Name: Method for Measurement of Viral Fusion Kinetics at the Single Particle Level (Video) | JoVE Journal
Uploaded: 2009-09-07T16:00:00.000+00:00
Duration: 14 min 59 s
Description: Learn a two-color fluorescence assay to visualize viral fusion kinetics at the single particle level. This method monitors hemifusion and pore formation using fluorophore-labeled virus particles docked to a lipid bilayer.

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The work was supported by NIH grants AI57159 (to S.C.H.) and AI72346 (to A.M.v.O.). S.C.H. is a Howard Hughes Medical Institute Investigator.

Glass cover slip functionalization
The planar bilayer used in the fusion assay is supported on a hydrated film of dextran. Dextran acts as a spacer between the planar bilayer and glass surface. This prevents membrane components from becoming stuck on the glass surface and also provides space in to which the contents of a virus particle can escape upon fusion. Glass coverslips are functionalized through treatment with an epoxy silane, which allows us to chemically bond dextran to the glass (Elender, et al. 1996).
<ol>
<li>Arrange 25 mm coverslips in a ceramic staining rack, and place the rack into a jar or beaker.</li>
<li>Fill the container with a 10% solution of laboratory grade glassware detergent. Place the container in an ultrasonic cleaner for 30 minutes.</li>
<li>Rinse the container and coverslip rack with deionized water, and refill with 1M postassium hydroxide. Return the container to the bath sonicator for another 30 minutes.</li>
<li>Repeat this cleaning procedure using acetone and ethanol.</li>
<li>Rinse the coverlips in water and dry.</li>
<li>Finally, clean the coverslips with an oxygen plasma stripper for three minutes.</li>
<li>The last cleaning step oxidizes the surface, making the glass uniformly hydrophilic and reactive in the following silanization steps.</li>
<li>Prepare a 0.2% solution of 3-glycidoxypropyltrimethoxy silane in isopropanol. Immerse the coverslips in this solution for five minutes.</li>
<li>Discard the silane solution and rinse the coverslips several times in isopropanol.</li>
<li>The coverslips are now coated with a layer of silane, but they must be cured to allow the silane to covalently bond to the glass.  Place the coverslips in an oven set to 80 degrees Celsius for one hour.</li>
<li>While the coverslips are curing, weigh out dextran 500 and prepare a 30% w/v solution. Preheating the water will aid dissolution. We will use approximately 1 ml of this solution per coverslip. Once the dextran has dissolved, defoam the solution in a vacuum desiccator.</li>
<li>When the silanized coverslips have finished curing, remove them from the ceramic rack and arrange them flat on the inside of a plastic freezer box. Use a 1 ml pipette to cover the top surface of each coverslip with ~ 1 ml of the dextran solution. Close the freezer box and leave in a quiet place for 24-36 hours.</li>
<li>While grasping the coverslips with plastic forceps, pour off the excess dextran and replace them into the ceramic rack. Rinse the coverslips several times in water, and allow the coverslips to soak in water for 48 hours. The jar may be gently agitated on a table-top rotator.</li>
<li>Dry the coverslips in an oven set to 80 degrees Celsius and store in a vacuum desiccator.</li>
</ol>
Liposome Preparation
Supported lipid bilayers are formed by adsorbing liposomes to a dextran functionalized coverslip. The adsorbed liposomes fuse with eachother until membrane ruptures and spreads flat over the surface.
<ol>
<li>A typical liposome formulation used in the assay consists of 1,2,dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, bovine brain Disialoganglioside (GD1a), and N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine in a ratio of 4:4:2:0.1:5x10-5. All components are stored in chloroform or chloroform and methanol solutions. </li>
<li>Prepare a mixture containing two micromoles of lipid by transferring volumes of each component into a glass test tube. Evaporate the solvent under a stream of argon or nitrogen gas. Remove the remaining solvent by leaving the test tube in a vacuum desiccator for two hours.</li>
<li>Resuspend the dried lipid film in 400 microliters of HNE buffer (5 mM HEPES, 145 mM NaCl, 0.1 mM EDTA). </li>
<li>Transfer the suspension to a plastic microcentrifuge tube. Freeze the suspension in a liquid nitrogen bath and thaw in a warm water bath. Repeat this freeze/thaw cycle four times.</li>
<li>Assemble a lipid extruder with a 100 nm polycarbonate membrane filter, and extrude the lipid suspension 25 times. The suspension should appear to be more transparent after extrusion.</li>
<li>Liposomes should be used on the day they are prepared. They may be stored at room temperature until used.</li>
</ol>
Microfluidic flow cell preparation
A simple microfluidic flow cell is made by sandwiching double stick tape between a quartz slide and a functionalized coverslip.
<ol>
<li>Obtain a 20x20x3 mm quartz slide. Use a diamond burr to drill two 1 mm holes on opposite sides.</li>
<li>Cut a 20 mm square from a sheet of double stick tape, and using a razor blade, cut out a 15 x 2 mm section from the center. </li>
<li>Peel off one side of the tape backing, and adhere the tape to the quartz slide. Adhere the other side to a functionalized coverslip.</li>
<li>Cut two 20 cm  lengths of polyethylene tubing and insert them into the holes in the quartz slide.</li>
<li>Seal the flow cell with 5 minute epoxy glue.</li>
<li>When the glue has cured, prime the flow cell and tubing with buffer. </li>
<li>A supported lipid bilayer can be formed at this stage by drawing approximately 80 microliters of the liposome suspension into the channels.</li>
</ol>
Virus particle labeling
<ol>
<li>H3N2 influenza A (X-31) is typically used for the fusion assay, but other flu strains and other viruses have been used successfully.</li>
<li>Virus is labeled with up to two different fluorescent dyes to allow detection of hemifusion and pore-formation. </li>
<li>Dissolve sulforhodamine b (SRB) in HNE buffer to make a 20 mM solution. Combine 20 microliters of the dye solution with 10 microliters of the viral suspension, and leave at room temperature for 20 hours. During this period, dye will slowly accumulate inside the virus particles.</li>
<li>Remove the excess dye by passing the the virus suspension through a PD-10 desalting column. If enough viral material is used, a colored band can be seen in the column. This band contains labeled virus and can be collected as it is eluted.</li>
<li>If no band is visible, collect 200 microliter fractions and determine which fractions contain labeled virus by measuring 565 nm absorption on a spectrophotometer. The virus should elute in a total volume of approximately 0.8 ml. </li>
<li>Label the viral envelope by adding 13 microliters of a 2 mM dimethyl formamide (DMF) solution of octadecyl rhodamine 110 (Rh110C18) to the SRB labeled virus suspension.</li>
<li>Stir the suspension by attaching the tube to a laboratory rotator and leave for 3 hours.</li>
<li>Purify the virus from excess Rh110C18 using a PD-10 desalting column as described before.</li>
</ol>
Microscope configuration
The experiment is performed on a fluorescence microscope equipped with a high NA oil immersion objective suitable for total internal reflection fluorescence microscopy. The 488 and 568 nm lines from an argon/krypton gas laser are used to excite the Rh110C18 and SRB labeled virus. Simultaneous imaging of each label is accomplished by splitting the green and red fluorescence emission with a dichroic mirror and independently focusing the images onto separate halves of a electron multiplying CCD camera.
Performing the Assay
Execution of the fusion assay involves a stepwise assembly of the biochemical components within the flow cell: assembly of the planar lipid bilayer, docking of labeled virus particles, coating the surface with fluorescein, and initiation of the reaction with low pH buffer (Figure 1A).
<ol>
<li>Mount the flow-cell onto the microscope stage, and attach the outlet tubing to a syringe pump set to flow at a rate of 40 microliters per minute.</li>
<li>Clear the flow channels of excess liposomes by flowing in 300 microliters of HNE.</li>
<li>Focus the microscope and adjust the laser power to illuminate the surface with 350 W/cm2 of 488 nm light and 70 W/cm2 of 568 nm light. If you are using a 1.45 NA 60x oil immersion objective, set the laser intensities to 100 micro Watts and 20 micro Watts, respectively.</li>
<li>Pump labeled virus (diluted 1/100) into a flow cell. As the virus diffuses to the bottom surface of the flow channel, particles will begin to stick to the ganglioside incorporated in the  lipid bilayer. </li>
<li>When a suitable density of docked virus particles has been attained (up to around 1000 particles per field of view). Switch the inlet tubing to a solution containing two micrograms per milliliter fluorescein labeled streptavidin. The labeled streptavidin will bind to biotin coupled lipid molecules in the bilayer, and eventually a dim green background will be visible.</li>
<li>Wash the flow channel with 300 microliters of HNE.</li>
<li>Choose an imaging area, focus the microscope and prepare the CCD camera for time-lapsed image acquisition. Set the exposure time to 100 ms and collect frames at a rate of 10 Hz.</li>
<li>Initiate fusion by flowing in an acidic buffer containing 10 mM sodium citrate and 140 mM NaCl. The pH of the buffer should be adjusted to be below the critical pH of the virus being assayed. X-31 fuses at pH’s below 5.5.</li>
</ol>
Representative Results
Figure 1B shows snapshots of bilayer docked virions before and during fusion. Each diffraction limited spot represents an individual virus particle. Red and green fluorescence have been separately imaged onto the top and bottom halves of a CCD camera, allowing simultaneous observation of the viral envelope and content labels. There are typically twice as many spots in the green channel compared to the red, and this reflects the lower efficiency of labeling by the content dye compared to the envelope label. The green background fluorescence emitted from surface bound fluorescein disappears upon arrival of the citrate buffer and allows determination of the start time of the fusion reaction. Hemifusion events are detected as bursts of green fluorescence as the quenched Rh110C18 in the viral envelope diffuses across the hemifusion stalk and is diluted in the planar membrane. An outward expanding fluorescent cloud can be seen around hemifused particles, demonstrating free diffusion of the fatty-acyl linked dye in the planar membrane. Pore formation is observed as the decay of red fluorescence from SRB trapped within the interior of the viral particles. The opening of a fusion pore allows the dye to escape into the aqueous space below the planar bilayer and out of the observation region.
Fluorescence intensity trajectories for each particle, plotted from the integrated intensities of individual particles, facilitate determination of the yield and times of hemifusion and pore formation (Fig. 1C). Hemifusion and pore formation event times are determined as the point at which the maximum slope of a Rh110C18 fluorescence burst or SRB signal decay occurs. In a successful experiment, about 50 percent of the particles labeled with Rh110C18 yield dequenching signals, and 30 percent of the SRB labeled particles give useable signal. Of the particles labeled with both dyes, approximately 10 percent show both lipid mixing and pore formation signals.
Fig. 2A-C shows the distribution of hemifusion and pore formation lag times from a typical experiment. Fig.2D-F shows event distributions of events combined from five experiments performed under the same conditions. Even with a modest number of observations, the shape of the histograms is apparent. Because each experiment is synchronized by detection of the pH drop time, multiple experiments can be combined and detailed information about rate limiting intermediate steps can be obtained.
<img src="/files/ftp_upload/1484/1484fig1a.jpg" border="0" alt="Influenza virus microscopy diagram; fluorescence dye labeling; lipid bilayer interaction; time-lapse." data-altupdatedat="1761735611000" data-alt="figure 1"> <img src="/files/ftp_upload/1484/1484fig1b.jpg" border="0" alt="Fluorescence vs. Time graph; pore formation, hemifusion, pH sensor; experimental kinetics." data-altupdatedat="1761735611000" data-alt="figure 1"> Figure 1. Experimental design. A) Virus particles are labeled with two fluorescent dyes to monitor the kinetics of hemifusion and fusion pore formation. Fluorescence is collected by a high-NA microscope objective and imaged onto a CCD. B) Fluorescence images before (left) and during (right) the fusion of individual viral particles. Top and bottom half of each image correspond to the red and green fluorescence, respectively, of the same ~ 50 x 100 mm2 area of the supported bilayer. C) The fluorescence intensity of the red SRB viral content tracer (upper trace), the green Rh110C18 membrane dye (middle trace), and the fluorescein pH sensor (lower trace) provide exact times elapsed between pH drop, hemifusion, and fusion. Please <a href="https://www.jove.com/files/ftp_upload/1484/1484fig1.jpg">click here</a> to see a larger version of figure 1.
<img border="0" src="/files/ftp_upload/1484/1484fig2tmb.jpg" alt="Hemifusion and pore formation histogram charts; frequency vs. time; fusion process analysis." data-altupdatedat="1761735611000" data-alt="figure 2"> Figure 2. Fusion kinetics of fluorescently labeled virus. Distributions of the time elapsed between the pH drop and hemifusion (A, D) and pore formation (B,E). The rise and decay of the distributions indicate the presence of multiple intermediate steps. The transition between hemifusion and opening of a fusion pore for individual particles is exponentially distributed, suggesting a single rate-limiting step. Panels A-C show the results of a single experiment. Panels D-F are compiled from five separate experiments performed under the same conditions. Please <a href="https://www.jove.com/files/ftp_upload/1484/1484fig2.jpg">click here</a> to see a larger version of figure 2.

<ol><li>Elender, G., Kuhner, M., Sackmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functionalisation+of+Si%2FSiO2+and+glass+surfaces+with+ultrathin+dextran+films+and+deposition+of+lipid+bilayers%5BTitle%5D)+AND+%22Biosens+Bioelectron%22%5BJournal%5D)">Functionalisation of Si/SiO2 and glass surfaces with ultrathin dextran films and deposition of lipid bilayers.</a> Biosens Bioelectron. 11, 565-577 (1996).</li>
<li>Floyd, D. L., Ragains, J. R., Skehel, J. J., Harrison, S. C., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-particle+kinetics+of+influenza+virus+membrane+fusion%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Single-particle kinetics of influenza virus membrane fusion.</a> Proc Natl Acad Sci U S A. 105, 15382-15387 (2008).</li>
</ol>

Preparation of fluid and continuous supported lipid bilayers can be challenging. Trace amounts of contaminating material or surface defects will prevent spreading of bilayers. Careful cleaning and deposition of a uniform layer of dextran are essential.
Prolonged imaging of virus particles can bleach the fluorescent labels or cause them to become inactivated. Photo-damage of this kind is well known in the bio-imaging and single molecule fields and is generally believed to stem from the generat...

<table><tbody><tr><td>Corning cover glass squares, 25 mm</td><td>Sigma-Aldrich</td><td>CLS286525</td><td></td></tr><tr><td>Fused silica slides</td><td>Technical Glass</td><td></td><td></td></tr><tr><td>Staining rack</td><td>Thomas Scientific</td><td>8542E40</td><td></td></tr><tr><td>(3- glycidoxypropyl)trimethoxysilane</td><td>Gelest Inc.</td><td>SIG5840.1</td><td></td></tr><tr><td>Dextran T-500</td><td>Pharmacosmos</td><td>5510 0500 4006</td><td></td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DOPC)</td><td>Avanti Polar Lipid, Inc</td><td>850375</td><td></td></tr><tr><td>1-palmitoyl-2oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (POPC)</td><td>Avanti Polar Lipid, Inc</td><td>850457</td><td></td></tr><tr><td>Cholesterol</td><td>Avanti Polar Lipid, Inc</td><td>700000</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosph&amp;#8211;thanolamine, triethylammonium salt (biotin-X DHPE)</td><td>Invitrogen</td><td>B1616</td><td></td></tr><tr><td>Streptavidin, fluorescein conjugate</td><td>Invitrogen</td><td>S-869</td><td></td></tr><tr><td>Disialoganglioside GD&lt;sub&gt;1a&lt;/sub&gt; from bovine brain</td><td>Sigma-Aldrich</td><td>G2392</td><td></td></tr><tr><td>Mini Extruder</td><td>Avanti Polar Lipid, Inc</td><td>610000</td><td></td></tr><tr><td>0.1 micron polycarbonate membrane filters</td><td>Whatman, GE Healthcare</td><td>800309</td><td></td></tr><tr><td>10 mm drain discs (membrane supports)</td><td>Whatman, GE Healthcare</td><td>230300</td><td></td></tr><tr><td>Sulforhodamine b sodium salt</td><td></td><td>S1402</td><td></td></tr><tr><td>Octadecyl rhodamine 110 (Rh110C18)</td><td></td><td></td><td>&lt;em&gt;See&lt;/em&gt; Floyd &lt;em&gt;et al.&lt;/em&gt; 2008 for synthesis protocol</td></tr><tr><td>PD-10 desalting columns</td><td>GE Healthcare</td><td>17-0851-01</td><td></td></tr><tr><td>Nikon fluorescence microscope</td><td>Nikon Instruments</td><td>TE2000-U</td><td></td></tr><tr><td>Plan Apo TIRF 60x 1.45 NA objective</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Innova 70C Spectrum Ar/Kr laser</td><td>Coherent Inc.</td><td></td><td></td></tr><tr><td>Syringe Pump</td><td>Harvard Apparatus</td><td>702213</td><td></td></tr></tbody></table>

method for measurement of viral fusion kinetics at the single particle level

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

The cover slips used in this assay are functionalized with dextrin then incorporated into microfluidic flow cells. Next, each flow cell is incubated with a suspension of liposomes, which fuse to form a lipid bilayer atop the hydrated dextrin. Under the fluorescence microscope, fluoro four labeled virus and fluorescein labeled streptavidin are added and the virus is allowed to dock to the bilayer.An influx of acidic buffer initiates viral fusion with the bilayer and a digital camera records the changes in fluorescence throughout the assay. Hi, I'm Daniel Floyd and I'm in the laboratory of Anine Vanian in the Department of Biological Chemistry and Molecular Pharmacology at Harvard University. Today I'm gonna show you a method for visualizing viral fusion kinetics at the single particle level.We use this procedure in the lab to study the way the virus is getting to host cells. So let's get started. Begin this protocol by preparing the dextrin functionalized cover slips.The first step is to sonicate the cover slips in solutions of 10%detergent, one molar potassium hydroxide acetone, and 100%ethanol sonicate for 30 minutes per solution. And rinse with deionized water between ations. After the last sonication, rinse the cover slips in water, then dry them in the 80 degrees Celsius incubator.Next, put the cover slips in an oxygen plasma stripper for three minutes. This step oxidizes the surface of the slides, making the glass uniformly hydrophilic and reactive in the following siloization steps. Next, immerse the cover slips in a fresh solution of 0.2%gly oxy propyl trimeth oxypro trim methoxy in isopropanol and incubate for five minutes.Then rinse the cover slips several times with isopropanol. Place the Slan coated cover slips at 80 degrees Celsius for one hour. To cure this step allows the slan to covalently bond to the glass.After curing, arrange the cover slips flat on the inside of a plastic freezer box. Cover the top surface of each cover slip with about one milliliter of a dextrin solution. 30%weight per volume in water.Close the freezer box and leave the cover slips in a quiet place for 24 to 36 hours. The next day pour off the excess dextrin, grasping the cover slips with plastic forceps and put them back into the ceramic rack. Rinse them with water several times and let them soak in water for 48 hours.The container may be gently agitated on a tabletop rotator dry the cover slips in an oven set to 80 degrees Celsius and store in a vacuum desiccate until you are ready for the next step. Coating the cover slips with a mixture of liposomes to form a planer bilayer. The next step is to prepare the liposomes to form the lipid bilayer in the hood, combine all of the lipids dissolved in chloroform in a glass test tube.The liposome formulation includes dia ganglia side, which binds virus particles and a biotinylated lipid, which binds the streptavidin conjugated pH indicator fluorescein. Next, evaporate the solvent under a stream of argon or nitrogen gas. And remove the remaining solvent by leaving the test tube in a vacuum desiccate for two hours.Re suspend the dried lipid film in 400 microliters HNE buffer. Then transfer the suspension to a plastic micro centrifuge tube. Freeze the suspension in a liquid nitrogen bath and thaw in a warm water bath.Repeat this freeze thaw cycle four times after the freeze thaw cycles. Use a lipid extruder fitted with a 100 nanometer membrane filter to extrude the lipid suspension. 25 times after extrusion, the suspension should appear to be more transparent.Store the liposomes at room temperature and use the day of preparation. Now that the lipids are ready to go, prepare the microfluidic flow cell. First, take a 20 by 20 by three millimeter quartz slide and use a diamond burr to drill two one millimeter holes on opposite sides.Cut a 20 millimeter square from a sheet of double stick tape and using a razor blade. Cut out a 15 by two millimeter section from the center. Peel off one side of the tape backing and adhere the tape to the court slide.Adhere the other side to a functionalized cover slip now cut to 20 centimeter lengths of polyethylene tubing and insert them into the holes in the quartz slide. Finally, seal the flow cell with five minute epoxy glue. Spread the glue around the sides of the quartz slide to bond it to the cover slip.Use two more drops of glue to seal the connection between the tubing and the quartz slide. When the glue has cured, prime the flow cell and tubing with HNE buffer. Next to create the supported lipid bilayer, take the extruded liposome suspension and draw about 80 microliters into the channels on the day prior to the fusion experiment.Begin labeling the virus particles to label the viral interior red. Combine 20 microliters of freshly prepared sulfur rumine or SRB solution with 10 microliters of the viral suspension and leave at room temperature for 20 hours. The next day, remove the excess dye by passing the virus suspension through a PD 10 desalting column and diluting with 800 microliters of HNE buffer.The column should have an obvious colored band, which contains the labeled virus. Collect this fraction during the ellucian step next, label the viral envelope green by adding 13 microliters of Octa decal, RUMINE one 10 or RH one 10 C 18 to the SRB labeled viral suspension. Stir the suspension by attaching the tube to a laboratory rotator and leave for three hours.Next, remove excess RH one 10 C 18 using a PD 10 desalting column. As before now the virus is ready for the fusion assay. Perform the fusion kinetics assay using a fluorescence microscope equipped with a high numerical aperture oil immersion objective.An Argonne krypton laser excites the fluoro fours in the viral membrane and interior. A dichroic mirror then splits the red and green fluorescence emissions and independently focuses the images onto separate halves of an electron multiplying CCD or charge coupled device camera. Mount the cover slip on the microscope stage and attach the outlet tubing to a syringe pump set to flow at 100 microliters per minute.Clear the flow channels of excess liposomes by flowing in 300 microliters of HNE. Focus the microscope and adjust the laser power to illuminate the surface with 350 watts per centimeter squared of 488 nanometer light to excite the red flora, four and 70 watts per centimeter squared of 568 nanometer light to excite the green flora.Four. Now that the microscope is ready, pump labeled virus diluted one to a hundred into the flow cell.As the virus diffuses to the bottom surface of the flow channel particles begin to dock. Sticking to the DIA ganglia side, incorporated into the cover slips, planar bilayer. When the density of docked particles reaches about several hundred particles per field of view, begin to flow in a solution of two micrograms per milliliter.Fluorescein labeled strep streptavidin, a pH sensitive fluorophore as the labeled streptavidin binds to biotin coupled lipid molecules in the bilayer. A dim green background appears again, wash the flow channel with 300 microliters of HNE. Now that the viruses have docked and the bilayer surface is coated with fluorescein, it is time to begin the assay.Choose an imaging area, focus the microscope and prepare the CCD camera for time lapsed image acquisition. Set the exposure time to 100 milliseconds and collect frames at a rate of 10 hertz. Initiate virus membrane fusion by flowing in an acidic sodium citrate buffer.As the pH drops the fluorescein fluorescence dims pinpointing the time of the acidification. The CCD camera records the data throughout the assay, which one can use to determine time of hemi fusion and poor formation as documented by the CCD camera. After the pH drop, there are bursts of green fluorescence as the viral and planer membranes fuse, the lipids mix and the green dye in the viral envelope is diluted in the planar membrane.In contrast, there is a decay in the red fluorescence after the pH drop as the viral and lipid membranes form pores allowing the red dye from the viral interior to escape into the aqueous space below the planar bilayer and out of view, these trends can also be visualized by fluorescent snapshots before and during fusion. Also graph the fluorescence intensity trajectories for each particle as shown in this example, Hemi fusion is defined as the point at which the slope of green fluorescence intensity is at its steepest pore formation as the point at which the red fluorescence decay occurs. Combined data from multiple experiments to generate histograms of hemi fusion and pore formation lag times as shown here, using the time of the pH drop as t equals zero to synchronize data from different experiments.I've just shown you a way to measure viral fusion kinetics at the single particle level. When doing this procedure. Make sure you use clean cover slips so that you have a homogeneous and fluid bilayer.Also, try to minimize the amount of light you use during fluorescence imaging so that you can minimize photo damage. So that's it. Thanks for watching and good luck in your experiments.

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We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Learn a two-color fluorescence assay to visualize viral fusion kinetics at the single particle level. This method monitors hemifusion and pore formation using fluorophore-labeled virus particles docked to a lipid bilayer.

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

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