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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+recruitment+of+nuclear+factors+to+productively+replicating+herpes+simplex+virus+genomes.">Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.</a> PLoS Pathog. 11, e1004939 (2015).</li><li>Dembowski, J. A., Dremel, S. E., DeLuca, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-Coupled+Recruitment+of+Viral+and+Cellular+Factors+to+Herpes+Simplex+Virus+Type+1+Replication+Forks+for+the+Maintenance+and+Expression+of+Viral+Genomes.">Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes.</a> PLoS Pathog. 13, e1006166 (2017).</li><li>Bjornberg, O., Nyman, P. 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><tbody><tr><td>MRC-5 cells</td><td>ATCC</td><td>CCL-171</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140-179</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>12800-082</td><td>Substituted with 10% FBS, 2 mM L-glutamine, 12 mM&amp;nbsp; (for growth in flasks) or 30 mM (for growth in dishes) sodium-bicarbinate</td></tr><tr><td>600 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture dish</td><td>Thermo Fisher Scientific</td><td>166508</td><td></td></tr><tr><td>Tris Buffered Saline (TBS), pH 7.4</td><td></td><td></td><td>137 mM NaCl, 5 mM KCl, 491 mM MgCl, 680 mM CaCl, 25.1 mM Tricine</td></tr><tr><td>HSV-1 stock</td><td></td><td></td><td>Stocks with titers greater than 1x10&lt;sup&gt;9&lt;/sup&gt; PFU/mL work best</td></tr><tr><td>Sephadex G-25&amp;nbsp; column (PD-10 Desalting Column)</td><td>GE Healthcare</td><td>17085101</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>D128-1</td><td></td></tr><tr><td>5&amp;acute;-Ethynyl-2&amp;acute;-deoxycytidine (EdC)</td><td>Sigma-Aldrich</td><td>T511307</td><td>Dissolve in DMSO to prepare 40 mM stock, aliquot, and store at -20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>2&amp;acute;-deoxycytidine (deoxyC)</td><td>Sigma-Aldrich</td><td>D3897</td><td>Dissolve in water to prepare 40 mM stock, aliquot, and store at -20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Nuclear Extraction Buffer (NEB)</td><td></td><td></td><td>Prepare fresh (20 mM Hepes pH 7.2, 50 mM NaCl, 3 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 300 mM Sucrose, 0.5% Igepal)</td></tr><tr><td>Cell scraper</td><td>Bellco glass</td><td>7731-22000</td><td>Autoclave before use</td></tr><tr><td>Trypan blue solution</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>PBS, pH 7.2 (10x)</td><td></td><td></td><td>1.37 M NaCl, 27 mM KCl, 100 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 18 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (dilute to 1x in sterile water before use)</td></tr><tr><td>Copper (II) sulfate pentahydrate (CuSO4-5H2O)</td><td>Fisher Scientific</td><td>C489</td><td>Prepare 100 mM stock and store at 4 &amp;deg;C for up to 1 month</td></tr><tr><td>(+) Sodium L-ascorbate</td><td>Sigma-Aldrich</td><td>A4034</td><td>Freshly prepare 100 mM stock and store on ice until use</td></tr><tr><td>Biotin azide</td><td>Invitrogen</td><td>B10184</td><td>Prepare 10 mM stock in DMSO, aliquot, and store at -20 &amp;deg;C for up to 1 year&amp;nbsp;</td></tr><tr><td>Click Reaction Mix</td><td></td><td></td><td>Prepare immediately before use by adding reagents in the indicated order (10 mL: 8.8 mL 1x PBS, 200 mL 100 mM CuSO&lt;sub&gt;4&lt;/sub&gt;, 25mL 10 mM Biotin Azide, 1 mL 100 mM sodium ascorbate)&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Complete Protease Inhibitor Cocktail</td><td>Roche</td><td>11697498001</td><td>Dissolve in 1 mL water to prepare 50x stock, can store at 4 &amp;deg;C for up to 1 week, or directly add 1 pill to 50 mL buffer</td></tr><tr><td>Freezing buffer</td><td></td><td></td><td>Prepare fresh (7 mL 100% glycerol, 3 mL NEB, 200 &amp;mu;L 50x protease inhibitor)</td></tr><tr><td>Buffer B1</td><td></td><td></td><td>Prepare fresh (25 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1% Igepal, 1x protease inhibitor)</td></tr><tr><td>Buffer B2</td><td></td><td></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><td>Buffer B3</td><td></td><td></td><td>Prepare fresh (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1x protease inhibitor)</td></tr><tr><td>Vibra Cell Ultra Sonic Processer equipped with a 3 mm microtip probe</td><td>Sonics</td><td>VCX 130</td><td></td></tr><tr><td>Cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>Dynabeads MyOne Streptavidin T1</td><td>Life Technologies</td><td>65601</td><td></td></tr><tr><td>DynaMag-2 Magnet</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>Mini-Tube Rotator</td><td>Fisher Scientific</td><td>260750F</td><td></td></tr><tr><td>2x Laemmli sample buffer</td><td></td><td></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&amp;nbsp; &amp;deg;C for up to 6 months. Before use add 1 M DTT to a final concentration of 200 mM.</td></tr><tr><td>Cap locks for 1.5 mL tube</td><td>Fisher Scientific</td><td>NC9679153</td><td></td></tr><tr><td>Standard western blotting reagents</td><td></td><td></td><td></td></tr><tr><td>NOVEX Colloidal Blue Staining Kit</td><td>Invitrogen</td><td>LC6025</td><td></td></tr><tr><td>12-well tissue culture dish</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-545-100</td><td>Autoclave before use</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-552-5</td><td></td></tr><tr><td>16% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Dilute to 3.7% in 1x PBS before use</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td>BP1605</td><td>Prepare 3% in 1x PBS</td></tr><tr><td>Permeabilization buffer (0.5% Triton-X 100)</td><td>Sigma-Aldrich</td><td>T8787</td><td>Prepare 0.5% Triton-X 100 in 1x PBS</td></tr><tr><td>Click reaction cocktail - Click-iT EdU Alexa Fluor 488 Imaging Kit</td><td>Molecular Probes</td><td>C10337</td><td>Prepare according to manufactorer&#039;s protocol</td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H1399</td><td>Prepare 10 mg/mL in water, can store at 4 &amp;deg;C for up to 1 year</td></tr><tr><td>Mouse anti-ICP8 primary antibody</td><td>Abcam</td><td>ab20194</td><td>Use a 1:200 dilution in 1x PBS</td></tr><tr><td>Mouse anti-UL42 primary antibody (2H4)</td><td>Abcam</td><td>ab19311</td><td>Use a 1:200 dilution in 1x PBS</td></tr><tr><td>Goat anti-mouse 594-conjugated secondary antibody</td><td>Life Technologies</td><td>a11005</td><td>Use a 1:500 dilution in 1x PBS</td></tr><tr><td>Immu-mount</td><td>Thermo Fisher Scientific</td><td>9990402</td><td></td></tr><tr><td>2x SDS-bicarb solution</td><td></td><td></td><td>2% SDS, 200 mM NaHCO3</td></tr><tr><td>Phenol:chloroform:isoamyl alcohol</td><td></td><td></td><td>Mix 25:24:1 at least 1 day before use, store at 4 &amp;deg;C in the dark</td></tr><tr><td>chloroform:isoamyl alcohol</td><td></td><td></td><td>Mix 24:1 at least 1 day before use, store at room temperature in the dark</td></tr><tr><td>10x Tris EDTA (TE), pH 8.0</td><td></td><td></td><td>100 mM Tris, 10 mM EDTA (dilute to 1x before use)</td></tr><tr><td>Qiaquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>3 M sodium acetate pH 5.2</td><td></td><td></td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Invitrogen</td><td>Q32866</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Invitrogen</td><td>Q32851</td><td></td></tr><tr><td>Qubit assay tubes</td><td>Invitrogen</td><td>Q32856</td><td></td></tr><tr><td>Fluorescence microscope equipped with imaging software</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge for 1.5 mL tubes</td><td></td><td></td><td></td></tr><tr><td>Tabletop centrifuge for 15 and 50 mL tubes</td><td></td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td></td><td></td><td></td></tr><tr><td>Biosafety cabinet</td><td></td><td></td><td></td></tr><tr><td>Heat blocks</td><td></td><td></td><td>65 &amp;deg;C and 95 &amp;deg;C</td></tr></tbody></table>

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

Research

JoVE Journal

Immunology and Infection

13.5K Views.  University of Pittsburgh School of Medicine. Het doel van dit protocol is te specifiek tag en selectief isoleren viraal DNA van geïnfecteerde cellen voor de karakterisatie van het virale genoom geassocieerde eiwitten.

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

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. ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Biology

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

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

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