We acknowledge Hannah Fox for help in the preparation of this manuscript. This work was supported by the NIH grant R01AI030612.

1. Cell Culture, Viral Infection, and EdC Labeling (Figure 1)
The following protocol involves working with viruses. Please refer to your institution&#39;s bio-safety protocols regarding safe handling of viruses and other biological agents. This protocol was approved by the Institutional Review Board of the University of Pittsburgh.
<ol>
	<li>Trypsinize a confluent 150 cm2 tissue culture flask of MRC-5 cells and transfer the cells to a 600 cm2 tissue culture plate containing 100 mL DMEM plus 10% FBS. Incubate at 37 &#176;C in the presence of 5% CO2 for 3 - 4 days to reach confluency and stationary phase. A confluent 600 cm2 dish contains ~7 x 107 cells. Prepare 1 plate for each condition and 1 plate for each corresponding negative control. 
	NOTE: Cells must reach the stationary phase to inhibit cellular DNA replication to ensure that only vDNA is labeled and purified in subsequent steps. 
	NOTE: Each sample requires a complimentary unlabeled negative control prepared using the same infection conditions and time point without the addition of EdC. 
	NOTE: A scaled down version of this experiment should be carried out in tandem to test for the labeling of cellular DNA by imaging using comparable cell growth, infection, EdC labeling conditions, and time points (see Step 2).</li>
	<li>Dilute 7 x 108 PFU HSV-1 in 7 mL cold Tris-Buffered Saline (TBS). Remove growth medium and store at 37 &#176;C. To infect, add the 7-mL diluted virus to MRC-5 cells and rock for 1 h at room temperature. Following adsorption, remove inoculum, rinse with 50 mL room temperature TBS, and replace the original growth medium. Incubate at 37 &#176;C in the presence of 5% CO2. This step should be carried out for each experimental condition and negative control. 
	NOTE: Increased EdC labeling can be achieved using HSV-1 mutants defective for the expression of the viral dUTPase (UL50 gene) and/or uracil glycosylase (UL2 gene). HSV-1 dUTPase has low substrate specificity and can decrease the activation of several nucleoside analogs25,27.</li>
	<li>Label viral genomes with EdC. Follow the directions to label incoming genomes (1.3.1.), replicating genomes (1.3.2.), or viral replication forks (1.3.3.). 
	NOTE: Only Step 1.3.1, 1.3.2, or 1.3.3 should be carried out depending on whether incoming genomes, replicated genomes, or replication forks are to be analyzed in subsequent steps, respectively. 
	NOTE: EdU or f-ara EdU can be substituted for EdC. The toxicity of nucleoside analogs should be monitored and minimized.
	<ol>
		<li>Use prelabeled stocks to carry out infection in Step 1.2 and proceed to Step 2 or 3 within 4 h post infection (hpi) to investigate unreplicated vDNA (Figure 1A). 
		NOTE: Prepare prelabeled virus stocks using the previously described protocol25. Virus stocks must be passed through a G-25 column to remove any residual EdC.</li>
		<li>Label replicating vDNA by adding 5 - 25 &#181;M EdC to the cell culture medium of infected cells for 2 - 4 h (Figure 1B). Before adding, dilute EdC in 1 mL of growth medium.</li>
		<li>To label viral replication forks, after the onset of vDNA replication (&#8805;4 hpi), pulse label replicating vDNA with 5 - 25 &#181;M EdC for 5-20 min. To chase, rinse 3x with chase medium containing 25 - 100 &#181;M 2&#180;deoxycytidine (deoxyC), and then incubate in the presence of chase medium for an additional 20-40 min (Figure 1C). 
		NOTE: Pulse and chase medium should be equilibrated to 37 &#176;C and 5% CO2 before use. 
		NOTE: For pulse chase experiments, it is important to consider the amount of time it takes for nucleosides to enter the cell and be phosphorylated before they can be incorporated into replicating DNA. This must be determined empirically. 
		NOTE: To determine the resolution of pulse chase experiments, calculate the viral replication rate under the experimental conditions used. This can be determined by quantitative PCR of viral genomes during a single step growth time course26. 
		NOTE: For imaging viral genomes proceed to Step 2 and for purification of viral genomes proceed to Step 3.</li>
	</ol>
	</li>
</ol>
2. Imaging of EdC labeled DNA (Figure 2A)
NOTE: It is useful to carry out imaging in tandem with the viral genome purification method to verify that cellular DNA is not labeled in experiments. Imaging can also be used to visualize the nature of labeled vDNA and to validate proteomics data. Examples of cellular and viral DNA staining patterns are shown in Figure 3.
<ol>
	<li>Carry out infections and EdC labeling as indicated in Step 1 except using scaled down conditions on coverslips in a 12-well tissue culture dish containing 1 mL of growth medium.</li>
	<li>Fix cells with 1 mL 3.7% paraformaldehyde in 1x PBS for 15 min at RT. After fixation, rinse cells 2x with 1 mL 3% BSA in PBS. 
	Caution: Paraformaldehyde can cause serious or permanent injury. Follow safety precautions. 
	NOTE: The protocol can be paused here and coverslips can be stored at 4 &#176;C in the second wash for up to 3 days.</li>
	<li>Permeabilize cells with 1 mL permeabilization buffer [see Table of Materials] for 20 min at room temperature while rocking.</li>
	<li>Rinse with 1 mL of 3% BSA and then block with 1 mL 3% BSA in PBS for 30 min at room temperature while rocking.</li>
	<li>Add 1 mL of the click reaction cocktail [see Table of Materials] to coverslips to covalently conjugate Alexa Fluor 488 azide to EdC labeled DNA. Incubate at room temperature for 30 min while rocking. Aspirate and rinse two times with 1 mL of PBS.</li>
	<li>Stain cellular DNA with 1 mL of a 1:2,000 dilution of Hoechst in PBS for 30 min at room temperature while rocking. Aspirate and rinse two times with 1 mL of PBS.</li>
	<li>Stain with primary and secondary antibodies according to standard indirect immunofluorescence protocols25. 
	NOTE: Labeled vDNA colocalizes with ICP8, ICP4, or UL42 and labeled cellular DNA colocalizes with Hoechst stain.</li>
	<li>Mount coverslips onto slides and image cells using a fluorescence microscope. 
	NOTE: Slides can be stored at 4 &#176;C for several weeks.</li>
</ol>
3. Purification of vDNA and Associated Proteins
NOTE: Several aspects of this protocol have been adapted from Leung et al.22 
NOTE: All buffers and reagents should be chilled on ice before use and all steps should be carried out on ice unless otherwise indicated.
<ol>
	<li>Harvest nuclei. 
	NOTE: Before isolation of nuclei, formaldehyde can be used to crosslink proteins to DNA. More stringent wash conditions can then be used during DNA purification on streptavidin-coated beads. For crosslinking and wash conditions see Sirbu et al.21.
	<ol>
		<li>Replace growth medium with 20 mL Nuclei Extraction Buffer (NEB) and incubate for 20 min at 4 &#176;C with occasional rocking. Nuclei will become visible in the microscope.</li>
		<li>Scrape nuclei from the plate using a cell scraper and transfer to a 50 mL conical tube. Centrifuge for 10 min at 2,500 x g at 4 &#176;C to pellet nuclei, discard the supernatant. 
		NOTE: Trypan blue staining can be used to verify the isolation of nuclei from cells.</li>
		<li>Gently dislodge the nuclear pellet in 10 mL PBS, transfer to a 15-mL conical tube, and pellet by centrifuging for 10 min at 2,500 x g at 4 &#176;C. Completely remove PBS. 
		NOTE: Nuclei can be frozen and the protocol can be paused at this point. To do so, gently resuspend the nuclear pellet in 500 &#181;L freezing buffer and incubate the tube in an ethanol/dry ice bath. Store frozen nuclei at -80 &#176;C. Before use, thaw nuclei at room temperature, quickly transfer to ice, add 10 mL PBS, rock gently to mix, pellet by centrifugation for 10 min at 2,500 x g at 4 &#176;C, and completely remove PBS. Freezing nuclei may result in reduced yield.</li>
	</ol>
	</li>
	<li>Covalently conjugate biotin-azide to EdC labeled vDNA.
	<ol>
		<li>Resuspend the nuclear pellet in 10 mL click reaction mix [see Table of Materials] by gently pipetting up and down 5 times with a 10 mL pipet. 
		NOTE: It is important that the reagents included in the click reaction mix are added in the indicated order.</li>
		<li>Rotate for 1h at 4 &#176;C. While rotating prepare and chill Buffers B1, B2, and B3 [see Table of Materials].</li>
		<li>Pellet nuclei by centrifuging for 10 min at 2,500 x g at 4 &#176;C. Completely remove click reaction mix and wash the pellet by gently resuspending in 10 mL PBS and centrifuging for 10 min at 2,500 x g at 4 &#176;C.</li>
		<li>Gently resuspend the nuclear pellet in 1 mL PBS and transfer to a 1.5 mL microfuge tube using a large bore pipette tip. Pellet nuclei by centrifuging for 10 min at 2,500 x g at 4 &#176;C. Completely remove PBS and flash freeze in liquid nitrogen. 
		NOTE: The protocol can be paused at this point and frozen nuclei can be stored at -80 &#176;C. Freeze thaw helps with subsequent lysis so it is recommended that nuclei be flash frozen even when proceeding to Step 3.3 on the same day.</li>
	</ol>
	</li>
	<li>Lyse nuclei and fragment DNA.
	<ol>
		<li>Thaw nuclei on ice and resuspend in 500 &#181;L Buffer B1 by pipetting up and down. Incubate on ice for 45 min.</li>
		<li>Sonicate samples 6 times for 30 s each at 40% amplitude using a 3 mm microtip probe. Place samples on ice for 30 s between pulses. After sonication, samples should appear clear, not cloudy. 
		NOTE: Sonication conditions should be optimized for individual sonicators. For detailed instructions on how to optimize sonication conditions refer to Leung et al.22.</li>
		<li>Pellet cell debris by centrifuging at 14,000 x g for 10 min at 4 &#176;C. The pellet size should decrease substantially. Filter the supernatant through 100 &#181;M cell strainer and retain the flow through.</li>
		<li>Add 500 &#181;L Buffer B2 to the filtered supernatant. 900 &#181;L of this sample will be used in Step 3.4.2.</li>
		<li>Take an aliquot of each condition for DNA isolation (50 &#181;L or 1/20th volume) and add 50 &#181;L 2x SDS-bicarb solution [see Table of Materials]. Proceed to the DNA isolation protocol (Step 3.6).</li>
		<li>Take an aliquot of each condition for input protein (50 &#181;L or 1/20th volume) and add 50 &#181;L 2x Laemmli sample buffer, freeze in liquid nitrogen and store at -80 &#176;C. Use in Step 3.5.6.</li>
	</ol>
	</li>
	<li>Bind biotinylated DNA to streptavidin coated beads.
	<ol>
		<li>Prepare Streptavidin T1 magnetic beads by transferring 300 &#181;L of bead slurry to a 1.5 mL microfuge tube. Prepare one tube of beads per sample. Wash beads 3x with 1 mL Buffer B2 by vortexing to resuspend, applying a magnet to separate the beads, and aspirating the wash buffer. 
		NOTE: Optimized binding conditions result in maximized yield and minimal background binding. It was experimentally determined that Streptavidin T1 magnetic beads have a significantly greater binding capacity than agarose beads, as well as other Streptavidin beads, and less background binding than Streptavidin C1 beads (Figure 4A).</li>
		<li>Add 900 &#181;L of sample from Step 3.3.4. to the washed beads and rotate overnight at 4 &#176;C. 
		Note: Do not vortex beads once sample is added to them. 
		NOTE: If significant amounts of DNA or protein are isolated in the unlabeled negative control, this step can be reduced from overnight to 4 h to reduce background binding.</li>
	</ol>
	</li>
	<li>Wash beads and elute vDNA and associated proteins. 
	NOTE: It is recommended to use filter tips for the remaining steps.
	<ol>
		<li>Place samples into a magnetic microfuge tube rack, remove supernatant, gently resuspend in 1 mL Buffer B2, rotate at 4 &#176;C for 5 min.</li>
		<li>Repeat Step 3.5.1 three times.</li>
		<li>Place the samples into a magnetic microfuge tube rack, remove supernatant, gently resuspend in 1 mL Buffer B3, and rotate at 4 &#176;C for 5 min.</li>
		<li>Transfer 100 &#181;L bead mixture (1/10th volume) to a new tube for bound DNA isolation. Apply this tube to the magnet, remove supernatant, resuspend beads in 100 &#181;L 1x SDS-bicarb solution and proceed to the DNA isolation protocol (Step 3.6).</li>
		<li>Apply the tube containing the remaining 900 &#181;L bead mixture from Step 3.5.3. to the magnet, remove supernatant, and resuspend beads in 50 &#181;L 2x Laemmli sample buffer to elute protein and DNA-protein complexes.</li>
		<li>Boil samples at 95 &#176;C for 15 min, vortex, quickly spin in the microfuge, and apply magnet. Transfer eluate to a new tube, flash freeze, and store at -80 &#176;C. 
		NOTE: Use cap locks to ensure that tubes do not pop open while boiling. 
		NOTE: The protocol can be paused at this point and samples can be stored at -80 &#176;C for several weeks.</li>
		<li>Analyze protein samples by Coomassie Blue staining, western blotting, or mass spectrometry by standard procedures25. Representative results are shown in Figure 4 and Figure 5. 
		NOTE: To determine if protein yield is sufficient for mass spectrometry, 7.5 &#181;L of each sample is used for analysis by Coomassie Blue staining and 7.5 &#181;L for western blotting of a representative viral genome associated protein (ICP4, ICP8, or UL42). The same volume of lysates from Step 3.3.6. are run alongside to control for input protein levels. The remaining sample (35 &#181;L) is then analyzed by mass spectrometry as described previously25.</li>
	</ol>
	</li>
	<li>DNA isolation
	<ol>
		<li>Incubate the samples from Steps 3.3.5. and 3.5.4. at 65 &#176;C for 4-16 h. Remove sample from magnetic beads, if necessary.</li>
		<li>Extract samples with 150 &#181;L phenol:chloroform:isoamyl alcohol (PCI) and transfer the aqueous phase to a new tube.</li>
		<li>Extract the remaining PCI with 100 &#181;L Tris-EDTA (TE) and add the aqueous phase to a new tube.</li>
		<li>Extract samples with 200 &#181;L chloroform:isoamyl alcohol (24:1).</li>
		<li>Purify the aqueous phase using the PCR Purification Kit according to the manufacturer&#39;s protocol. 
		NOTE: The pH indicator in Buffer PB will turn purple when added to the sample. Add 10 &#181;L 3M NaOAc pH 5.0 to decrease the pH of the sample.</li>
		<li>Determine DNA concentration using a fluorometer. 
		NOTE: This protocol typically yields 100 - 400 ng bead-bound DNA. No DNA should be detected in the eluate of the negative control in which EdC was not added in Step 1.3.</li>
		<li>DNA can be stored in a low DNA binding tube at 4 or -20 &#176;C and can be used for real-time PCR or high throughput sequencing analysis.</li>
	</ol>
	</li>
</ol>

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dUTPases+from+herpes+simplex+virus+type+1+and+mouse+mammary+tumour+virus+are+less+specific+than+the+Escherichia+coli+enzyme.">The dUTPases from herpes simplex virus type 1 and mouse mammary tumour virus are less specific than the Escherichia coli enzyme.</a> J Gen Virol. 77 (Pt 12), 3107-3111 (1996).</li><li>Chiou, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-+and+protein-scission+activities+of+ascorbate+in+the+presence+of+copper+ion+and+a+copper-peptide+complex.">DNA- and protein-scission activities of ascorbate in the presence of copper ion and a copper-peptide complex.</a> J Biochem. 94, 1259-1267 (1983).</li><li>Kennedy, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+consequences+of+copper+complexes+used+to+catalyze+bioorthogonal+click+reactions.">Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions.</a> J Am Chem Soc. 133, 17993-18001 (2011).</li><li>Snel, B., Lehmann, G., Bork, P., Huynen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRING:+a+web-server+to+retrieve+and+display+the+repeatedly+occurring+neighbourhood+of+a+gene.">STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene.</a> Nucleic Acids Res. 28, 3442-3444 (2000).</li></ol>

The authors have nothing to disclose.

This protocol includes multiple steps which, if not followed carefully, can result in significantly reduced protein yield or contamination with cellular DNA. It is critical that stationary cells are used for all experiments to ensure that cellular DNA is not labeled and purified. This can be confirmed by the absence of cellular DNA polymerases in the protein sample because HSV-1 does not utilize cellular DNA polymerase for genome synthesis. During EdC labeling and nuclei harvesting steps, samples should be staggered to e...

Viruses have a limited capacity to carry out essential functions and therefore depend on host factors to facilitate critical aspects of infection including viral gene expression, replication, repair, recombination, and transport. The activities of these host factors are often augmented by virally encoded proteins. In addition, viruses must avoid detection and interference by cellular responses to viral infection. Therefore, virus host interactions dictate the outcome of infection. Of paramount importance is understanding how viruses alter the cellular environment to adapt the cellular machinery to facilitate viral processes. Of particular interest is identifying which factors and processes act on viral genomes throughout the infectious cycle.
Herpes simplex virus type 1 (HSV-1) is a double stranded DNA virus that infects a substantial proportion of the human population. Within the first hour of infection, the viral genome enters the nucleus, where an ordered cascade of viral gene expression ensues in coordination with viral DNA (vDNA) replication1. In the nucleus, genomes are subject to epigenetic regulation, undergo repair and recombination, and are packaged into capsids, such that the first progeny virions are produced within less than six hours. The comprehensive evaluation of viral genome associated proteins throughout the course of infection will lay the groundwork to investigate the molecular details of processes that act on viral genomes, and will provide insight into which viral and cellular factors are involved in various stages of infection.
Previous methods for the investigation of host factors involved in viral infection include affinity purification of viral proteins for the analysis of associated cellular proteins2,3,4,5,6,7,8,9. These assays have been instrumental for the identification of cellular factors involved in host antiviral responses, as well as viral chromatin modification, gene expression, and DNA repair. However, it is difficult to ascertain whether interactions depend on the association with vDNA, and proteomics only provide insight into interactions that occur as a function of a specific viral factor. Chromatin immunoprecipitation (ChIP) has been used to identify where specific viral and cellular proteins bind to viral genomes10,11,12,13,14,15 and fluorescent in situ hybridization (FISH) combined with immunocytochemistry has enabled the visualization of cellular factors that colocalize with vDNA16,17,18,19,20. These assays allow for spatial and temporal analysis. However, limitations include the need for highly specific antibodies, limited sensitivity, and the need for previous insight into virus host interactions. We therefore developed a method based on iPOND (isolation of proteins on nascent DNA)21 and aniPOND (accelerated native iPOND)22 to selectively label and purify vDNA from infected cells for the unbiased identification of viral genome associated proteins by mass spectrometry. iPOND has been instrumental for the investigation of cellular replication fork dynamics.
For the selective purification of viral genomes from infected cells, replicating vDNA is labeled with ethynyl modified nucleosides, 5-ethynyl-2&#180;-deoxyuridine (EdU) or 5-ethynyl-2&#180;-deoxycytidine (EdC) (Figure 1), followed by covalent conjugation to biotin azide via click chemistry to facilitate single step purification of viral genomes and associated proteins on streptavidin-coated beads (Figure 2B). Importantly, infections are carried out in stationary cells, which are not engaged in cellular DNA replication to enable specific labeling of vDNA. Furthermore, HSV-1 infection causes cell cycle arrest and inhibits cellular DNA replication23,24. Virus can be prelabeled before infection for the analysis of proteins associated with incoming viral genomes (Figure 1A) or labeled during DNA replication for the analysis of proteins associated with newly synthesized vDNA (Figure 1B)25. Furthermore, pulse chase analysis can be used to investigate the nature of proteins associated with viral replication forks (Figure 1C)26. In addition, ethynyl-modified vDNA can be covalently conjugated to a fluorophore for spatial investigation of protein dynamics (Figure 2A and Figure 3). Imaging allows for the direct visualization of vDNA and is a complimentary approach for the validation of vDNA-protein interactions, and can be adapted to track viral genomes throughout infection. We anticipate that these approaches could be further modified to study any aspect of herpesviral infection, including latency and reactivation, and to study other DNA viruses. Moreover, labeling with 5-ethynyl uridine (EU) could allow for the analysis of RNA viral genomes.

<table border="0" cellpadding="0" cellspacing="0" width="1930">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">MRC-5 cells</td>
			<td style="width:197px;">ATCC</td>
			<td style="width:101px;">CCL-171</td>
			<td style="width:1215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:197px;">Gibco</td>
			<td style="width:101px;">26140-179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:197px;">Gibco</td>
			<td style="width:101px;">12800-082</td>
			<td>substituted with 10% FBS, 2 mM L-glutamine, 12 mM&#160; (for growth in flasks) or 30 mM (for growth in dishes) sodium-bicarbinate</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:419px;">600 cm2 tissue culture dish</td>
			<td style="width:197px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:101px;">166508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tris Buffered Saline (TBS), pH 7.4</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>137 mM NaCl, 5 mM KCl, 491 mM MgCl, 680 mM CaCl, 25.1 mM Tricine</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:419px;">HSV-1 stock</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>stocks with titers greater than 1x109 PFU/mL work best</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Sephadex G-25&#160; column (PD-10 Desalting Column)</td>
			<td style="width:197px;">GE Healthcare</td>
			<td align="right" style="width:101px;">17085101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">D128-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">5&#180;-Ethynyl-2&#180;-deoxycytidine (EdC)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">T511307</td>
			<td>Dissolve in DMSO to prepare 40 mM stock, aliquot, and store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">2&#180;-deoxycytidine (deoxyC)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">D3897</td>
			<td>Dissolve in water to prepare 40 mM stock, aliquot, and store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">Nuclear Extraction Buffer (NEB)</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (20 mM Hepes pH 7.2, 50 mM NaCl, 3 mM MgCl2, 300 mM Sucrose, 0.5% Igepal)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Cell scraper</td>
			<td style="width:197px;">Bellco glass</td>
			<td style="width:101px;">7731-22000</td>
			<td>Autoclave before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Trypan blue solution</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">T8154</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">PBS, pH 7.2 (10x)</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4 (dilute to 1x in sterile water before use)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Copper (II) sulfate pentahydrate (CuSO4-5H2O)</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">C489</td>
			<td>Prepare 100 mM stock and store at 4 &#176;C for up to 1 month</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">(+) Sodium L-ascorbate</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">A4034</td>
			<td>Freshly prepare 100 mM stock and store on ice until use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Biotin azide</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">B10184</td>
			<td>Prepare 10 mM stock in DMSO, aliquot, and store at -20 &#176;C for up to 1 year&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">Click Reaction Mix</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare immediately before use by adding reagents in the indicated order (10 mL: 8.8 mL 1x PBS, 200 mL 100 mM CuSO4, 25mL 10 mM Biotin Azide, 1 mL 100 mM sodium ascorbate)&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">cOmplete Protease Inhibitor Cocktail</td>
			<td style="width:197px;">Roche</td>
			<td align="right" style="width:101px;">11697498001</td>
			<td>Dissolve in 1 mL water to prepare 50x stock, can store at 4 &#176;C for up to 1 week, or directly add 1 pill to 50 mL buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">freezing buffer</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (7 mL 100% glycerol, 3 mL NEB, 200 &#956;L 50x protease inhibitor)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Buffer B1</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (25 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1% Igepal, 1x protease inhibitor)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Buffer B2</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 0.5% Igepal, 1x protease inhibitor)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Buffer B3</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1x protease inhibitor)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibra Cell Ultra Sonic Processer equipped with a 3 mm microtip probe</td>
			<td style="width:197px;">Sonics</td>
			<td style="width:101px;">VCX 130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Cell strainer</td>
			<td style="width:197px;">Falcon</td>
			<td align="right" style="width:101px;">352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dynabeads MyOne Streptavidin T1</td>
			<td style="width:197px;">Life Technologies</td>
			<td align="right" style="width:101px;">65601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">DynaMag-2 Magnet</td>
			<td style="width:197px;">Life Technologies</td>
			<td style="width:101px;">12321D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Mini-Tube Rotator</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">260750F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">2X Laemmli sample buffer</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>Mix 400 mg of SDS, 2 mL 100% glycerol, 1.25 mL of 1 M Tris (pH 6.8), and 10 mg of bromophenol blue in 8 mL water. Store at 4&#160; &#176;C for up to 6 months. Before use add 1 M DTT to a final concentration of 200 mM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">cap locks for 1.5 mL tube</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">NC9679153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Standard western blotting reagents</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">NOVEX Colloidal Blue Staining Kit</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">LC6025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">12-well tissue culture dish</td>
			<td style="width:197px;">Corning</td>
			<td align="right" style="width:101px;">3513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Coverslips</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">12-545-100</td>
			<td>Autoclave before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Microscope slides</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">12-552-5</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">16% paraformaldehyde</td>
			<td style="width:197px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:101px;">15710</td>
			<td>dilute to 3.7% in 1x PBS before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Bovine serum albumin (BSA)</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">BP1605</td>
			<td>prepare 3% in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Permeabilization buffer (0.5% Triton-X 100)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">T8787</td>
			<td>prepare 0.5% Triton-X 100 in 1x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Click reaction cocktail - Click-iT EdU Alexa Fluor 488 Imaging Kit</td>
			<td style="width:197px;">Molecular Probes</td>
			<td style="width:101px;">C10337</td>
			<td>prepare according to manufactorer&#39;s protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Hoechst 33342</td>
			<td style="width:197px;">Life Technologies</td>
			<td style="width:101px;">H1399</td>
			<td>prepare 10 mg/mL in water, can store at 4 &#176;C for up to 1 year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">mouse anti-ICP8 primary antibody</td>
			<td style="width:197px;">Abcam</td>
			<td style="width:101px;">ab20194</td>
			<td>Use a 1:200 dilution in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">mouse anti-UL42 primary antibody (2H4)</td>
			<td style="width:197px;">Abcam</td>
			<td style="width:101px;">ab19311</td>
			<td>Use a 1:200 dilution in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Goat anti-mouse 594-conjugated secondary antibody</td>
			<td style="width:197px;">Life Technologies</td>
			<td style="width:101px;">a11005</td>
			<td>Use a 1:500 dilution in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Immu-mount</td>
			<td style="width:197px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:101px;">9990402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">2x SDS-bicarb solution</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>2% SDS, 200 mM NaHCO3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Phenol:chloroform:isoamyl alcohol</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>mix 25:24:1 at least 1 day before use, store at 4 &#176;C in the dark</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">chloroform:isoamyl alcohol</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>mix 24:1 at least 1 day before use, store at room temperature in the dark</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">10x Tris EDTA (TE), pH 8.0</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>100 mM Tris, 10 mM EDTA (dilute to 1x before use)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qiaquick PCR purification kit</td>
			<td style="width:197px;">Qiagen</td>
			<td align="right" style="width:101px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">3 M sodium acetate pH 5.2</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qubit 2.0 Fluorometer</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">Q32866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">Q32851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qubit assay tubes</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Fluorescence microscope equipped with imaging software</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
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			<td height="21" style="height:21px;width:419px;">Heat blocks</td>
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purification of viral dna for the identification of associated viral and cellular proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The use of click chemistry for the purification of DNA from cells was first accomplished by the iPOND method21. The purpose of iPOND is to purify cellular replication forks for the identification of associated proteins. We have adapted this technique to specifically study vDNA protein interactions during infection. Manipulation of the approach to label viral genomes with EdC (Figure 1), combined with synchronized infections, has allowe...

Watch this Scientific Journal Video about Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins at JoVE.com

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

purification-viral-dna-for-identification-associated-viral-cellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.4K Views.  University of Pittsburgh School of Medicine. The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins. 

Video: Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Summary

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Chapters in this video

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Preparation of Viral DNA from Nucleocapsids

Production and Titering of Recombinant Adeno-associated Viral Vectors

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay