Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging

Abstract

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.

Keywords: DNA condensation; soft matter; virology.

Fig. 1.

Motor stall–restart experiments. (A) Schematic diagram illustrating the experimental method. Packaging is initiated and followed to ∼75% prohead filling under a low applied force (5 pN) (steps 1–3). The motor is then stalled by locally injecting the nonhydrolyzable analog γS-ATP via a microcapillary tube (step 4). After a variable waiting time, packaging is restarted by reintroducing ATP (step 5). (B) Example packaging trajectory before (red) and after (blue) an imposed stall of 10.4 min. The gray plot shows the same data as in blue shifted back to the time of the stall, showing the acceleration. (C) Packaging rate vs. length of DNA packaged before (red) and after (blue) stalling for the event shown in B. The black lines are linear trend lines fit to data before and after the stall point. (D) Mean change in packaging rate (blue) and motor velocity (red, rate not including pauses and slips) after short stalls (<3.4 min; average, 1.5 min; n = 50 events), medium stalls (3.4–10 min; average, 5.7 min; n = 50), and long stalls (>10 min; average, 12.3 min; n = 20). The bars marked “No Stall” indicate control experiments where the motor was not stalled (n = 65). P values for significance of differences vs. the next lowest stall time are indicated above the bars, and P values for significance of differences vs. the no stall control are indicated below the bars.

Fig. 2.

Heterogeneity in packaging dynamics. (A) Normalized velocities measured in 3-s intervals vs. filling measured for an ensemble of packaging events (red, n = 85), events with perforated proheads (blue, n = 58), events measured after long stalls (green, n = 20), and events with 5 mM added spermidine (gray, n = 53). The velocities are normalized by the average velocity measured with <10% genome length packaged. (B) Index of dispersion (variance divided by mean) of normalized velocity vs. filling, labeled with the same colors as in A.

Fig. 3.

Motor pausing. (A) Pause duration and (B) pause frequency per DNA length vs. filling for standard events (red), events with short stalls (light blue), medium length stalls (dark blue), and long stalls (green), and with 5 mM spermidine (gray). Inset shows frequencies of long pauses (>5 s).