Slow and steady wins the race: physical limits on the rate of viral DNA packaging

Abstract

During the assembly of dsDNA viruses such as the tailed bacteriophages and herpesviruses, the viral chromosome is compacted to near crystalline density inside a preformed head shell. DNA translocation is driven by powerful ring ATPase motors that couple ATP binding, hydrolysis, and release to force generation and movement. Studies of the motor of the bacteriophage phi29 have revealed a complex mechanochemistry behind this process that slows as the head fills. Recent studies of the physical behavior of packaging DNA suggest that surprisingly long-time scales of relaxation of DNA inside the head and jamming phenomena during packaging create the physical need for regulation of the rate of packaging. Studies of DNA packaging in viral systems have, therefore, revealed fundamental insight into the complex behavior of DNA and the need for biological systems to accommodate these physical constraints.

Fig. 1.

Architecture of the phi29 DNA packaging motor. The pentameric packaging ATPase, gp16, (blue) is anchored to the capsid through the pentameric prohead RNA (pRNA) scaffold (magenta) and pushes DNA through the dodecameric connector portal (green). Reprinted from Mao et al. 2016 with permission from Elsevier.

Fig. 2.

Early single-molecule measurements of phi29 DNA packaging using laser tweezers. (a) Packaging complexes were tethered between two microspheres, with the prohead/motor complex attached to one bead (blue) by antibodies against the capsid protein and the free end of the DNA attached to a second bead (orange) via a biotin-streptavidin linkage. (b) The change of tether length over time was used to calculate the change in packaging velocity over the course of head filling (grey trace is raw data; red trace is decimated and filtered). Reprinted from Chemla et al. 2005 with permission from Elsevier (a) and Smith et al. 2000 with permission from Springer Nature (b).

Fig. 3.

Mechanochemistry of the phi29 DNA packaging motor. (a) Plot of DNA tether length vs time showing the dwell-burst cycle of DNA packaging using high-resolution single-molecule laser tweezers (grey trace is raw data; blue trace is decimated and filtered). (b) The corresponding mechanochemical scheme showing the order and timing of the ATPase cycle within the motor ring and DNA movement, where ADP is exchanged for ATP during the static dwell (red line) and mechanical stepping (consisting of four 2.5 bp substeps) occurs during the burst (green line). (c) Changes in dwell and burst duration over the course of head filling. As the head fills, much of the reduction in DNA translocation rate is due to the lengthening of the static dwell (red lines) rather than the dynamic burst (green lines). Reprinted from Liu et al. 2014 with permission from Elsevier.

Fig. 4.

Long relaxation time-scale and jamming during DNA packaging. (a) Experimental design, where packaging is initiated in situ by bringing prohead motor complexes in close contact with tethered DNA (steps 1 and 2). After packaging is initiated and the rate of DNA translocation determined (step 3), the phi29 motor is reversibly stalled (step 4) and, after a prescribed time, restarted to determine if the rate of packaging has increased relative to the rate before the stall (step 5). (b) Measurement of DNA tether length over time taken before (red trace) and after (blue trace) the analogue induced stall (green trace). When the post-stall trace is transposed to the point of the stall (grey trace), the inflection in the trace reveals the increase in packaging velocity after the stall. (c) An example trace where the motor velocity before (red trace) and after (blue trace) the stall are plotted relative to the amount of head filling, showing that the motor increased in velocity by 46.5% due to the stall. (d) The length of the stall time corresponds to increase in packaging rate, for both packaging velocity (red) and motor velocity (blue; edited for motor pauses/slips), indicative of a time-dependent process of DNA relaxation during the stall. (e) Jamming appears with the addition of spermine as a DNA condensing agent can be seen when observing packaging over time. At low concentrations of spermine, packaging rate increases compared to controls (blue vs black traces). Higher concentrations of spermine causes abrupt decelerations and translocation failures (red traces). Reprinted from Berndsen et al. 2014 (a-d), and from Keller et al. 2014 with permission from Elsevier (e).