This work was supported by NIH AI099854 and AI126742 to KEY.

1. Induce PFV IN Expression in E. coli
<ol>
	<li>Add 1 &#956;L of PFV IN expression plasmid (10 ng/&#956;L) to 20 &#956;L of E. coli BL21(DE3) pLysS (20 &#956;L aliquot of commercially available cells has &#62;2 x 106 CFU/&#956;g plasmid) in a 1.5 mL tube. Mix gently by finger tapping or flicking the tube. Incubate on ice 5 min.
	<ol>
		<li>Heat shock for exactly 30 s in a 42 &#730;C water bath and then immediately return to ice for 2 min.</li>
		<li>Add 80 &#956;L of room temperature super optimal broth with catabolite repression (SOC) media. Incubate for 1 h at 37 &#730;C with 225 revolutions per minute (rpm) shaking.</li>
		<li>Plate 25 &#956;L of the mixture on a 100 mm x 15 mm Petri dish containing 25 mL of Luria broth (LB) agar (1 L LB agar: 10 g bacto-tryptone, 10 g NaCl, 5 g yeast extract, 15 g agar) and 100 &#956;g/mL ampicillin (100 mg/mL stock solution).</li>
		<li>Plate the remaining 75 &#956;L of the transformed cells on a second identical plate. Incubate the plates with the lids down overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Retrieve the plates of transformed E. coli. Use a single colony to inoculate 3 mL of LB (1 L LB: 10 g of bacto-tryptone, 10 g of NaCl, 5 g of yeast extract) supplemented with 100 &#956;g/mL ampicillin in a 14-mL round bottom polypropylene culture tube. Incubate for ~8 h at 37 &#730;C with 225 rpm shaking.
	<ol>
		<li>Add the 3 mL of culture to 50 mL LB supplemented with 100 &#956;g/mL ampicillin and 34 &#956;g/mL chloramphenicol (34 mg/mL stock solution) in a 250 mL Erlenmeyer flask. Incubate the culture overnight at 37 &#176;C with 225 rpm shaking.</li>
		<li>Transfer the 53 mL of culture to 1 L LB, 100 &#956;g/mL ampicillin, and 34 &#956;g/mL chloramphenicol in a 4 L Erlenmeyer flask. Incubate at 37 &#176;C with 225 rpm shaking.</li>
		<li>Measure the optical density at 600 nm (OD600) of the culture periodically. Initially, check the culture OD600 every h. As the culture reaches the desired OD600, check every 15 min to not overgrow. Grow the culture to an OD600 between 0.9 and 1.0. Transfer the flask to an ice bath and swirl to reduce the temperature of the culture.</li>
	</ol>
	</li>
	<li>Transfer 1 mL of the culture to a 1.5 mL tube for later analysis by denaturing SDS-PAGE analysis.
	<ol>
		<li>Pellet the cells in the 1.5 mL tube by centrifugation for 1 min in a microfuge at 14,000 x g at room temperature. Discard the supernatant. Resuspend the pellet in 150 &#956;L phosphate buffered saline (PBS) by pipetting. The PBS may be either with or without CaCl2 and MgCl2.</li>
		<li>Add 150 &#956;L of 2x SDS-PAGE sample buffer (150 mM Tris-HCl, pH 6.8, 1.2% SDS, 30% glycerol, 15% &#946;-mercaptoethanol (&#946;ME), 0.0018% bromophenol blue) and mix thoroughly by pipet or vortex. Boil for 3 min. Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Add to the bacterial culture: a final concentration of 0.25 mM isopropyl-beta-D-thiogalactopyranoside (IPTG, 0.25 M stock solution) and 50 &#956;M ZnCl2 (50 mM stock solution). IPTG induces expression of the PFV IN gene from the T7 promoter and ZnCl2 is added as a supplement for the correct folding of an amino terminal zinc finger domain in PFV IN. Incubate the culture at 25 &#176;C with shaking at 225 rpm for 4 h. Immediately transfer the flask to an ice bath. Save 1 mL of culture for SDS-PAGE analysis following the same protocol as above in 1.3.</li>
	<li>Transfer the bacterial culture to appropriately sized centrifuge bottles. For example, a 1 L culture may be transferred to four sterile disposable 250 mL conical bottom polypropylene bottles. Spin the bottles in a refrigerated tabletop centrifuge at 3000 x g for 20 min at 4 &#730;C. Discard supernatants by pouring.
	<ol>
		<li>Resuspend all of the pellets from a 1 L bacterial culture in a total volume of 25 mL (6.25 mL per bottle) cold PBS (with or without CaCl2 and MgCl2) by pipetting. Combine and transfer the bacterial suspensions to one 50 mL conical tube. Spin in a refrigerated centrifuge at 3,000 x g for 20 min at 4 &#176;C. Discard the supernatant by pouring or pipetting.</li>
	</ol>
	</li>
	<li>Resuspend the bacterial pellet in 10 mL resuspension buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 500 &#956;M phenylmethylsulfanoxide (PMSF) protease inhibitor) by pipetting. Snap freeze the pellet by placing the tube in a Dewar flask with liquid nitrogen sufficient to submerge the 50 mL tube. Carefully remove the tube from liquid nitrogen and store at -80 &#176;C. 
	Note: Bacterial pellets may be stored for several months at -80 &#176;C. We have seen no loss of active protein following -80 &#176;C storage for two years.</li>
	<li>Analyze the pre-induction and post-induction samples by 8.3 cm wide, 7.3 cm high, 0.75 mm thick SDS-PAGE gels with 1.5 mL 6% polyacrylamide stacking layer (125 mM Tris-HCl, pH 6.8, 6% acrylamide, 0.1% SDS, 0.1% ammonium persulfate, 0.001% TEMED) and 3.5 mL 10% polyacrylamide resolving layer (375 mM Tris-HCl, pH 8.8, 10% acrylamide, 0.1% SDS, 0.1% ammonium persulfate, 0.001% TEMED)12.
	<ol>
		<li>After pouring the stacking gel, insert a 10 well comb. When the gel has polymerized, assemble the PAGE apparatus, and add sufficient SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS).</li>
		<li>Load 10 &#956;L of each sample and 4 &#956;L of prestained protein standards. Run at 16.5 V/cm until the dye front reaches the bottom of the gel, approximately 60 min.</li>
		<li>Disassemble the gel plates and transfer the gel to a plastic tub. Add Coomassie Brilliant Blue staining solution (0.1% Coomassie brilliant blue R-250, 40% methanol, 10% acetic acid) to generously cover the gel and stain for 20 min at ambient temperature.</li>
		<li>Remove the staining solution by pouring and replace with destain solution (40% methanol, 10% acetic acid) until bands are readily visible. Remove the destaining solution by pouring and replace with deionized water. PFV IN should be easily identified in the post-induction sample. PFV IN with a hexahistidine tag has a molecular weight of 47374 Da.</li>
		<li>If PFV IN is not visible, repeat the induction starting with a different colony from one of the transformation plates.</li>
	</ol>
	</li>
</ol>
2. Nickel Affinity Chromatography
<ol>
	<li>Thaw one bacterial pellet on ice. 
	Note: This will take significant time (approximately 2 - 3 h). When the pellet has thawed to a cell slurry, add an additional 500 &#956;M PMSF (100 mM stock solution).</li>
	<li>Keep the 50 mL tube of bacteria slurry on ice. Sonicate the bacteria at 30% amplitude for 30 s (1 s on, 1 s off) per pellet derived from 1 L culture using a sonicator with a 0.5 inch diameter bio horn with a 0.125 inch diameter tapered microtip, a frequency of 20 kHz and maximum power of 400 W.</li>
	<li>Transfer the cell sample to a cold ultracentrifuge tube. Use polycarbonate bottle assemblies (25 mm diameter, 89 mm height) compatible with a Type 60 Ti fixed angle rotor. Alternative tubes and rotors may be used if the same gravitation force is achieved. Spin 120,000 x g for 60 min at 4 &#176;C. There should be an obvious pellet. The supernatant may have a yellow color.</li>
	<li>Transfer the supernatant by pouring or pipetting to a cold 50 mL conical tube. Note the volume; this volume should be approximately the volume of resuspension buffer used prior to snap freezing. If the supernatant is viscous, it may be contaminated by bacterial DNA which could clog the chromatography column. In this case, repeat the ultracentrifugation to pellet the bacterial DNA.</li>
	<li>Prepare 500 mL Buffer A (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 500 &#956;M PMSF) and 500 mL Buffer B (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 500 &#956;M PMSF, 200 mM imidazole). 
	Note: In this protocol, keep the fast protein liquid chromatography (FPLC) system, all buffers and the sample at 4 &#176;C in a cold room.
	<ol>
		<li>Sterile filter Buffers A and B with 0.2 &#956;m disposable filter units. Depending on the FPLC system, wash pump A and pump B with Buffer A and Buffer B, respectively. Flow 90% Buffer A and 10% Buffer B through the system until the conductivity and UV readings stabilize. The maximum flow rate will depend on the instrument; use a flow rate of 5 mL/min with a maximum pressure limit of 1.0 MPa.</li>
	</ol>
	</li>
	<li>Prepare a 110 mm long, 5 mm diameter FPLC column with 1.5 mL nickel-charged resin (maximum binding capacity 50 mg/mL, maximum pressure 1 MPa). The column may be prepared the day before purification and stored at 4 &#730;C.</li>
	<li>Connect the column to the FPLC instrument. Equilibrate the column with 20 mL of 90% Buffer A and 10% Buffer B (20 mM imidazole) at a flow rate of 0.5 mL/min with a maximum pressure limit of 0.5 MPa. The instrument should be collecting real time data of conductivity and UV absorbance at 280 nm (A280). The conductivity and UV readings should stabilize. If these readings have not stabilized, continue to flow buffers through the column until they are stable.</li>
	<li>Load the protein sample (~10 mL per pellet) to the column at a flow rate of 0.15 mL/min with a maximum pressure limit of 0.5 MPa. The flow rate and column pressure remains the same throughout the purification. Collect the flow through in a 50 mL tube.
	<ol>
		<li>Wash the column with 20 mL 90% Buffer A and 10% Buffer B. Collect the wash in a 50 mL tube. Elute the proteins with a 20 mL linear gradient of 10% to 100% Buffer B (20 mM to 200 mM imidazole).</li>
		<li>Finally, wash the column with 5 mL 100% Buffer B (200 mM imidazole). Collect the gradient and final wash in 0.27 mL fractions.</li>
	</ol>
	</li>
	<li>Combine 15 &#956;L of 2x SDS-PAGE sample buffer with 15 &#956;L of the flow through, wash, and peak A280 fractions. Boil the samples for 3 min.
	<ol>
		<li>Prepare two 10% SDS-PAGE gels as described previously in step 1.6 except with 15 well combs. Load 10 &#956;L of each sample to the gels and 4 &#956;L of prestained protein standards. Run, stain, and analyze the gels as described in step 1.6.</li>
		<li>Combine fractions that appear to contain nearly pure PFV IN based on visual inspection. Measure the volume by pipetting, typically 8 - 10 mL.</li>
		<li>Using a spectrophotometer, measure the A280 of the combined fractions and calculate the total amount of PFV IN protein; our typical yield is ~10 mg per L of induced culture. The extinction coefficient of PFV IN with the hexahistidine tag is 59360 cm-1 M-1.</li>
	</ol>
	</li>
	<li>To remove the hexahistidine tag, supplement the PFV IN with 10 mM dithiothreitol (DTT, 1 M stock solution) and 0.1 mM EDTA, pH 8.0 (0.5 M stock solution). Add 15 &#956;g of human rhinovirus (HRV) 3C protease per mg PFV IN. Incubate at 4 &#730;C overnight. Cleavage reduces the PFV IN molecular weight from 47374 Da to 44394 Da and may be verified by 10% SDS-PAGE analysis.</li>
</ol>
3. Heparin affinity chromatography
<ol>
	<li>Prepare 250 mL Buffer C (50 mM Tris-HCl, pH 7.5, 10 mM DTT, 0.1 mM EDTA, 500 &#956;M PMSF) and 250 mL Buffer D (50 mM Tris-HCl, pH 7.5, 10 mM DTT, 0.1 mM EDTA, 500 &#956;M PMSF, 1 M NaCl). 
	Note: In this protocol, the FPLC system, buffers, and sample are kept in a cold room at 4 &#730;C.
	<ol>
		<li>Sterile filter Buffers C and D with 0.2 &#956;m disposable filter units. Depending on the FPLC system, wash pump A and pump B with Buffer C and Buffer D, respectively. Flow 80% Buffer C and 20% Buffer D through the system until the conductivity and UV readings stabilize. The maximum flow rate will depend on the instrument; we routinely use a flow rate of 5 mL/min with a maximum pressure limit of 1.0 MPa.</li>
	</ol>
	</li>
	<li>Prepare an 80 mm long, 5 mm diameter FPLC column with 1 mL of heparin sepharose resin (maximum binding capacity 2.0 mg of bovine antithrombin III per mL resin; maximum pressure 1.4 MPa). The column may be prepared the day before purification and stored at 4 &#176;C. Connect the column to the FPLC instrument.
	<ol>
		<li>Equilibrate the column with 20 mL of 80% Buffer C and 20% Buffer D (200 mM NaCl) at a flow rate of 0.5 mL/min with a maximum pressure limit of 1 MPa. The instrument should be collecting real time data of conductivity and UV absorbance at 280 nm (A280). The conductivity and UV readings should stabilize. If these readings have not stabilized, continue to flow buffers through the column until they are stable.</li>
	</ol>
	</li>
	<li>Reduce the NaCl concentration of the PFV IN sample to 200 mM by adding 1.5 volumes Buffer C. For example, add 15 mL of Buffer C to 10 mL PFV IN. Our total volume is typically ~25 mL.</li>
	<li>Load the diluted PFV IN sample to the heparin sepharose column at 0.5 mL/min flow rate with maximum pressure limit 1.0 MPa. The flow rate and column pressure remains the same throughout the purification. Collect the flow through in a 50 mL conical tube.
	<ol>
		<li>Wash the column with 20 mL of 80% Buffer C and 20% Buffer D (200 mM NaCl), collecting the wash in a 50 mL conical tube. Elute the column with a 30 mL linear gradient of 20% to 100% Buffer D (200 mM to 1 M NaCl).</li>
		<li>Wash the column with 5 mL of 100% Buffer D. Collect the gradient and final wash in 0.37 mL fractions.</li>
	</ol>
	</li>
	<li>Analyze load, flow through, wash, and fractions by 8% SDS-PAGE as described in 2.9. PFV IN following cleavage of the hexahistidine tag has a molecular weight of 44394 Da and the extinction coefficient remains 59360 cm-1 M-1 without the hexahistidine tag. Store all fractions at 4 &#730;C until the completion of a nuclease assay.</li>
</ol>
4. Nuclease assay
<ol>
	<li>Identify heparin sepharose fractions that appear to contain nearly pure PFV IN. Combine 3 &#956;L of heparin fraction and 50 ng of 3 kb supercoiled plasmid DNA in reaction buffer (10 mM HEPES, pH 7.5, 110 mM NaCl, 5 mM MgSO4, 4 &#956;M ZnCl2, 10 mM DTT) with a final volume of 15 &#956;L. Incubate at 37 &#730;C for 90 min. Include a negative control with no PFV IN.
	<ol>
		<li>Stop the reaction with 1 mg/mL proteinase K (20 mg/mL stock solution) and 0.5% SDS (10% stock solution). Incubate at 37 &#176;C for 1 h. Samples may be stored at -20 &#176;C for later analysis.</li>
	</ol>
	</li>
	<li>Prepare a 125 mL gel of 1% weight per volume (w/v) agarose in 1x&#160;TAE buffer (40 mM Tris-acetate, 1 mM EDTA) with 0.1 &#181;g/mL ethidium bromide (10 mg/mL stock solution)13. Melt the agarose solution and pour to a 15 cm x 10 cm gel casting tray. Insert a 15 well comb with 5 mm wide, 0.75 mm thick wells. Thin wells yield better band resolution compared to 1.5 mm thick wells. Allow the gel to solidify at ambient temperature.
	<ol>
		<li>Immerse the gel in 1x&#160;TAE with 0.1 &#181;g/mL ethidium bromide. Add 3.5 &#956;L of 6x loading dye (50% glycerol, 0.15% Orange G dye) to the nuclease assay reactions. Load the entire volume to the gel. Run the gel at constant voltage, 10 volts per cm (100 V) at ambient temperature for 1 h or until the Orange G dye front reaches the end of the gel.</li>
	</ol>
	</li>
	<li>Immediately image the agarose gel with a fluorescent scanner set to detect ethidium bromide. Linear DNA should run true to size at 3 kb, supercoiled DNA should run faster (~2 kb), and relaxed circle DNA should run slower (~3.5 kb).
	<ol>
		<li>Using image analysis software, calculate the pixel volume of the supercoiled, linear, and relaxed circle plasmid in each lane. Call the sum of the pixel volumes for these three DNA forms the &#34;total DNA&#34; value and use it to calculate the percentage of linear and relaxed circles. For example, the pixel volume of relaxed circles is divided by the total DNA pixel value in that lane, multiply this number by 100 to determine a percentage. Fractions that contain contaminating nuclease display a higher percentage of linear and relaxed circles compared to the negative control.</li>
	</ol>
	</li>
	<li>Combine heparin sepharose fractions that do not display contaminating nuclease activity. Dialyze in 10 mm 6 - 8 kDa molecular weight cutoff (MWCO) dialysis tubing at 4 &#176;C overnight against 1 L of 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 50 &#956;M ZnCl2, 10 mM DTT, 20% glycerol.</li>
	<li>Recover the nuclease-free PFV IN sample from dialysis tubing. Measure the A280 and calculate the final protein concentration. Aliquot and snap freeze in liquid nitrogen. Store aliquots at -80 &#176;C.</li>
</ol>

<ol>
	<li>Lopez, M. A., Mackler, R. M., Yoder, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+nuclease+contamination+during+purification+of+recombinant+prototype+foamy+virus+integrase.">Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase.</a> J Virol Methods. 235, 134-138 (2016).</li><li>Senavirathne, G., 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=Widespread+nuclease+contamination+in+commonly+used+oxygen-scavenging+systems.">Widespread nuclease contamination in commonly used oxygen-scavenging systems.</a> Nat Methods. 12 (10), 901-902 (2015).</li><li>Gunn, K. H., Marko, J. F., Mondragon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthogonal+single-molecule+experiment+reveals+multiple-attempt+dynamics+of+type+IA+topoisomerases.">An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases.</a> Nat Struct Mol Biol. 24 (5), 484-490 (2017).</li><li>Valkov, E., 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=Functional+and+structural+characterization+of+the+integrase+from+the+prototype+foamy+virus.">Functional and structural characterization of the integrase from the prototype foamy virus.</a> Nucleic Acids Res. 37 (1), 243-255 (2009).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Li, M., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+viral+DNA+ends+channels+the+HIV-1+integration+reaction+to+concerted+integration.">Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration.</a> J Biol Chem. 280 (32), 29334-29339 (2005).</li><li>Li, M., Mizuuchi, M., Burke, T. R., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+DNA+integration:+reaction+pathway+and+critical+intermediates.">Retroviral DNA integration: reaction pathway and critical intermediates.</a> EMBO J. 25 (6), 1295-1304 (2006).</li><li>Sinha, S., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+immunodeficiency+virus+type+1+integrase+exhibits+a+capacity+for+full-site+integration+in+vitro+that+is+comparable+to+that+of+purified+preintegration+complexes+from+virus-infected+cells.">Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells.</a> J Virol. 79 (13), 8208-8216 (2005).</li><li>Goodarzi, G., Im, G. J., Brackmann, K., Grandgenett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+integration+of+retrovirus-like+DNA+by+human+immunodeficiency+virus+type+1+integrase.">Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.</a> J Virol. 69 (10), 6090-6097 (1995).</li><li>Vora, A. C., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+catalytic+properties+of+retrovirus+integrase-DNA+complexes+capable+of+efficiently+performing+concerted+integration.">Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.</a> J Virol. 69 (12), 7483-7488 (1995).</li><li>Jones, N. D., 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=Retroviral+intasomes+search+for+a+target+DNA+by+1D+diffusion+which+rarely+results+in+integration.">Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.</a> Nat Commun. 7, 11409 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li></ol>

The authors have nothing to disclose.

Recombinant proteins that interact with DNA, such as DNA repair proteins, oxygen scavengers for single molecule microscopy applications, or retroviral integrases, should be free of contaminating bacterial nucleases2,3. These contaminants may confuse the interpretation of results during bulk biochemical or single molecule assays.
We have found that a bacterial nuclease frequently co-purifies with PFV IN. However, PFV IN displays an affinity for heparin sepharose that is readily distinguishable from the bacterial nuclease contaminant1. The key to preparation of nuclease-free PFV IN is the careful quantitation of nuclease activity in each fraction following gradient elution from heparin sepharose. The gradient must be sufficiently shallow to allow the separation of PFV IN from the nuclease contaminant; a steep gradient would not be compatible with efficient separation. In addition, the plasmid DNA substrate should have a low percentage of relaxed circles to reduce the background of the nuclease assay. If the plasmid DNA substrate has a high background of relaxed circles, a new purification of plasmid DNA should be performed. The nuclease activity assay reveals the presence of the contaminating nuclease and fractions that should be discarded.
In some cases, size exclusion chromatography (SEC) may be an effective method for removal of a contaminating nuclease2. Previous groups have used SEC during purification of PFV IN4. SEC has a limited load volume and tends to reduce the final protein concentration. The major advantage to affinity and ion exchange chromatography is the ability to load a significantly greater volume than SEC.
Testing for nuclease activity may be adapted to different proteins that co-purify with a bacterial nuclease. Here we identify that PFV IN and a contaminating nuclease may be separated by heparin sepharose chromatography. We have previously shown that PFV IN has a similar profile to the nuclease with Mono-S cation and Mono-Q anion exchange chromatography making these resins unsuitable for this protein. Depending on the affinity characteristics of the desired protein in relation to the nuclease, this protocol may be used with affinity or ion exchange resins and gradient elution. The key to effective removal of nuclease contamination from a protein of interest is a difference in affinity for a resin. Each fraction must be assayed for nuclease activity. The chromatography resins of this protocol may not be directly applied to all proteins that co-purify with bacterial nucleases. However, the overall concept of using an affinity or ion exchange resin to separate the nuclease from a protein of interest and assaying fractions for nuclease activity may be broadly applicable.

Biochemical and single molecule studies of protein interactions with DNA require exceptionally pure recombinant proteins. Contaminating nucleases from bacteria can obscure the results of these assays. A contaminating nuclease has been found in preparations of recombinant proteins oxygen scavenger protocatechuate-3,4-dioxygenase (PCD) and prototype foamy virus (PFV) integrase (IN) isolated from Escherichia coli (E. coli)1,2,3.
Retroviral integration assays rely on the conversion of supercoiled DNA to nicked or linear products as a measure of IN activity4. During cellular infection IN joins the two ends of a viral cDNA to the host chromatin5. Each end joining reaction is termed strand transfer. Assays of recombinant IN activity may join two DNA oligomers mimicking the viral cDNA ends to a target DNA in a concerted integration reaction4,5,6,7,8. Alternatively recombinant IN may join only one DNA end in a non-physiologically relevant half site integration reaction9,10. When supercoiled plasmid DNA is the target of integration, concerted integration products are linearized DNA and half site integration products are relaxed circles. These reaction products are identified by their relative mobility during agarose gel electrophoresis1. If the recombinant IN has a contaminating nuclease, there will be spurious relaxed circles or a possibly linearized plasmid confusing the experimental results. Viral DNA oligomers may be fluorescently labeled to conclusively identify integration products, as opposed to nuclease products. However, IN greatly favors supercoiled DNA targets; any loss of supercoiled plasmid to relaxed circles or linear DNA by contaminating nuclease could skew results and interpretation of data11. Thus it is imperative to remove bacterial nucleases from retroviral IN preparations.
PFV IN has a different affinity for heparin sepharose compared to the bacterial nuclease1. PFV IN and the nuclease may be separated by a linear gradient elution from heparin sepharose. The nuclease is not readily detected by an ultraviolet (UV) absorbance at 280 nm peak or by analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Instead, the nuclease is detected by a nuclease activity assay employing the conversion of a supercoiled plasmid to relaxed circles or linear products. Each fraction following heparin sepharose chromatography is tested for nuclease activity. PFV IN and the nuclease contaminant have no difference in affinity for Mono-Q anion resin. There is a small difference in affinity for Mono-S cation resin. However, the Mono-S resolution of bacterial nuclease and PFV IN would not allow efficient separation of the proteins. Ultimately, heparin sepharose affinity purification offers the best separation of bacterial nuclease from PFV IN and has the advantage of unlimited load volume.
Testing for contaminating nuclease activity may be adapted to other proteins. The protein of interest will likely have alternative affinity characteristics than PFV IN; the difference in binding characteristics of the protein of interest and the nuclease contaminant must be empirically determined. This methodology for identifying nuclease contamination may be adapted to other resins including Mono-S cation or Mono-Q anion exchange resins. Affinity and ion exchange resins may offer a reliable method to isolate a recombinant protein of interest from contaminating nucleases with no limits on the volume of protein during chromatography.

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BL21/DE3 Rosetta E. coli</td>
			<td style="width:180px;">EMD Millipore</td>
			<td style="width:119px;">70956-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB broth</td>
			<td style="width:180px;">EMD biosciences</td>
			<td style="width:119px;">1.10285.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:180px;">Amresco</td>
			<td style="width:119px;">0339</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloramphenicol&#160;</td>
			<td style="width:180px;">Amresco</td>
			<td style="width:119px;">0230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG&#160;</td>
			<td style="width:180px;">Denville Scientific</td>
			<td style="width:119px;">CI8280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZnCl2</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">208086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Ultra Pure</td>
			<td style="width:180px;">Gojira Fine Chemicals</td>
			<td style="width:119px;">UTS1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:180px;">P212121</td>
			<td style="width:119px;">RP-S23020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PMSF&#160;</td>
			<td style="width:180px;">Amresco</td>
			<td style="width:119px;">0754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imidazole&#160;</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">I0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT&#160;</td>
			<td style="width:180px;">P212121</td>
			<td style="width:119px;">SV-DTT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure EDTA</td>
			<td style="width:180px;">Invitrogen/Gibco</td>
			<td style="width:119px;">15575</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4</td>
			<td style="width:180px;">Amresco</td>
			<td style="width:119px;">0662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose&#160;</td>
			<td style="width:180px;">Denville Scientific</td>
			<td style="width:119px;">CA3510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethidium bromide</td>
			<td style="width:180px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP1302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">G37-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ni-NTA Superflow</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:119px;">30430</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Heparin Sepharose 6 Fast Flow</td>
			<td style="width:180px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0998-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HRV14 3C protease&#160;</td>
			<td style="width:180px;">EMD Chemicals</td>
			<td style="width:119px;">71493-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant PFV IN is often contaminated with a bacterial nuclease1. Biochemical integration assays depend on the quantitation of the conversion of supercoiled plasmid DNA to relaxed circles and linear products. The presence of a contaminating nuclease could lead to spurious quantitation of these assays. Expression of PFV IN with a hexahistidine tag is induced in E. coli (Figure 1) and first purified by nickel affinity chromatography (Figure 2). The fractions with nearly pure PFV IN are combined and the hexahistidine tag is cleaved by a protease. We have determined that PFV IN and the contaminating nuclease have different affinities for heparin sepharose chromatography (Figure 3)1. PFV IN fractions following heparin sepharose chromatography are incubated with a supercoiled plasmid to assay for relaxed circles and linearized plasmid, the products of nuclease activity (Figure 4). This assay allows the identification of protein fractions without the nuclease (Figure 5). These fractions may be combined, dialyzed, and frozen for future experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56605/56605fig1.jpg" /> 
Figure 1: Induction of PFV IN in E. coli . Cells from cultures before (lane 2) and after (lane 3) induction are analyzed for the presence of PFV IN, 47374 Da. Samples were separated by 10% SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, molecular weight markers (kDa). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56605/56605fig2.jpg" /> 
Figure 2: Nickel affinity chromatography fractions. Soluble bacterial lysates were separated by nickel affinity chromatography. PFV IN with a hexahistidine tag was eluted with a linear gradient of 20 mM to 200 mM imidazole. Fractions were evaluated by 10% SDS-PAGE and stained with Coomassie brilliant blue. PFV IN, 47374 Da, is readily apparent in the sample loaded to the column (load), but is less apparent in the flow through or wash. Fractions that elute early in the imidazole gradient commonly display a slower mobility contaminant near 75 kDa as well as some faster mobility contaminants at or below 25 kDa. At higher concentrations of imidazole there are no readily apparent contaminants and PFV IN appears to be relatively pure. In this example, fractions #33 through #84 were combined and further purified. Molecular weight markers (kDa) are on the left of each gel. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56605/56605fig3.jpg" /> 
Figure 3: Heparin sepharose chromatography of PFV IN 1 . Following nickel affinity and protease removal of the hexahistidine tag, PFV IN (44394 Da) was fractionated by heparin sepharose. Proteins were eluted with a linear gradient from 200 mM to 1 M NaCl. Even fractions #12 to #44 were evaluated by 8% SDS-PAGE stained with Coomassie blue. Molecular weight markers (kDa) are on the left of each gel. These fractions were tested for nuclease activity. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56605/56605fig4.jpg" /> 
Figure 4: Nuclease assay of heparin sepharose fractions1 . Each heparin sepharose fraction was added to supercoiled plasmid DNA. Fraction numbers correlate to those shown in Figure 3. Following incubation, the nuclease reactions were deproteinated and separated by 1% agarose with ethidium bromide. Negative controls include plasmid DNA with no protein (Target Only) and a previous purification of wild type PFV IN known to be free of nuclease (PFV IN WT). A positive control for contaminating nuclease activity is a previous purification of catalytically inactive PFV IN that is known to have contaminating nuclease (PFV IN D128N). DNA size markers are shown in kb. Supercoiled plasmid (SC), linear plasmid (L), and relaxed circles (RC) are indicated. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56605/56605fig5.jpg" /> 
Figure 5: Quantitation of nuclease assays1. Agarose gels were scanned and pixel values were quantified for supercoiled (SC), linear (L), and relaxed circle (RC) bands in each lane. Fraction numbers correlate to those shown in Figures 3 and 4. The percentages of linear and relaxed circles were calculated using the pixel values and the equation shown. For example, the pixel values for the bands of fraction #12 are: 817636 supercoiled plasmid, 50467 linear plasmid, and 112052 relaxed circles. The total DNA pixel value for fraction #12 is the sum of these values, 980155. The percentage of linear DNA is 50467 multiplied by 100 for the percentage and divided by the total DNA value equaling 5.1%. The percentage of relaxed circles is 112052 multiplied by 100 and divided by the total DNA value equaling 11.4%. The sum of linear and relaxed circle percentages is 16.5%. Negative control plasmid DNA with no protein (No IN, bottom graph) indicates that this preparation of plasmid included 4.4% of the total DNA as linear or relaxed circles. A second negative control was a previous purification of wild type PFV IN (WT) that displayed 8.6% of the total DNA as linear or relaxed circles. These two control lanes indicate the background level of nicked or linearized plasmid present with the substrate alone and with PFV IN. A positive control is a previous purification of catalytically inactive PFV IN (D128N) which had 23% of the total DNA as linear and relaxed circles. The DNA exposed to heparin sepharose fractions #12 through #22 resulted in &#62;10% of the DNA as linear and relaxed circles. In this example, fractions #24 to #42 displayed &#60;10% conversion to linear and relaxed circles. These fractions were combined and dialyzed. Aliquots were frozen and stored at -80 &#176;C for future use. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase at JoVE.com

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

detection-removal-nuclease-contamination-during-purification

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.0K Views.  The Ohio State University College of Medicine. Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

Video: Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...