1 A cell membrane derived platform to study virus binding kinetics and

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A cell membrane derived platform to study virus binding kinetics and diffusion with single particle sensitivity Nadia Peerboom, Eneas Schmidt, Edward Trybala, Stephan Block, Tomas Bergström, Hudson P. Pace, and Marta Bally ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00270 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 29, 2018

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ACS Infectious Diseases

A cell membrane derived platform to study virus binding kinetics and diffusion with single particle sensitivity

Nadia Peerbooma1, Eneas Schmidta1, Edward Trybalab, Stephan Blockc, Tomas Bergströmb, Hudson P. Pacea and Marta Ballya,d*

a

Department of Physics, Chalmers University of Technology, Fysikgränd 3, 412

96 Göteborg, Sweden b

Department of Infectious Diseases, Section for Clinical Virology, Institute of

Biomedicine, University of Gothenburg, Guldhedsgatan 10B, 413 46 Göteborg, Sweden c

Department of Chemistry and Biochemistry, Freie Universität Berlin,

Takustraße 3, 141 95 Berlin, Germany d

Department of Clinical Microbiology, Umeå University, NUS Målpunkt R,

901 85 Umeå, Sweden 1

These authors contributed equally to this work

*

corresponding author [email protected]

Discovery and development of new antiviral therapies essentially rely on two key factors: an in-depth understanding of the mechanisms involved in viral infection and the development of fast and versatile drug screening platforms. To meet those demands, we present a biosensing platform to probe virus-cell membrane interactions on a single particle level. Our method is based on the formation of supported lipid bilayers from cell membrane material. Using total internal reflection fluorescence microscopy, we report the contribution of viral and cellular components to the interaction kinetics of herpes simplex virus type 1 with the cell membrane. Deletion

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of gC, the main viral attachment glycoprotein, or deletion of heparan sulfate, an attachment factor on the cell membrane, leads to an overall decrease in association of virions to the membrane and faster dissociation from the membrane. In addition to this, we perform binding inhibition studies using the antiviral compound heparin to estimate its IC50 value. Finally, single particle tracking is used to characterize the diffusive behavior of the virus particles on the supported lipid bilayers. Altogether, our results promote this platform as a complement to existing bioanalytical assays, being at the interface between simplified artificial membrane models and live cell experiments. Keywords: virus-membrane interactions, supported lipid bilayers, herpes simplex virus, binding kinetics, single particle tracking, biomimetic sensing platform.

Recent outbreaks of viral diseases have demonstrated the need for more efficient and readily available antiviral treatments. The development of such therapies relies among others on two key aspects: A fundamental and in-depth understanding of the viral infection cycle on the one hand and the development of simple bioanalytical tools for antiviral drug screening on the other. One central step of the viral life cycle to focus on in this context is the interaction between the virus and the plasma membrane, which needs to be crossed both during virus entry and egress. Despite intense research in this field a complete picture of the mechanisms employed by many viral species to modulate the biomolecular interactions between viral and cellular components during initial attachment or release from the cell surface1 is still lacking. This missing knowledge is of importance for identifying new viral inhibitors as antiviral drug candidates and will, together with fast and efficient drug screening tools, facilitate progress in antiviral research.


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In recent years, there has been an increased demand for biomolecular binding assays to study virus-cell membrane interactions both for fundamental applications and for drug testing. The simplest of these platforms is based on the immobilization of a cell receptor of interest to a sensor surface in order to characterize the interaction of virus particles or viral proteins using surface-sensitive techniques2,3 or to measure the effect of virus inhibitors in competition assays4,5. While such receptor-based assays allow for a precise control of the surface composition and determination of reaction constants, the cell receptor lacks its natural lipid environment, which has been shown to play an important role for the functionality of a variety of biomolecules, in particular membrane proteins6. These platforms also appear unsuited for the study of multivalent interactions, a characteristic common to virus-membrane interactions7, which could rely on mobile cell membrane components to form multiple bonds between viral proteins and cell membrane receptors8. In order to better mimic the structure of the cell membrane, and to preserve the mobility of the receptors, a number of studies employ supported lipid bilayers (SLBs), in which the receptors of interest are incorporated to study their interactions with viruses9,10. These minimalistic biomimetic systems exhibit a high degree of flexibility and allow for a tight control of the surface properties, while providing structural similarities to the native cell membrane. However, traditional SLBs remain highly simplified model membranes, whose compositional complexity is far from that of native membranes. To study virus-cell membrane interactions in their most natural environment, a number of studies rely on live cells11,12. Cell-based assays are also the most commonly used approach for viral inhibition tests13,14. These studies provide a high degree of compositional complexity; however, it becomes difficult to dissect the initial attachment steps of the virus to the cell membrane and to decouple the contributions



of cell membrane components from those of other cellular factors. A needed element in the library of platforms to study virus-cell membrane interactions is therefore a system that combines the compositional complexity of native membranes with the control and flexibility of traditional SLBs. A number of reports demonstrate the possibility of incorporating native membrane material from mammalian, insect, or bacteria cells, into planar bilayers, thereby creating complex supported lipid bilayers15–19. Such platforms contain the whole milieu of native cell membrane components (e.g., lipids, proteins, and carbohydrates) without the experimental complications related to working with living cells. Furthermore, they are compatible with surface-based sensing techniques. In this paper, we demonstrate the potential of SLBs containing native membrane material as tools to probe virus-membrane interactions. These SLBs are derived from native-membrane vesicles (NMV) extracted from the plasma membrane, and therefore herein referred to as NMV-derived SLBs (nSLB). To construct the platform, we took advantage of a method based on the creation of hybrid vesicles containing both native and synthetic membrane material19 to form nSLBs via spontaneous rupture of the hybrid vesicles. We chose to use nSLBs in combination with total internal reflection fluorescence microscopy (TIRFM) to probe virus-cell membrane interactions and particle mobility on a single particle level (Figure 1). The advantage of this approach over more traditional biosensing assays based on ensembleaveraging, is that single particle detection with TIRFM allows us to resolve binding behaviors of subpopulations, a feature that is of importance when studying highly heterogeneous virus suspensions20,21. We focused our study on the herpes simplex virus type 1 (HSV-1), a widespread human pathogen commonly known for causing blisters on the skin or mucosa of the


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oral or genital area. In particular, we studied the contribution of different cellular and viral components to HSV-1 attachment and detachment kinetics. Furthermore, we performed binding inhibition studies using heparin as a sample antiviral compound. Finally, we performed single particle tracking to characterize the diffusive behavior of the virus particles on the nSLBs.

Evanescent field

TIRF illumination

Figure 1: Probing single fluorescent virus particles interacting with NMV-derived supported lipid bilayers with TIRFM. Thanks to the evanescent field created by the TIRFM illumination, only particles in close proximity to the surface will be detected.

Results and Discussion To probe binding kinetics and mobility of individual HSV-1 particles interacting with plasma membrane components in a native-like environment, we produced supported lipid bilayers in which we incorporated cell membrane material extracted from mechanically disrupted green monkey kidney (GMK) AH1 cells, a cell-line commonly used for HSV culture and infection studies22. The NMVs, obtained from the disrupted GMK cells, do not spontaneously rupture into planar SLBs, likely due to the high percentage of proteins, gel-phase lipids, and cholesterol, all of which have been reported to inhibit SLB formation23–26. We therefore took advantage of a previously described method19 relying on sonication-induced fusion of the NMVs



with synthetic vesicles containing PEGylated lipids (PEG_POPC) to form hybrid vesicles. These hybrid vesicles can, up to a critical ratio of native membrane material, spontaneously rupture upon contact with the glass surface to form a planar bilayer. Besides facilitating spontaneous SLB formation, the PEGylated lipids have also been shown to increase the percentage of mobile transmembrane proteins in the resulting nSLBs19. The successful fusion of native NMVs and synthetic PEG_POPC vesicles was verified using a Förster Resonance Energy Transfer (FRET)-based assay (Section S1 in the Supporting Information). Through real time observation of the bilayer formation process using TIRFM, we further verified successful SLB formation (see figure S3), and chose a suitable ratio of native vs. synthetic material for our experiments. As further discussed in section S3 of the Supporting Information, the 6% native nSLBs presented low numbers of unruptured vesicles after SLB formation (estimated from the data presented in Figure S3 to cover ~3% of the total surface area), which was completed within a short time (10 min). Most importantly, these nSLBs were found to contain sufficient native membrane material to obtain statistically relevant numbers of HSV-1 binding events. The 6% ratio, which is expressed in terms of the contribution from native cell material to the total surface area of the nSLB (Section S2 in the Supporting Information), was therefore used for all further experiments. We further used Fluorescence Recovery After Photobleaching (FRAP) to assess the quality of the 6% native SLBs. We estimated lipid mobility in the nSLBs to 0.83 ± 0.09 µm2/s with an immobile fraction of 15.9 ± 1.9%. For comparison, the lipid mobility of fully synthetic SLBs (made of 100% PEG_POPC) was estimated to 1.73 ± 0.09 µm2/s with an immobile fraction of 0.2 ± 0.1%. The lower lipid mobility of the nSLBs in comparison to PEG_POPC is in line with previous results19 and can be


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explained by the presence of native cell membrane components, which leads to a higher number of non-ruptured vesicles in the nSLB. While it is at this stage not clear how the presence of these vesicles influences the binding behavior of the virus, we expect their impact on our study to be minor, as we estimate the non-ruptured vesicles to represent only ~3% of the total surface area.

HSV-1 binding kinetics To illustrate the potential of our bioanalytical platform in fundamental interaction studies and virus inhibition studies, we probed binding kinetics of single HSV-1 virions to nSLBs obtained from GMK AH1 cells. More precisely, we quantified association and dissociation of the virions to and from the surface to investigate how specific viral or cellular components influence the binding and release propensity of the virus. Initial attachment of HSV-1 to the cell membrane occurs mainly via interaction between the viral envelope glycoprotein gC27, but also by a redundant function provided by gB28, and cell-surface glycosaminoglycans (GAGs) heparan sulfate (HS)29 and chondroitin sulfate (CS)30. This is then followed by internalization after interaction between other viral glycoproteins and a number of cellular entry receptors of protein and carbohydrate nature31.

Influence of viral glycoproteins To quantify the contribution of the viral glycoprotein gC to the virus binding kinetics and avidity, we compared the nSLB binding kinetics of a mutant HSV-1 strain, deficient in gC expression, to the wild-type (WT). Figure 2 shows measured binding kinetics for the two viral strains on GMKderived nSLBs together with negative controls performed on SLBs from PEG_POPC



vesicles only. The association kinetics, presented as the cumulative number of bound particles over time (Figure 2A), reveal that binding of the gC-deficient mutant to the nSLBs is reduced to ~25% of the WT level (Figure 2C), while no association events could be detected on the PEG_POPC bilayers. While a determination of association rate constants is complex for our system which involves multivalent binding of the HSV-1 virions to native membrane receptors, a direct qualitative comparison of apparent , i.e. the on-rate constant for the binding of a virus particle to the nSLB, for the two virus strains is nevertheless possible under the given experimental conditions, which ensure that the measurement is reaction limited and that the number of occupied receptors is low as compared to the total number of receptors (see section S4 in the Supporting Information for further information). Thus, this comparison shows that the apparent for the gC negative virus is ~4 times lower than for the WT. Figure 2B shows dissociation curves for WT and mutant particles. These curves typically showed a fast dissociating population of particles and an irreversibly bound fraction (in the time frame of our experiments). This complex dissociation behavior was therefore approximated using a single exponential decay function plus constant (see section S4 and Figure S5 in the Supporting Information for a more detailed discussion on the fits). From these fits we extracted apparent dissociation rate constants for the reversibly bound subpopulations and irreversibly bound virus fractions. The latter ones did not appear to be significantly affected by the removal of gC (65.3% ± 10.7% for KOSc WT vs. 73.3% ± 9.0% for gC def.) As summarized in Figure 2D, apparent values for the mutant strain were increased ~2 fold in comparison to WT level. One can therefore conclude that in absence of gC in the


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virion, the overall apparent affinity of the virus to the nSLB is reduced by a factor of ~8, as compared to the WT. This result reflects the commonly accepted notion that gC acts as the main attachment protein of HSV-127. We attribute residual binding of gC-deficient particles to the nSLBs to either glycoprotein gB or other minor receptors on the virus28. Interestingly, previous studies on live cells showed that significant changes in attachment of gC-deficient mutants in comparison to the WT only became visible after relatively mild pre-treatment of cells with heparinase, an enzyme degrading HS32, thereby reducing both size and amount of HS in the cell membrane33,34. This might indicate that gC-deficient virions rely on a high GAG expression to successfully bind to the cell surface via gB, which is not the main attachment protein. This observation therefore promotes the use of our model system to study the contribution of specific glycoproteins to the initial attachment to the cell membrane. Indeed, in our system, cellular lipids, proteins and other receptors are more diluted as compared to the crowded native cell membrane. While it cannot be excluded that this dilution could alter the characteristics of the probed interaction (a lower receptor density could effectively reduce the number of bonds between virus and membrane, which affects, for example, the overall affinity), it can also be beneficial to resolve differences in binding kinetics of viral mutants of compromised functionality (as shown here). Indeed, promiscuous viruses have higher chances to find alternative pathways for interaction when exposed to dense native cell membranes, which makes differences in functionality less visible.



A

B

C

D

Figure 2: Binding kinetics of WT (KOSc) vs. gC-deficient HSV-1 particles on GMK AH1 cells derived nSLBs. A) Association kinetics: Cumulative plot of associated particles over time including linear fits and association rates (AR) obtained from the fits for a representative dataset. The curves were normalized using the surface area and the final virus concentration. B) Dissociation kinetics: Fraction of bound particles over time including exponential decay fits and values obtained from the fits for a representative dataset. C) Relative association rates (slopes of linear fits of the association curves) normalized with respect to the WT virus for each dataset. Averages of normalized association rates were calculated over ≥ 3 datasets and presented with standard deviations. D) Relative dissociation constants (koff) (obtained from exponential decay fits of the dissociation curves) normalized with respect to the WT virus for each dataset. The averages of the normalized values were calculated over ≥ 3 datasets and presented with standard deviations.

Effect of heparinase treatment on HSV-1 binding kinetics While the previous section focused on probing the effect of alternations of the virus glycoprotein composition on binding kinetics, we show here the possibility to

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alter the composition of the nSLBs. As an example, we reduced the display of HS on the cell surface by treating the GMK AH1 cells with heparinase prior to NMV production. To allow for a direct comparison of the HSV-1 association rates to nSLBs from treated and non-treated cells, we formed nSLBs containing the same amount of native membrane material (6%) from both NMV suspensions. SLB formation characteristics were qualitatively very similar for the two batches of NMVs (Figure S4). Successful enzymatic treatment with heparinase was verified by qualitative comparing the amount of HS displayed in the nSLB by immunostaining of the HS chains (using a primary anti-HS antibody and a fluorescent secondary antibody). For the heparinase treated nSLBs, the amount of fluorescent signal in the image (determined by the signal-to-background value, see Methods section) was reduced by a factor 2.75 ± 0.55 ( = 3). In control experiments performed in absence of primary antibodies signal and background were undistinguishable. This confirms that the heparinase treatment effectively reduced the amount of HS in the nSLBs. We proceeded with equilibrium fluctuation analysis to study the effect of the heparinase treatment on HSV-1 binding kinetics (Figure 3). Modified presentation of HS in the nSLBs translated into a decrease in association rates to ~ 60% (Figure 3A & C) and a ~ 2.5 fold increase of dissociation rate constants (Figure 3B &D), while the irreversible bound fraction of particles appeared unaffected (62.0% ± 16.7% and 66.7% ± 5.5% for non-treated and heparanase treated nSLBs, respectively). The observed change in association rates could be a consequence of either a reduction of the number of available binding sites in the nSLB, a change in the association rate constant of individual HSV-HS bonds, caused directly by the heparinase treatment, or a combination of both. While this complicates the comparison of relative values, and thereby the calculation of the change in apparent affinities, our results



nevertheless provide a qualitative comparison of the binding propensities of the virus to the different nSLBs. We concluded that binding and release of the virus to/from the nSLBs is strongly influenced by the amount of HS, which is in line with previous reports demonstrating reduced HSV-1 attachment to heparinase-treated cells33,35 and reflects the role of HS in recruiting HSV-1 to the cell surface29. Residual binding may be related to incomplete degradation of the HS chains by heparinase, but also to the interaction of HSV-1 with other cellular components besides HS. Indeed, it is known that HSV-1 also binds to CS30. In addition, HSV-1 also interacts with a series of entry receptors (for example HVEM (herpes virus entry mediator) and nectin-1 and nectin231), which are receptor proteins for viral glycoprotein gD facilitating entry of viral particles by the gB-mediated fusion between lipids of the viral envelope and the cell plasma membrane.


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A

B

C

D

Figure 3: Effect of heparinase treatment of GMK AH1 cells on HSV-1 binding kinetics. A) Association kinetics: Cumulative plot of associated particles over time including linear fits and association rates (AR) obtained from the fits for a representative dataset. The curves were divided by the image area and the final virus concentration. B) Dissociation kinetics: Fraction of bound particles over time including exponential decay fits and values obtained from the fits for a representative dataset. C) Relative association rates (slopes of linear fits of the association curves) normalized with respect to the non-treated nSLB for each dataset. Averages of normalized association rates were calculated over ≥ 3 datasets and presented with standard deviations. D) Relative dissociation constants (koff) (obtained from exponential decay fits of the dissociation curves) normalized with respect to the non-treated nSLB for each dataset. The averages of the normalized values were calculated over ≥ 3 datasets and presented with standard deviations.

Inhibition of HSV-1 binding by heparin To demonstrate the potential of our platform in antiviral drug screening and in particular in the context of testing the efficiency of binding inhibitors, we performed



HSV-1 binding inhibition studies using the nSLBs. Binding inhibitors are compounds that prevent virus attachment to the cell membrane. As a model compound, we used heparin, a highly sulfated glycosaminoglycan known for its inhibitory effect on HSV infection36. HSV-1 virions were incubated with heparin for 10 min at different concentrations. Time-lapse movies were recorded for each condition and analyzed with equilibrium fluctuation analysis to determine the respective association rates (Figure 4A). Association rates were then plotted against the inhibitor concentration (Figure 4B). The characteristic sigmoidal shape37 of the dose-response curve in Figure 4A indicates successful inhibition; at highest heparin concentration (1 mg/mL) the relative association rates were reduced to ~ 25% of the non-inhibited case. From the fit of the curve, we obtained an IC50 value of 5.5 µg/mL, which represents the half maximal inhibitory concentration and serves as a measure of the inhibiting efficiency of the studied compound. The IC50 value is in good agreement with values reported from inhibition studies based on cell culture experiments38,39. Incomplete inhibition of HSV-1 binding and HSV-1 infectivity even at high heparin concentrations is also usually observed in cell culture-based experiments35,39. This may indicate that viral glycoproteins which are not occupied by binding to heparin retain their ability to interact with their specific cell surface receptors, thereby underscoring the importance of using a platform, which displays the full milieu of cellular membrane components for investigating antiviral compounds.


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A

B

Figure 4: HSV-1 binding inhibition by heparin. A) Cumulative plot of bound particles over time for different inhibitor concentrations for a representative dataset. B) Dose-response curve representing the relative association rate (obtained from the slope of the linear fits in A) over inhibitor concentrations. Calculated averages of association rates ( ≥ 3) were normalized with respect to the association rate at 0 µg/mL. The data was fitted with a sigmoid function using the equation y(x) = A + B/(1 + C × exp (Dx)).

Single Particle Tracking To characterize the diffusive behavior of single HSV-1 particles bound to the nSLBs, we further performed single particle tracking analysis of the recorded timelapse movies. From our analysis of the particle trajectories (Figure 5A) we concluded that the mobile virus particles were best described using a model for anomalous diffusion, representative for confined particle motion (see section S5 in the Supporting Information for further details about SPT analysis). Figure 5B shows histograms of diffusion coefficients for particles undergoing anomalous diffusion, which are dominated by a peak at an apparent diffusion coefficient (D value) of ~ 1 × 10-6 µm2/s. As reported previously40, this population can be assigned to the fraction of immobile particles; the non-zero value is a consequence of the localization noise of the SPT analysis41. In addition to this immobile HSV-1 fraction, the histogram shows a second peak appearing on average at 3.8 × 10-4 ± 2.3 × 10-4 µm2/s and ranging up to



0.1 µm2/s, which is several orders of magnitude higher than the apparent D value of the immobile fraction, and therefore a clear indication of mobile tracks. Our results show that nSLB-bound HSV-1 virions are either immobile or exhibit anomalous diffusion. The corresponding distribution of the confinement length LC of the anomalously diffusing particles shows a mono-exponential decay with a decay length around 2 µm (Figure S6B). We have recently reported a similar diffusive behavior for HSV-1 virions bound to end-grafted GAGs40, for which we reported diffusion coefficients that were in the same range as the ones observed for the nSLBs. The here observed diffusion of the virions is likely to originate both from the virus itself, through an exchange of bonds (as discussed in40), but also from the mobile components in the nSLB. As transmembrane proteins often show anomalous diffusion in cell membranes, which has also been observed in complex cell membrane-derived SLBs16, it is therefore reasonable to hypothesize that the observation of anomalously diffusing HSV-1 particles at least partially originates from anomalously diffusing nSLB-incorporated receptor structures. Diffusion of membrane-bound viruses is an important aspect when studying virus-cell membrane interactions, as a number of studies have shown that viruses diffuse on the cell membrane during initial attachment12,42,43. Since our nSLB platform is compatible with SPT studies, it appears as a good complement to live-cell studies, which require complicated experimental procedures and advanced tracking algorithms. The provided platform allows related experiments to be conducted in a less complicated experimental setting, offering the perspective to characterize virus diffusion in a near-native environment. Furthermore, single recorded tracks are longer than in live-cell experiments, where the virus can be readily internalized by the cell, and therefore contain more information.


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A

B

Figure 5: Single particle tracking of nSLB-bound HSV-1 particles. A) Trajectories of single WT HSV1 particles undergoing lateral diffusion on a representative nSLB surface. B) Histograms of diffusion coefficients for anomalous diffusion calculated from fits of the MSD curves. The histogram shows an immobile peak at 1 × 10-6 µm2/s and a mobile peak at 3.8 × 10-4 ± 2.3 × 10-4 µm2/s ( = 3).

Conclusions The cell membrane-derived platform we presented in this paper fills the gap between artificial model-systems and live cell experiments, by combining the flexibility and control provided by simplified model systems with the compositional complexity of a native system. It appeared to be a useful bioanalytical system to probe binding kinetics and mobility of HSV-1 for fundamental interaction studies and for antiviral drug tests. Our nSLBs represent a planar version of the cell membrane in which the native components are more dilute. Although this dilution could alter the natural interaction characteristics of the virus, we showed that it could also be of advantage when studying the contribution of single glycoprotein types to attachment and release. The nSLBs represent a snapshot of the composition of the cell membrane at a specific time point and the expression of membrane components in the nSLBs is therefore fixed. This is different from the case of live cells, where, for example, GAG chains are constantly degraded and recycled by the cell44. The platform is compatible



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with surface-sensitive techniques and single-particle based methods, which are essential to characterize highly heterogeneous virus samples. Another strength of our platform lies in the small sample consumption; we typically use volumes as little as 10 µL per dataset, which is an advantage, for example, when working with samples relying on tedious purification methods. Furthermore, our assay platform is compatible with standard 96-well plates and can be combined with automated sample preparation and imaging37, towards increased throughput, the main limiting factor being the time needed to acquire time-lapse movies of sufficient statistical significance. Increased throughput would be a valuable asset for antiviral drug screening applications37. Finally, an advantage of our method is its versatility, as it can, in principle, be easily adapted to different virus strains, inhibitors, or nSLBs derived from different cell lines, including mammalian and insect cells19. Indeed, the methodology introduced herein could be used to elucidate previously unknown hostpathogen receptors on the cell line of choice or study specific host-pathogen interactions by using a series of genetically modified cell lines. We believe that the versatility of our platform is a needed feature to meet the challenges of future viral outbreaks and new emerging virus strains.

Methods Materials – All materials were purchased from commercial sources, unless stated otherwise. Synthetic lipids (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), lissamine rhodamine B-DOPE (Rho-DOPE), 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)

(NBD-DOPE)

and

PEG5000-ceramide (PEG5Kce) were obtained from Avanti Polar Lipids Inc., USA. Phosphate buffered saline (PBS, tablets), PKH26 red fluorescent cell linker, and


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heparinase I were purchased from Sigma-Aldrich, Germany. The 10E4 (anti- heparan sulfate) antibody was from AMS Biotechnology Ltd, United Kingdom (Product # 370255-S, Lot # 10E4020216-290216), and Alexa Fluor 488 –labeled goat antimouse IgG / IgM (H+L) secondary antibody was from Thermo Fisher Scientific Inc. (Product # A-10680 # 1857664), Sweden. The buffer used throughout all experiments, unless stated differently, was PBS prepared using deionized water (filtered using a Milli-Q system, MILLIPORE, France) from tablets using the manufacturer’s instruction and then passed through 0.2 µm WHATMAN® filters (GE Healthcare, UK) prior to use. IllustraMicrospinTMs-200 HT columns were obtained from GE Healthcare, Sweden. Preparation of native membrane vesicles (NMV) – NMVs were prepared from GMK AH1 cells22 according to the protocol described in19. Briefly, the cells were cultivated in Eagle’s minimum essential medium (supplemented with 2% fetal calf serum and 0.05% Primaton RT substance). After rinsing, the cells were harvested using cell scrapers and harvesting buffer (2 × PBS supplemented with complete® protease inhibitor cocktail, (Roche)), and pelleted via 5 min centrifugation at 4 °C and 1000 × g. Cell disruption was performed using a dounce homogenizer (Kimble). Unbroken cells were separated from cell debris by centrifugation, re-suspended and disrupted using the same procedure until no visible cell pellet was left. Purification of the NMV suspension was obtained by 10 min centrifugation at 9000 × g, followed by 90 min. ultracentrifugation at ~180 000 ×g at 4°C. The final pellet was dissolved in 2 × PBS containing 20% glycerol. The protein content of the NMV suspensions was estimated using the CBQCA Protein Quantitation Kit (Thermo Fisher Scientific Inc., Sweden), according to manufacturer’s instructions, and a FLUOstar OPTIMA microplate reader (BMG Labtech, Germany) (excitation wavelength 440 nm;



emission wavelength 520 nm). This measurement yielded 1.22 ± 0.24 mg/mL of protein ( = 3). Heparinase treatment of cells – T75 cell culture flasks containing confluent monolayers of GMK AH1 cells were rinsed twice with PBS and 5 mL of heparinase I (2 units/mL) was added to the flasks. The flasks were incubated for 4 h at 37°C before proceeding with NMV preparation as described above. The protein content of these suspensions was estimated to 0.86 ± 0.16 mg/mL ( = 3). Preparation of synthetic vesicles – All lipids were dissolved and stored in chloroform. Lipids were mixed in round glass flasks to the following ratios: POPC vesicles: 100% POPC; PEG_POPC vesicles: POPC + 0.5 mol % PEG5Kce; FRET_POPC vesicles: 98 mol % POPC + 1 mol % Rhod-DOPE + 1 mol % NBDDOPE; Rhod_POPC vesicles (1 mol % Rhod-DOPE + 99 mol % POPC); PEG_Rhod_POPC vesicles (0.5 mol % PEG5Kce + 1 mol % Rhod-DOPE + 98.5 mol % POPC). Chloroform solvent was evaporated under N2 flow followed by >1 h evaporation under vacuum. Lipids were hydrated in PBS to a final concentration of 1.3 mM, vortexed (~1 min) and extruded 11 times through a 100 nm polycarbonate membrane (WHATMAN®) using a mini extruder (Avanti Polar Lipids Inc.). Creation of hybrid vesicles via sonication – A bath sonicator (Elmasonic S40H, Germany) was used to induce lipid mixing and vesicle fusion to produce hybrid vesicles. 12 µL of NMV suspension was mixed with 48 µL of PEG_POPC vesicles (1.3 mM) and sonicated at 30°C for 10 min (this ratio of NMV to PEG_POPC corresponds to 6% native vesicles; 3% native: 6 µL NMV + 54 µL PEG_POPC; 9% native: 18 µL NMV + 42 µL PEG_POPC; 14% native: 24 µL NMV + 36 µL PEG_POPC).


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FRET assays – A FRET assay monitoring decrease in FRET upon vesicle fusion was used to monitor the fusion between NMVs and synthetic vesicles containing a FRET pair and to quantify the NMV suspensions (see section S1 and S2 in the Supporting Information for more details). The fluorescent emission spectrum of the sonicated mixtures was measured at an excitation wavelength of 460 nm, using a spectrofluorometer (QM-4/2005, Photon Technology International Inc.). TIRFM – Microscope cover glasses (Fisher Scientific, USA) were boiled in a 10% 7X cleaning solution (MP Biomedicals, Santa Ana, USA) for at least 2 h, then rinsed and stored in water until use. The cover glasses were rinsed again with water and dried under N2 flow shortly before the experiment. Microwells were formed using a PDMS piece with punched chambers (2-3 mm diameter). TIRFM experiments were carried out using a Nikon Eclipse Ti-E inverted microscope (Nikon Corporation, Japan) and a 60× oil immersion objective (NA = 1.49). The microscope was equipped with a mercury lamp, Andor Ixon+ EMCCD camera (Andor Technology, Ireland) and TRITC and FITC filter cubes. Tracer vesicle experiments – To monitor nSLB formation in TIRFM tracer vesicles were used as previously described19. Briefly, 1 µL of tracer vesicles (Rhod_POPC) at 2.6 µM were mixed with 2 µL of hybrid vesicles at ~ 1.3 mM and 7 µL of PBS to produce a suspension with a final vesicle concentration of ~ 130 µM. 10 µL of this mixture was added to the PDMS microwells mounted on the microscope. Time-lapse movies of 0.2 fps were recorded and analyzed with in-house written MATLAB scripts to count the number of fluorescent vesicles per frame. FRAP measurements – To assess lipid mobility within the formed nSLBs, we performed Fluorescence Recovery After Photobleaching (FRAP) using a diode pumped solid-state 532 nm laser with 100 mW output (B&W TEK Inc.). NMVs were



sonicated with PEG_Rhod_POPC vesicles. nSLBs were formed as stated above. Excess vesicles were rinsed away after 15 min incubation with PBS followed by rinsing. Laser illumination of ~2 s was used to create a bleached spot in the fluorescent nSLB and time-lapse movies were recorded for 20 min at 0.2 fps. For comparison, we applied the same procedure of sonication, SLB formation and laser illumination to PEG_Rhod_POPC vesicles only. In this case time-lapse movies were recorded for 10 min at 1 fps. Lipid diffusions and immobile fractions were evaluated using the Hankel transform method described in45. Data analysis with this method can be carried out using a MATLAB software available for download45. Virus purification and fluorescent labeling – HSV-1 KOS (ATCC, VR1493)46 was cultured in GMK AH1 cells and purified from infectious cell medium through a three-step discontinuous sucrose gradient, as previously described35. In addition, the viral mutant HSV-1 KOS-gCdef, lacking glycoprotein gC due to a frameshift-causing mutation (deletion of cytosine at position 366 of gC gene), was used47,48. The concentrations of the virus suspensions were determined by extraction of viral DNA using the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics, Mannheim Germany), followed by detection and amplification of a 118-nucleotide segment of the highly conserved gB region of HSV-1 with a pair of primers and a gBspecific probe using real-time quantitative PCR49. A specific cycle threshold (Ct) value was obtained for each sample and Ct-values were then related to a standard curve with known concentrations of DNA copies. In addition, we determined the PFU counts for these virus samples using a viral plaque titration assay35. Briefly, confluent monolayers of GMK AH1 cells were incubated for 1 h with the virus suspension (HSV-1 KOS or HSV-1 KOS-gCdef) at different dilutions (range 10-3 – 10-7). After another 72 h of incubation with methylcellulose (1% in cell culture medium) at 37°C,


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the plaques were stained with crystal violet solution and counted manually using an optical microscope. DNA and PFU counts of the virus suspensions used in this study are given in Table 1. Viral envelopes were fluorescently labeled with PKH26 red fluorescent cell linker. To this end, 3 µL of dye (0.5 mM in ethanol) was added to 10 µL of virus stock solution with 200 µL of diluent C diluting agent (provided in the labeling kit), and incubated for 10 min. Excess dye was removed using IllustraMicrospinTMs-200 HT columns after exchanging the buffer solution of the spin columns to PBS. Table 1: HSV-1 virus variants used in this study

Used for

DNA count

PFU count

HSV-1 KOS batch1

Figure 2, 4, 5

7.99 × 1010 mL-1

8.1 × 109 mL-1

HSV-1 KOS batch2

Figure 3

6.55 × 1010 mL-1

1.5 × 1010 mL-1

HSV-1 KOS gCdef

Figure 2

1.11 × 1011 mL-1

2.6 × 109 mL-1

Equilibrium fluctuation analysis – nSLBs were formed on cleaned cover glasses after incubating the surface with a hybrid vesicle suspension for 15 minutes followed by thorough rinsing with PBS. Fluorescent virus solution was injected (10 µL of concentrated virus solution added to ~5 µL of PBS buffer) and time-lapse movies were recorded ~60 min after virus injection. For the heparin inhibition experiments, 5 µL of the fluorescent virus solution was pre-incubated with 5 µL of heparin solution in PBS at appropriate concentration for 10 min, before adding the mixture to the well. The TIRFM movies for these experiments (Figure 4) were recorded for 1500 s at a frame rate of 0.1 fps, while all other TIRFM experiments were carried out at 0.067 fps over a duration of 1 hour. Recorded movies were analyzed with in-house written MATLAB scripts and a method called equilibrium



fluctuation analysis50,51, to quantify the rate of arrival and release of virus particles to and from the surface. Briefly, for each frame, the software counts the number of newly bound and released particles and determines the residence time for each particle. Particle detection is based on a three-level threshold (high – medium –low) algorithm: particles of intensity higher than the high threshold are counted as bound if present for at least 3 frames, and considered dissociated if their intensity drops below the low threshold. The medium threshold serves to discern particles with gradual intensity loss (i.e. bleached particles) from particles of abrupt intensity loss (i.e. dissociated particles). From plots of the cumulative number of newly associating particles over time, association rates are obtained by calculating the slope of the linear fit of the curve. These association rates are directly proportional to the apparent , i.e. the on-rate constant for the binding of a virus particle to the lipid membrane, under our experimental conditions. The obtained association rates were divided by the total surface area of the images, and by the virus concentration (in molar) based on the viral DNA count. Note that the first 13% of frames disregarded for the fit, to exclude initial faster association rates, which are an artifact of the software, originating from an underestimation of the number of particles in the first frames. Dissociation plots were constructed based on the histograms of the residence times of the particles and represent the fraction of bound particles over time. Only particles landing in the first half of the movie were considered in this case, to avoid an overrepresentation of short residence times. By applying a single exponential (plus constant) fitting model to the dissociation curves, we obtained apparent dissociation rate constants and fractions of irreversibly bound particles (fraction of particles that did not dissociate within the experiment time). Using the association rates (normalized by the image area and the virus concentration) and , the overall


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affinity, i.e. the avidity of the overall HSV-membrane interaction could be determined using = ⁄ , where a high value represents a low avidity). More detailed information on the extraction of binding rate constants from the association and dissociation plot is provided in section S4 of the Supporting Information. Single particle tracking – The recorded TIRFM movies were further analyzed to characterize particle mobility using in house written MATLAB scripts for single particle tracking, as previously described in40. Briefly, for each trajectory (constructed from detected particles on each frame), the software calculates the mean squared displacement (MSD), which is plotted against the lag time (∆ ) (maximum lag time limited to 10%). MSD curves were fitted using both a model for normal diffusion and a model for anomalous diffusion (see section S5 in the Supporting Information for more detailed information). Accordingly, trajectories were assigned to either diffusion mode and histograms of diffusion coefficients were generated separately for both. Peak coefficients (D values) were determined using log-normal fitting40 and all average D values were calculated for = 3 datasets and shown with standard deviations. Antibody binding to nSLBs – To verify that the heparinase I treatment was successful and lead to a reduction of HS, the freshly formed nSLBs were thoroughly rinsed with low ionic strength PBS buffer (10 mM phosphate, 2.7 mM KCl, 50 mM NaCl, pH 7.4), and incubated for 2 hours with the anti-heparan sulfate antibody at a final concentration of 13 µg/ml. After thorough rinsing in PBS, the well was incubated with the Alexa Fluor 488-labelled secondary (13 µg/ml) for 1 hour, rinsed and imaged by TIRFM. To provide an estimate of the background signal, a bleached spot was produced as described above (for FRAP analysis). The average intensity of the bleached spot (background) and of the rest of the image (signal) was measured in



ImageJ.

The

signal-to-background

was

evaluated

Page 26 of 41

by

taking

S/B=(signal-

background)/background.

Supporting Information Additional information on FRET for monitoring of vesicle mixing and quantification of NMV suspensions, nSLB formation, binding kinetics, and single particle tracking.

Abbreviations SLB, supported lipid bilayer; NMV, native membrane vesicle; nSLB, NMVderived supported lipid bilayer; TIRFM, total internal reflection fluorescence microscopy; HSV, Herpes Simplex Virus; WT, wild-type; GAG, glycosaminoglycan; HS, heparan sulfate; CS, chondroitin sulfate; gC, glycoprotein C; gB, glycoprotein B

Author Information Corresponding Author *E-mail: [email protected] Author Contributions M.B., H.P., and T.B. conceived and coordinated this study. The experiments were designed by N.P. and M.B. and performed by E.S. and N.P.. E.T. provided guidance for the enzyme treatment and antibody binding studies, S.B. helped with data analysis and interpretation of the single particle tracking. N.P. and M.B. wrote the manuscript with contributions from all authors. All authors read and approved the manuscript.


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Conflict of Interest The authors declare no conflict of interest.

Acknowledgements The Swedish Research Council (Vetenskapsrådet, 612–2012-5024, and 5212011-3297), the Area of Advance (Materials for Health, Chalmers), and Sahlgrenska University Hospital (ALF-Gbg 145-841) are acknowledged for financial support. We would like to thank Maria Johansson for the preparation and of the virus samples, Fredrik Höök for fruitful discussions, and Karin Norling for help with the protein quantification of the NMV samples.

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For Table of Contents Use Only HSV-1 wild type

gC-deficient HSV-1

enzymatic removal of heparan sulfate

binding inhibitor

PEG

A cell membrane derived bioanalytical platform to probe virus binding kinetics and dif fusion


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ACS Paragon Plus Environment

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HSV-1 wild type


gC-deficient HSV-1

enzymatic removal of heparan sulfate

binding inhibitor

membrane derived bioanalytical platform to probe virus binding kinetics and diffusion


1 A cell membrane derived platform to study virus binding kinetics and

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