colors and symbols indicate the different ionic conditions studied. (B and C) Internal forces with either 50% (B) or 80% (C) of the genome packaged versus ionic condition. The colors matched those used in A. The y axis in B and C share the same units as the plots in A, and B has the same x axis label as C.