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Portal Stability Controls Dynamics of DNA Ejection from Phage Krista G. Freeman, Manja Annette Behrens, Kiril A Streletzky, Ulf Olsson, and Alex Evilevitch J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b04172 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 14, 2016

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The Journal of Physical Chemistry

Portal Stability Controls Dynamics of DNA Ejection from Phage Krista G. Freeman,1 Manja A. Behrens,2 Kiril A. Streletzky,3 Ulf Olsson2 and Alex Evilevitch1,4,*

1

Carnegie Mellon University, Department of Physics, Pittsburgh, Pennsylvania, USA 2

3

4

Lund University, Division of Physical Chemistry, Lund, Sweden

Cleveland State University, Department of Physics, Cleveland, Ohio, USA

Lund University, Division of Biochemistry and Structural Biology, Lund, Sweden

*Corresponding author: A. Evilevitch, email: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT Through a unique combination of time-resolved single-molecule (cryo-EM) and bulk measurements (light scattering and small angle X-ray scattering), we provide a detailed study of the dynamics of stochastic DNA ejection events from phage λ. We reveal that both binding with the specific phage receptor, LamB and thermal destabilization of the portal vertex on the capsid are required for rapid initiation of ejection of the pressurized λ-DNA from phage. Specifically, we found that a measurable activation energy barrier for initiation of DNA ejection with LamB present, Ea = (1.2 ± 0.1) x 10-19 J/phage (corresponding to ~28 kT/phage at 37°C), results in 15 times increased rate of ejection event dynamics when the temperature is raised from 15 to 45°C (7.5 minutes versus 30 s average lag-time for initiation of ejection). This suggests that phages have a double fail-safe mechanism for ejection— besides receptor binding, an activation energy barrier must also be thermally overcome for DNA to eject. This ensures that viral genome ejection into cells occurs with high efficiency only when the temperature conditions are favorable for genome replication. At lower sub-optimal temperatures, the infectious phage titer is preserved over much longer times since DNA ejection dynamics is strongly inhibited even in the presence of solubilized receptor or susceptible cells. This work also establishes a light scattering based approach to investigate the influence of external solution conditions, mimicking those of the bacterial cytoplasm, on the stability of the viral capsid portal, which is directly linked to dynamics of virion deactivation. INTRODUCTION Viral fitness1-2 is a quantifiable measure of the ability of a virus to transmit from host to host through infection, replication, and survival in the extracellular environment. Improvements in viral fitness are guided by evolutionary trade-offs, like balancing virion stability between


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infection events against the virions’ ability to rapidly uncoat during infection3-5. The way these trade-offs are balanced determines the timescales of each step of the viral replication cycle. Consider for example the optimization of host-lysis time; short lysis times promote the rapid spread of infection, but longer lysis times allow more new virions to form in each infection cycle6. Thus the timescales present in the viral replication cycle influence the success of viral infections, and studies of each dynamic process within the cycle contribute to our understanding of how viruses spread7-11. Understanding of these dynamic processes has been advanced through in vitro experiments, which allow for isolation of particular steps in the replication cycle7,

11-14

.

Bacteriophage λ, a long-tailed phage, is often used for in vitro studies of viral DNA ejection dynamics since its membrane receptor, LamB, can be successfully isolated while preserving its function15-18. When bound to LamB, phage λ ejects its 48.5 kbp of double-stranded (ds) DNA by harnessing the tens of atmospheres of DNA pressure stored in its capsid19-20. Using time-resolved light scattering (LS), we previously measured the dynamics of DNA ejection by adding LamB to a population of phage λ particles and monitoring the resulting decrease in scattered light intensity15-16. While we found an equilibration time of tens of minutes, both phage-receptor binding times

13, 21

and the time for complete DNA translocation from a single λ phage were

shown to be on the order of seconds17-18. These order of magnitude time-scale discrepancies suggest that LS may measure the dynamics of DNA ejection events within the population of DNA-filled phages rather than the dynamics of single-phage DNA translocation. Interpreting the data this way suggests a stochastic delay in initiation of DNA ejection, resulting in a timescale of tens of minutes for genome ejection from an entire population of phages. This timescale is comparable to viral host-cell lysis times in vivo6, 22, suggesting that stochasticity of DNA ejection



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events may be one of the rate-limiting steps in the spread of infection. However, the mechanism causing this stochastic ejection behavior remains unclear. To facilitate genome packaging and its subsequent ejection for infection, many bacteriophages and eukaryotic dsDNA viruses (e.g., herpesviruses) utilize a specialized portal complex at a unique vertex, which forms a narrow channel through the capsid wall23. We have recently shown that, when exposed to high temperatures, in the absence of virus receptor, both phages λ and P22, as well as Herpes Simplex virus type 1 (HSV-1), eject their DNA when the portal fails due to thermo-mechanical destabilization24. We investigated the effect of DNA pressure on the portal vertex’s thermal stability by packaging variable DNA lengths in the capsid. We found that the energy required for heat-triggered ejection is smaller for the same virus with larger DNA pressure, which demonstrates mechanical destabilization of the portal vertex5, 24. These findings suggest a link between an energy barrier at the portal and temperaturedependent dynamics of DNA ejection events which occur stochastically despite the addition of excess LamB-receptor, as determined by LS measurements in this work. Overcoming this energy barrier through destabilization of the portal structure leads to a conformational change (or its failure) with subsequent DNA ejection. In this work we combine time-resolved cryo-electron microscopy (cryo-EM) with timeresolved small angle X-ray scattering (SAXS) to explain the LS-measured DNA ejection dynamics data. We confirm experimentally, for the first time for phage λ, that the bi-exponential decay in the light scattering intensity observed when LamB is added to phage λ is caused primarily by stochastic DNA ejection events. We use this LS-assay to quantify the dynamics of stochastic ejections at different temperatures and find that the rate of ejection initiation is increased 15-fold when the temperature is raised from 15 to 45°C. Our results show that receptor


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binding significantly lowers the threshold for critical portal destabilization (compared to the heat-triggered ejection without receptor). Despite the receptor binding we measured a remaining energy barrier for DNA ejection, which causes a strong temperature dependence of ejection event rates. Our data suggest that dynamics of initiation of DNA ejection is regulated by a delicate balance between thermal stability of the portal and internal DNA capsid pressure. The increase in temperature studied in this work leaves internal DNA pressure unchanged25, allowing us to isolate the effect of thermal destabilization of the portal vertex on ejection rates. Thermally regulated portal stability improves viral fitness by preventing viral genome ejection at temperatures too low for efficient replication within the host cell, but ensuring efficient ejection at physiological temperatures for infection. Furthermore, this work provides an LS based approach to assess virus portal stability and its effects on the DNA ejection dynamics central for viral replication.

MATERIALS AND METHODS Phage λ and LamB purification WT bacteriophage λ cI85, with a genome length of 48.5kb, was produced by thermal induction of lysogenic E. coli strain AE1 derived from the S2773 strain. Phage purification details are described elsewhere19. As a final purification step, phages (at a final titer of ~1013 virions/mL) were dialyzed against and stored in TM buffer (50mM Tris, 20mM MgCl2, pH 7.4). The receptor was the LamB protein purified from pop 154, a strain of E. coli K12 in which the LamB gene has been transduced from Shigella sonnei 3070. The detailed preparation of LamB was previously described26. LamB was concentrated to 0.36mg/mL and stored in the same TM buffer plus 1% oPOE (n-Octyl-oligo-oxyethylene) to aid solubility of the receptor.



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Light scattering spectroscopy Light scattering experiments were performed on a home built system by directing a 1W Argon Ion laser (wavelength λ = 514.5nm, Spectra-Physics 2017) into a Brookhaven Instruments setup comprised of a goniometer (BI-200SM), photomultiplier (BI-DS2) and correlator (BI9000)27. Both incident and measured scattered light were vertically polarized, as ensured by a Glan-Laser calcite polarizer (Thorlabs, GL10) and a Precision Linear Polarizer (Newport 20LPVIS-B). All experiments were performed at a scattering angle of 120°. Light scattering samples were prepared by diluting bacteriophage λ stock to 1.2 x 1011 pfu/mL in a 1.2mL volume of TM+1% oPOE buffer. This diluted phage sample was filtered (0.2µm polycarbonate filter, Whatman) directly into a pre-cleaned28 glass cell then incubated for at least 10 minutes in the temperature controlled sample holder, which is maintained within 0.1°C by a Neslab RTE-110 water bath, heat exchange coils, and a decalin-filled quartz vat that surrounds the sample cell. The sample is then briefly removed to mix in a small volume of concentrated LamB solution (such that λ:LamB = 1:100 to ensure rapid binding), gently mixed, and reinserted into the experimental apparatus after ~30s so that the intensity can be monitored for the next 100 minutes. Cryo-EM Cryo transmission electron microscopy (cryo-EM) samples were prepared by mixing, in a water bath held at 20°C, 20µL of ~2 x 1013 pfu/mL bacteriophage λ solution with 30µL of 0.36mg/mL LamB solution. The samples were incubated at 20°C for a specified time (between 1 and 100 minutes) then vitrified in a controlled environment vitrification system by loading onto a glow discharged lacey carbon film supported by a copper grid, blotting with filter paper to create a thin film, then plunging the grid into liquid ethane at its freezing point. The vitrified samples


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were stored under liquid nitrogen and imaged with an electron microscope (Philips CM 120 BioTWIN Cryo) equipped with an energy filter imaging system (Gatan GIF 100) and a digital multiscan CCD camera (Gatan 791). One grid was prepared for each timepoint studied, and for each grid at least 20-30 micrographs of unique 750nm X 750nm square areas (containing at least 500 unique phages total) were imaged. These micrographs were analyzed and averaged to measure the fraction of DNA-filled capsids remaining in the sample at a given time. The average fractions of filled particles versus total number of particles were normalized between 0 and 1 to account for phages that either were empty before the experiment began or damaged DNA-filled phages which do not trigger DNA ejection. This normalization was accounted for in propagation of the standard errors [standard error σSE = σSD/sqrt(#micrographs), where σSD is the standard deviation of the fraction of DNA-filled phages across all of the micrographs at a particular timepoint], which are plotted with the average fraction of DNA-filled phages at each timepoint in Figure 1A. SAXS Small angle X-ray scattering (SAXS) measurements were carried out at the I911-4 beamline at the MAX IV Laboratory in Lund, Sweden (wavelength λ = 0.91Å, scattering vector range of 0.006-0.475 Å-1). The sample was 20µL of ~2 x 1013 pfu/mL bacteriophage λ solution which, just before loading, was mixed with 30µL of 0.36mg/mL LamB solution to trigger DNA ejection. The sample was then immediately injected into a flow through capillary held at 20°C. The intensity was monitored for 100 minutes, with higher resolution during the first 1.5-6.5 minutes (10 consecutive 30s exposures) followed by alternating 2 minute exposures and 3 minute breaks. The intensity versus scattering angle was plotted for each time and the curves



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analyzed with linear combination fitting, as described in the text, to yield the fraction of filled phages as a function of time. SAXS data core-shell modelling SAXS has a unique dependence of I versus q on the scattering length density distribution, ρ(r), of scattering objects29: = 4

. For a core-shell system, a spherical shell

(radius Rs, uniform scattering length density ρs) with a filled core (radius Rc, uniform ρc), in solvent with ρsolvent this simplifies to

)

− %&' " # ( (Eq. 1). ∝ − " # +

Here j1(qR), the first spherical Bessel function, is the source of the unique q-dependence observed experimentally for both DNA-filled and empty bacteriophage capsids (Figure 2B). Filled and empty capsids can be coarsely modeled as core-shell systems with the capsid as the protein shell and a core of either DNA (filled capsid) or solution (empty capsid). In this work we compare the I versus q dependence for our time-resolved experimental data with two ejection models utilizing the core-shell modelling described by Eq. 1. The two ejection models are: 1) stochastic ejection modelled with linear combinations of DNA-filled and solvent-filled protein shells (Figure 2C middle, weighting of each curve is described by the ratio “Filled:Empty”); and 2) synchronized ejection modelled by changing the density of DNA filling the core (Figure 2C bottom, DNA density described by “% DNA Inside”).

RESULTS AND DISCUSSION Light scattering measures stochastic initiation of DNA ejection from phage


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In an LS measurement of bulk DNA ejection dynamics from a bacteriophage population, the intensity (I) of light scattered by DNA-filled phage particles decreases immediately after mixing with an excess of the solubilized phage receptor15-16,

30

. Figure 1A shows the LS

measured DNA ejection process from wild-type DNA length (48.5 kbp) phage λ at 20°C in TMbuffer (50mM Tris, 20mM MgCl2, pH 7.5). The figure shows that the normalized decay in scattering intensity versus time, ∆ =

+'+,-./0

+-.-1-/0 +,-./0

, does not equilibrate for at least 100 minutes.

At the temperatures tested in this study, spontaneous non-receptor triggered ejections account for a negligible fraction of all ejections occurring within 100 minutes (only 0.0003% at 15°C and just over 1% at 45°C). These numbers were determined by extrapolating the rates of heattriggered ejection at different temperatures from our recently published data5. Therefore, the LSmeasured dynamics reflects the receptor-triggered ejection process and is not influenced by spontaneous, receptor-less DNA ejections. Below we show that intensity decays with time as a result primarily of stochastic DNA ejection events, but also due to a significantly slower second parallel process possibly associated with relaxation-diffusion of the ejected DNA. The normalized intensity curve shown in Figure 1A is well described by a bi-exponential fit, ∆ = 23

'5 4,/1

+ 1 − 23

'5 4078 ,

with two characteristic time constants τfast = 6.0 ± 0.1

minutes and an order of magnitude slower τslow = 49.5 ± 2.0 minutes. The pre-exponential factor A is ~0.75, showing that 75% of the scattering intensity change is associated with the much faster process described by τfast, while the process described by τslow contributes only 25% of the overall intensity change. In this work, we focus on the primary scattering intensity decay described by τfast, which we demonstrate experimentally for the first time is caused by stochastic DNA ejection events occurring after binding of LamB receptor to phage λ. The decrease in I due to DNA ejections can be explained by one of two scenarios (Figure 2A): synchronized ejection 9 ACS Paragon Plus Environment


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from all phages with slow DNA translocation dynamics; or non-synchronized, stochastic ejection from individual phages with rapid DNA translocation. Because in vitro translocation of DNA from phage λ has been shown to complete within seconds for the temperature and buffer conditions tested here17, it is more likely that the observed τfast is associated with stochastic ejections. We demonstrate this by comparing the LS intensity versus time normalized between 0 and 1 with the fraction of DNA-filled phage particles obtained from the time-resolved cryo-EM analysis (Figure 1B). Cryo-EM snapshots of phage λ particles were taken at time intervals after introducing LamB receptor. Analysis of the micrographs provides the fraction of DNA-filled phages compared to the total number of phages with time (blue circles in Figure 1A), see details in the Materials and Methods section. The cryo-EM data shows a single exponential decrease in the fraction of DNA-filled phages with a characteristic time constant of ~ 5.6 ± 1.1 minutes, which agrees well with the LS measured τfast ~ 6.0 ± 0.1 minutes. Therefore we attribute the faster decaying LS process to stochastic DNA ejection events. Since τfast quantifies the mean lifetime of this exponential process, it can be considered to be the average lag time between receptor binding and a DNA ejection event. We use time-resolved SAXS measurements to provide additional support for the stochastic ejection behavior observed with LS for phage λ. Like LS, SAXS measured scattering intensity is sensitive to the changing density of DNA (for SAXS it is electron density, which is proportional to mass density) during the ejection and relaxation processes. For small particles, SAXS intensity I at scattering vector q≈0 is proportional to MW, and thus this I(q≈0) value decreases as the DNA ejection from phage and DNA coil relaxation-diffusion processes proceed. We plot the normalized change in I(q≈0) versus time, which agrees well with the LS data (both LS and SAXS data are plotted in Figure 1A). More importantly, SAXS also provides detailed


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information on the composition of the sample through its unique dependence of I versus q on the scattering length density distribution, ρ(r), of scattering objects29. For bacteriophage capsids, this dependence is best described by core-shell models (explained in Eq. 1 of the Materials and Methods section), which result in the unique intensity curves observed experimentally (Figure 2B). In Figure 2C we compare the q-dependence near the first minimum for our time-resolved experimental SAXS data with two ejection models (synchronized and stochastic ejection) utilizing the core-shell modelling (all modelling details are described in Materials and Methods). The two ejection models are: 1) stochastic ejection modelled with linear combinations of DNAfilled and solvent-filled protein shells (Figure 2C middle, weighting of each curve is described by the ratio Filled:Empty); and 2) synchronized ejection modelled by changing the density of DNA filling the core (Figure 2C bottom, DNA density described by “% DNA inside”). Several important observations can be made from this comparison. First, we notice a systematic shift toward lower q-values in the position of the first minimum in the synchronized ejection model (Figure 2C bottom) which is not present in either the experimental data or the stochastic ejection model (Figure 2C upper and middle, respectively). Furthermore, we observe so-called isosbestic points, or points of constant intensity at fixed q values, on both sides of the first minimum in both the experimental data and the stochastic ejection model (indicated with dotted lines in Figures 2C upper and middle), but not in the synchronized ejection model. Isosbestic points indicate that a sample contains a changing mixture of two distinct scattering species31 (here, DNA-filled capsids and empty capsids), where changes in the concentrations of each species are linearly related (i.e. one DNA filled-capsid becomes one empty capsid). The existence of isosbestic points in the experimental data and the stochastic ejection model strongly support our conclusion that the sample is an evolving mixture of populations of DNA-filled and empty



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capsids rather than a uniform sample of synchronously DNA-ejecting particles (i.e., identical partially filled capsids). We now turn to the secondary, much slower LS-measured intensity decay process described by τslow, by considering Eq. 2 which is a small-molecule approximation of the Zimm equation32 that highlights the dependence of I on molecular weight (MW) and radius of gyration (Rg) of a scattering object:

∝ 9: 51 + ) #; ) + ⋯ where q is the scattering vector =

=>&? @

(Eq. 2)

D

sin ), with index of refraction n0, wavelength λ, and

scattering angle θ. Using Eq. 2, we estimate the theoretical contributions to intensity from a single DNA molecule and phage capsid (two scattering objects with approximately equal MW) as a function of the DNA’s radius of gyration (Figure 3). Initially, DNA is encapsidated and the capsid (MW,capsid = 31 x 106 Da)33 and DNA (MW,DNA = 32 x 106 Da)34, both with Rg ~ 31nm (λcapsid radius)35, contribute nearly equally to the total scattering intensity (Figure 3, point A on the black curve). After binding with receptor, DNA is ejected from the capsid into the surrounding buffer solution, so that the lower mass empty capsid becomes the dominant scattering object (Figure 3, point C on the black curve). However, the rapidly ejected DNA, while considerably less dense than encapsidated DNA, likely contributes transiently to the scattering signal since it can exist initially in a semi-condensed state before relaxing into the buffer (condensed ejected DNA coils are observed occasionally in cryo-EM micrographs as shown in Figure 3). This condensed ejected DNA state (e.g. with Rg as small as 55nm estimated from Figure 3, see cryo-EM image corresponding to point B on the black curve) is created when the ~20µm contour length of DNA is rapidly ejected from the tip of the phage tail within seconds17, which is significantly faster than DNA diffusion away from the phage16. Therefore, 12 ACS Paragon Plus Environment

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the ejected DNA contributes to the intensity until relaxing to its random coil state (~1µm36-37). At this point the DNA contributes negligibly to the scattering signal and the total intensity plateaus at around I ~ 0.5, with the empty capsid alone as the constant source of scattered light intensity (Figure 3, point C on the black curve). Thus, based on Eq. 2 and illustrated in Figure 3, each molecule of DNA likely contributes to a change in intensity as it relaxes from ~30nm (while encapsidated) to ~1µm (fully relaxed). This intensity decay process is caused by a single DNA molecule relaxation, and thus happens for each stochastically occurring phage ejection. The experimentally measured intensity, then, should reflect the sum of all DNA ejections and subsequent relaxations. Indeed, the total intensity measured by LS does decay to 0.5 of its initial value where it equilibrates, as predicted by Eq. 2 (see I/Iinitial plot, Figure 4B). Because of low contrast between vitreous ice and DNA, only highly condensed DNA can be visualized by cryo-EM. Therefore, for the thousands of imaged phage particles, the ejected DNA coils seen in Figure 3B are often not visible. However, free larger condensed DNA aggregates consisting of ejected DNA from multiple phages were consistently observed in cryoEM images. Therefore, it is likely that all ejected DNA molecules are at least loosely condensed immediately after ejection and contribute to the scattering intensity before fully relaxing in solution (as shown in Figure 3). The relaxation dynamics of the ejected DNA may be as slow as reptation dynamics of an entangled DNA chain and can thus be estimated with a self-diffusion coefficient of ~ 6 x 10-3 µm2/s38-39. Therefore, the relaxation-diffusion of a single ejected DNA from ~55nm (tightly coiled) to ~1µm (relaxed random-coil) could take up to ~35 minutes. This suggests that the LS-measured τslow of ~49 minutes could be indeed associated with the relaxation/self-diffusion of ejected DNA molecules.



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To summarize, Figure 1A shows that three independent experiments all suggest that the LS-measured fast exponential decay time constant, τfast, corresponds to the average lag-time for stochastic initiation of DNA ejection from phage λ. Similar observations of LS-measured stochastic DNA ejection events in vitro have also been made for bacteriophage T521. T5, however, pauses during its genome translocation process40, unlike λ which ejects continuously. The minutes-long stochastic delay in phage λ genome ejection (despite essentially instantaneous phage-receptor binding13, 21) is orders of magnitude longer than the time necessary for λ DNA translocation17 and is on a similar time-scale as the one-step phage replication cycle7. It may therefore play a significant role in the overall phage population dynamics during infection. Yet, the mechanism leading to the stochasticity has not been investigated and remains unclear. In the next section we investigate the nature of the ejection stochasticity by using our LS-based assay to analyze the effect of portal vertex stability5, 24 on dynamics of viral ejection events.

Force balance between portal vertex stability and internal DNA pressure controls the dynamics of DNA ejection events The results of the previous section, summarized in Figure 1A, demonstrate that the LSmeasured time constant τfast corresponds to the average lag-time for initiation of DNA ejection. We now use our LS-assay to investigate the mechanism leading to the minutes-long delay in dynamics of initiation of DNA ejection events. As described above, pressure driven DNA ejection out from bacteriophage capsids occurs through a portal vertex, which connects the phage tail to the capsid41. The same portal vertex is also the pathway for motor-assisted DNA packaging during virion assembly42. It has been suggested that once the DNA is packaged, the pressurized genome is retained within the capsid by the action of a portal “plug” protein43-45


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and/or by the portal ring/neck structure forming a valve blocking the DNA from exiting46-49. Here we refer to these structures as a portal vertex or portal that prevents spontaneous DNA loss which results in inactivation of phage particles. We have recently found that portal vertex stability is determined by the delicate force balance between the portal vertex strength and the internal DNA pressure24. We showed that this force balance can be perturbed either mechanically, by an increase in the capsid’s DNA pressure, or thermally by destabilizing the portal structure at higher temperatures24. At a critical level of thermo-mechanical destabilization, the portal opens and DNA is ejected, even in the absence of a specific phage receptor5. In our previous study5, we found that such heat-triggered DNA ejection from wild-type (WT) DNA length phage λ occurs at ~70°C. On the other hand, the temperature for heat-triggered DNA ejection for an underpackaged λ mutant (with 78% of WT DNA length, resulting in a lower DNA pressure of 15 atm compared to 25 atm in a WT DNA phage λ20) is nearly 5°C higher5. Since the mutant is otherwise identical to the WT phage, this shift up in DNA ejection temperature suggests that the heat-triggered ejection mechanism relies on mechanical destabilization of the portal exerted by DNA pressure in the capsid. In the same study5, we measured heat-triggered ejection from WT phage P22 and a P22 mutant with a weakened portal complex (keeping the packaged DNA length and therefore pressure unchanged). Indeed, the temperature for heat-triggered ejection was ~5°C smaller for the weakened-portal P22. This findings strongly suggest that heat-triggered ejection occurs when the portal opens due to critical destabilization by heat and DNA pressure. In the following section we argue that receptortriggered ejection is also sensitive to the critical thermal destabilization necessary to open the portal. However receptor binding significantly lowers the threshold for this critical



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destabilization, allowing DNA ejection events to proceed at a rate high enough for efficient infection. The details supporting this argument are below. In this work the DNA ejection through the portal from WT phage λ is triggered by addition of LamB receptor under physiological conditions mimicking those of in vivo infection. As described in the previous section, despite the addition of a specific phage λ receptor, the DNA ejection does not occur instantaneously and simultaneously from all phage particles. Instead we found a significant stochastic lag-time preceding ejection events. In this section we vary the temperature to thermally destabilize the portal vertex and investigate how the inherent portal stability correlates with receptor-triggered DNA ejection dynamics. While temperature destabilizes the portal vertex structure against the internal DNA stress5, 24, it does not affect the internal DNA pressure itself. Figure 4A shows our recently reported25 SAXS data for DNA-DNA interaxial spacing measured inside WT phage λ between 10 and 37° at identical buffer conditions with LS-measurements. DNA interaxial distance inside the capsid is set by DNA-DNA repulsions and bending stress, which in turn determine the DNA pressure on the capsid walls19, 50-51

. Therefore this plot suggests that DNA pressure is unchanged with temperature, since DNA-

DNA spacing remains essentially constant at 27.6Å. Thus, by increasing the temperature we isolate the effect of thermal destabilization of the portal vertex on ejection dynamics while keeping the capsid DNA pressure unaffected. Figure 4B shows LS-measured normalized intensity I/Iinitial versus time for the process of phage λ DNA ejection triggered by LamB receptor in the temperature range 15-45°C. As has been explained above, we fit the scattering data to a sum of two exponentials (with exponential time constants τfast and τslow), /F&F'FG = HI + 2 exp −M/NOG' + 2) exp −M/NP . Here we focus only on the much faster intensity decay process shown above to correspond to stochastic


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DNA ejection events, with τfast reflecting the average lag-time for initiation of ejections. [We separately confirmed, at each temperature, that τfast is the dominant process since it is an order of magnitude larger than τslow. Furthermore, normalizing the data between 0-1 as in Figure 1A and analyzing it such that the coefficients of the exponentials must sum to 1, the τfast process is shown to contribute to 0.75-0.85 of the measured scattering signal in the measured temperature range]. At the same time, the much slower secondary process τslow shows very weak temperature dependence and remains close to a value of ~35min (data not shown). This observation agrees well with the proposed above post-ejection DNA coil diffusion process, where temperature dependence of diffusion rates is very weak compared to measured here temperature dependence of DNA ejection events from phage. Having established above the physical interpretation of τfast, we now convert it to the more biologically relevant half-life values given by N/) =

ln2 x NOG' , a measure of the time required for half of the phage population to eject their

genomes. Figure 4C (main plot), showing τ1/2 versus temperature, reveals that the half-life decreases 15 times when the temperature is raised from 15 to 45°C (τ1/2 = 7.5 minutes versus 30 seconds). Since this increase in temperature thermally destabilizes the portal while leaving DNA pressure unchanged, this striking decrease in DNA-filled phage half-life demonstrates the major effect of temperature regulated portal vertex stability on dynamics of phage DNA ejection events. To further explore the dependence of phage λ DNA ejection dynamics on temperature, we plot ln(1/τfast) versus 1/kT in Figure 4C (inset) to derive from the slope the activation energy Ea provided by the Arrhenius rate equation: 1/NOG' = 2exp−SG ⁄TU, where A is a prefactor, T is the temperature, and k is Boltzmann constant. The measured activation energy value for initiation of receptor-triggered DNA ejection is Ea = (1.2 ± 0.1) x 10-19 J/phage (corresponding to



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~28 kT/phage at 37°C). This energy is required for the critical thermo-mechanical destabilization that causes the portal to open. It is interesting to note that this energy barrier is 3 times smaller than that associated with the heat-triggered DNA ejection without a receptor5, indicating that receptor binding significantly lowers the threshold for critical thermo-mechanical destabilization of the portal. Our data shows that at 40°C (close to the optimum temperature for infection of ~37°C), the introduction of receptor leads to a 600 times decrease in the half-life of infectious phage λ particles (without LamB, τ1/2 = 7.5 hours5, while with LamB τ1/2 = 45 seconds, see Figure 4C). Despite the receptor binding, the energy barrier for portal opening and initiation of DNA ejection remains an order of magnitude larger than the average molecular thermal energy52 (~1.5 kT), which prevents instantaneous and synchronized DNA ejection from all phage particles. Thus, there is apparently a fail-safe double mechanism to keep DNA inside the capsid – ejection requires both receptor binding and portal opening caused by critical thermo-mechanical destabilization of the portal. This portal complex opening may be associated with removal of the so-called portal plug structure leading to ejection43, 45, 47 or with other conformational changes. Since keeping the DNA inside the virus is of the utmost importance for viral survival between infection events, it is not surprising that there is an energy barrier for DNA ejection, even in the presence of susceptible cells. A small energy barrier for ejection initiation, which can be overcome by thermal energy, ensures that DNA ejection events occur at a high rate at physiological temperatures but are limited at low temperatures where cellular replication machinery is inefficient. Indeed, it is interesting to note that the activation energy measured here for receptor-triggered phage DNA ejection (~28kT) is similar to the activation energy (measured in the same temperature range) of ~22kT regulating the growth of the λ host, E. coli53. This


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suggests that the phage is tuned to respond to temperature changes the same way as its host does—ensuring that rates of infection are highest when the host reproductive machinery is most efficient. Virion metastability is one of the central concepts in virology54. It implies that the virus, in order to successfully replicate, must be sufficiently stable to prevent spontaneous release of its genome outside the cell between infection events, and at the same time be unstable enough to release its genome during infection. Viral particles are therefore not inert structures and have not attained the minimum free energy conformation, separated by an energetic or kinetic barrier, prior to cell attachment and entry54. Thus viral structure, and in particular the delicate force balance between DNA pressure and portal strength, plays an active role in genome delivery to the host cell. The metastable state of the portal vertex in the capsid provides a paradigm of physical adaptation of viruses to the physiologic environment of their host. CONCLUSIONS In this work we employ a unique combination of time-resolved techniques involving both single-molecule (cryo-EM) and bulk (LS and SAXS) analysis to reveal the stochastic behavior of DNA ejection events from phage λ. Specifically, we quantify the average lag-time between receptor binding and initiation of DNA ejection from a population of phage. The lag-time is described by a characteristic time constant which is derived from the bi-exponential fit to the time-resolved LS data. While DNA translocation from a single phage λ particle occurs on the time scale of seconds17, our measured lag-time for deactivation of a phage population is on the order of minutes. This slower rate of DNA ejection events likely influences the overall replication dynamics of the infectious cycle in vivo, which is on a minutes to hours time-scale6-11. Therefore, we investigate the mechanism leading to this delayed and stochastic genome ejection



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behavior. We measure population dynamics of DNA ejection events at variable temperatures between 15 and 45°C. By thermally destabilizing the portal vertex on the capsid, through which DNA ejection occurs, we found that ejection dynamics increases by 15 times when the temperature in increased (lag-time decreases from ~7.5 minutes to ~30 seconds). We have previously found that portal vertex stability is influenced by both temperature and internal pressure of the encapsidated DNA5, 24. In our investigated temperature interval, the internal DNA pressure remains unaffected25 while the portal is thermally destabilized. Furthermore, due to the strong temperature dependence of DNA ejection rate we are able to deduce the activation energy required to initiate ejection. This energy barrier is several times lower than the energy barrier to release phage DNA through thermal portal destabilization alone without phage LamB receptor present (Ea ≈ 28 kT versus 82 kT at 37°C). Nevertheless, even with receptor binding, the energy barrier for ejection it is an order of magnitude higher than the thermal molecular energy52. These findings suggest a fail-safe double protection mechanism at the portal vertex preventing spontaneous DNA ejection under sub-optimum conditions for infection, such as at lower temperatures. Besides the requirement for susceptible cells with phage receptors, the thermal regulation of the portal stability also plays a significant role for viral genome delivery. That is, temperature must be sufficiently high (e.g. ~37°C corresponding to favorable replication environment in E. coli cells) in order for virus uncoating to take place with high efficiency. This facilitates rapid spread of infection, improving the viral fitness1, 3, 6. ACKNOWLEDGEMENTS We would like to greatly acknowledge Gunnel Karlsson for help with cryo-EM imaging experiments and Sherwood Casjens for important discussions. This research was supported by the National Science Foundation Grants (CHE-1152770 and CHE-1507694 to AE), Swedish


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Research Council Grant (622-2008-726 to AE) and the National Science Foundation Graduate Research Fellowship Grant (DGE-1252522 to KGF). Figure Captions: FIG. 1. (A) λ phages initiate DNA ejection stochastically after addition of LamB receptor at 20°C. Time-resolved LS data (black points) shows a bi-exponential decay, where ~80% of the intensity decay is caused by the faster exponential process quantified by τfast. τfast measures the average lag time between receptor binding and DNA ejection (both instantaneous events on this timescale), since DNA ejection occurs stochastically after addition of LamB. This stochastic ejection behavior is confirmed by analysis of cryo-EM snapshots during the ejection process and is also apparent in time-resolved SAXS scattering intensity measurements (data from all three experiments shown in the plot). (B) A series of cryo-EM micrographs shows the stochastic nature of DNA ejection events. For these images, LamB was introduced to a solution of λ phages at t = 0min and the sample was incubated at 20°C until loading and vitrification at the specified time. FIG. 2. A theoretical estimate, based on Eq. 2, of DNA’s changing contribution to intensity through the ejection process from a single phage. Point A represents the DNA-filled capsid, where the equally massive DNA and capsid contribute equally to the total intensity. The total intensity is normalized by this point, when both the capsid and DNA have Rg~31nm. Point B represents the intensity immediately after ejection, when DNA may be tightly condensed (Rg~55nm, estimated from the cryo-EM micrograph corresponding to point B and therefore still a significant contributor to the total intensity. Finally, point C represents the final scenario when DNA has fully relaxed and no longer contributes to the total intensity. At this point the intensity is due to empty capsids alone, which contribute constant intensity throughout the ejection process. FIG. 3. (A) An illustration of stochastic ejection events versus synchronized ejection. (B) The measured SAXS I versus q data shows that I(q≈0) decreases during the DNA ejection and diffusion processes, indicating that the average molecular weight of scattering particles decreases as the system progresses toward one with all empty capsids and diffuse DNA. (C) The presence of fixed intensity points at certain q values (isosbestic points) and an unchanged q-position of the first minimum in the experimental SAXS curves progressing with time indicates that the sample is a mixture of DNA-filled and empty phages. The experimental SAXS curves versus time are compared to core-shell models of stochastic ejection events and synchronized ejection. Our data agrees well with the stochastic ejection model. FIG. 4. (A) DNA pressure in the phage λ capsid is unchanged with temperature, as indicated by an essentially constant interaxial DNA-DNA spacing determined by SAXS (data reprinted from our recent publication25). (B) LS-measured normalized intensity for stochastic DNA ejection and DNA relaxation-diffusion from phage λ population mixed with LamB. The LS-data is fit with a bi-exponential curve to quantify τfast, which is the average lag time for initiation of stochastic DNA ejection. (C) τfast values obtained from the bi-exponential fit to the LS-measured data in Figure 3A are converted to the more biologically relevant half-life values for DNA-filled phage population, τ1/2. τ1/2 depends strongly on temperature, an effect caused by the activation energy 21 ACS Paragon Plus Environment


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barrier, Ea, required for critical destabilization of the portal vertex leading to DNA ejection. Ea is determined from the Arrhenius plot (inset).

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Portal Stability Controls Dynamics of DNA Ejection from Phage - The

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