Two-Stage Dynamics of In Vivo Bacteriophage Genome Ejection

Biopolymer translocation is a key step in viral infection processes. The transfer of information-encoding genomes allows viruses to reprogram the cell fate of their hosts. Constituting 96% of all known bacterial viruses [A. Fokine and M. G. Rossmann, Molecular architecture of tailed double-stranded DNA phages, Bacteriophage 4, e28281 (2014)], the tailed bacteriophages deliver their DNA into host cells via an “ejection” process, leaving their protein shells outside of the bacteria; a similar scenario occurs for mammalian viruses like herpes, where the DNA genome is ejected into the nucleus of host cells, while the viral capsid remains bound outside to a nuclear-pore complex. In light of previous experimental measurements of in vivo bacteriophage $\lambda$ ejection, we analyze here the physical processes that give rise to the observed dynamics. We propose that, after an initial phase driven by self-repulsion of DNA in the capsid, the ejection is driven by anomalous diffusion of phage DNA in the crowded bacterial cytoplasm. We expect that this two-step mechanism is general for phages that operate by pressure-driven ejection, and we discuss predictions of our theory to be tested in future experiments.

Popular Summary

Viruses replicate by injecting their genomes into host cells, which, in turn, churn out more copies of the virus’ genetic code. In experiments, researchers have watched as bacteriophages—viruses that attack only bacteria—eject their DNA into their host while leaving behind their protein shells. But the underlying physical mechanism of DNA ejection remains unclear. In some bacteriophages, DNA-processing enzymes underlie genome ejection. In others, ejection seems to be driven by the high pressure stored in the densely packed viral DNA. But the counteracting internal pressure of the cells poses a conceptual problem for this strategy. To find the missing pieces of the puzzle, we have identified a novel mechanism for virus genome delivery that originates from anomalous diffusion of the viral DNA in the crowded interior of bacterial cells.

We applied our theory to bacteriophage lambda, a type of virus that attacks the bacteria species E. coli. This bacteriophage is the only one for which researchers have been able to measure the ejection from a single virus into a host cell, as well as the ejection driving force. The measured dynamics is consistent with our model, in which DNA is initially driven into the host cell by a release of pressure within the bacteriophage head and then completes its journey by anomalous diffusion in the bacterial cytoplasm.

These results resolve the mystery of how complete viral genome injection is possible without the need for DNA-processing enzymes. Our formulation is general and applicable to a broad class of viruses that deliver genomes without catalytic reactions, and it has implications for important biopolymer translocation processes such as bacterial conjugation, protein trafficking, or messenger RNA export through the nuclear pore.

Figure 1

Data on in vivo phage ejection and macromolecular diffusion. (a) Speed of phage in vivo ejection as a function of the amount of DNA ejected. The data from Ref. [15] are plotted on a log-log scale, suggesting two stages of behavior. A linear fit to the later stage gives $\log \mathrm{v}=(-3.99\pm 1.67)\log N+(20.2\pm 7.7)$. The two $\lambda$ phage mutants, cI60 and b221, were used for their different genome lengths. In the inset, the data plotted on a linear scale are compared with the results (solid curves) calculated for the early-stage ejection using different parameter sets in Eqs. (1)–(3), as explained in the text. The red and blue curves are phage b221 and cI60, respectively, with the same parameter values (pv) choice. Cyan and purple curves show how the phage cI60 curve changes when the parameters change. For pv1, $p_0=6.58\times {10}^4\mathrm{kPa}$, $P_b=4.28\mathrm{kPa}$; for pv2, $p_0=1.2\times 6.58\times {10}^4\mathrm{kPa}$, $P_b=4.28\mathrm{kPa}$; and for pv3, $p_0=6.58\times {10}^4\mathrm{kPa}$, $P_b=5\times 4.28\mathrm{kPa}$. (b) Loci tracking of diffusing macromolecules in the live cells reported in the literature. The mean-squared displacement (MSD) as a function of time is plotted on a log-log scale. Black dot symbols represent bacterial chromosomal loci [32], $\alpha =0.4$, $D_{\mathrm{app}}={10}^{-2}{\mu \mathrm{m}}^2/{\mathrm{s}}^{\alpha }$. Red and pink left pointed triangle symbols represent plasmid (6 kbp, circular) [33]; red represents the subdiffusive regime, $\alpha =0.54$, $D_{\mathrm{app}}=8\times {10}^{-3}{\mu \mathrm{m}}^2/{\mathrm{s}}^{\alpha }$. Blue and purple times symbols represent M2G (crescentin-GFP protein complex) [33]; blue represents the subdiffusive regime, $\alpha =0.44$, $D_{\mathrm{app}}=9\times {10}^{-3}{\mu \mathrm{m}}^2/{\mathrm{s}}^{\alpha }$. Yellow, green, and cyan circle symbols represent phage $\lambda$ DNA (50 kbp, circularized in the cell) searching for its specific integration site on the bacterial genome [34]; green represents the subdiffusive regime, slope $\alpha =0.5$, $D_{\mathrm{app}}=6\times {10}^{-3}{\mu \mathrm{m}}^2/{\mathrm{s}}^{\alpha }$. Yellow represents constrained diffusion of the phage DNA that remains anchored to the entry site after ejection. For reference, the two gray lines have slopes of 0.5 and 1, respectively. The logarithms are with base 10. Inset: The ejecting phage genome.