This research was supported by grants NIH/NIAID AI112423 and NIH/NIGMS GM113885 to Benjamin K. Chen and NIH/NIAID K08-AI120806 to Talia H. Swartz. We would like to thank the Icahn School of Medicine at Mount Sinai Dean&#39;s flow Cytometry CORE.

1. Generation of Target Cell Lines
NOTE: This step is optional; if using an existing RG target cell line, start at step 2.
<ol>
	<li>Cotransfect a 70% confluent 10 cm plate of 293T cells11 [in 10 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% fetal bovine serum (FBS)] with pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE10 and pCL-10A112 (packaging plasmid) in a 1:1 ratio (20 &#181;g total) using calcium-based transfection13.</li>
	<li>Harvest 10 mL of viral supernatant 48 h post-transfection by pipetting the media from the plate into a 15 mL conical tube and centrifuging the tube at 3,000 x g for 5 min at 23 &#176;C to pellet any cell debris.</li>
	<li>Attach a 0.45 &#181;m filter to a 10 mL syringe. After centrifugation, take the entire supernatant and load the syringe. Run the sample through into a clean 15 mL tube.</li>
	<li>Aliquot the filtered viral supernatant into appropriate sizes (usually, 0.5&#8211;1 mL aliquots). These can be used immediately in the next step or can be frozen at -80 &#176;C and stored.</li>
	<li>Use 50&#8211;100 &#181;L of viral supernatant to infect a cell line of choice in a 96-well plate. Culture the cells at 37 &#176;C with 5% CO2. The RFP signal should appear in as soon as 24 h under a fluorescence microscope (40X magnification, 532 nm excitation, 588 nm emission) but may take up to 72 h. 
	NOTE: In these experiments, Jurkat cells were used, but other types may be used as well.</li>
	<li>Select transduced cells with puromycin by setting up a series of 10 wells and adding puromycin to each, leaving at least 1 well untreated as a control (use a range of concentrations from 0.5 &#181;g/mL to 5 &#181;g/mL; this may vary per cell type). Monitor the cell viability (cell lysis will occur in cells without puromycin resistance) over the course of several days compared to the untreated control and use a concentration of puromycin where only RFP-expressing cells survive. 
	NOTE: Select the concentration where untransfected cells are killed while transfected cells survive.</li>
	<li>Using either single-cell flow cytometry sorting or a limiting dilution (see steps 1.8&#8211;1.10), grow&#160;cultures derived from single cells1 to develop a clonal cell line (this may take several weeks to grow).</li>
	<li>If using the dilution method, count the cells using a hemocytometer and dilute the sample to approximately 500 cells/mL.</li>
	<li>In a 96-well plate, pipette 50 &#181;L of the cell dilution into 50 &#181;L of media [Roswell Park Memorial Institute (RPMI) medium with 10% FBS and 2% penicillin-streptomycin, if using Jurkat cells] and perform 1:1 serial dilutions into 11 wells containing 50 &#181;L of media. For the best results, perform at least 10 replicates.</li>
	<li>Monitor the cell growth via microscopy (40X) of the plate in a tissue culture incubator (37 &#176;C with 5% CO2) or&#160;approximately 4 weeks. Choose cultures from the lowest puromycin concentration with growth (as seen via microscope, 40X) for the clonal cultures.</li>
	<li>Freeze the aliquots by centrifuging 20 mL of the culture (500,000 cells/mL, counted with a hemocytometer) at 800 x g for 5 min at 23 &#176;C and resuspending it in 500 &#181;L of FBS with 10% DMSO. Place it at -80 &#176;C using a cell-freezing container and then store it in liquid nitrogen.</li>
</ol>
2. Cell-to-cell Virus Transmission
<ol>
	<li>Preparation of target cells
	<ol>
		<li>Thaw one 500 &#181;L vial of Jurkat RG reporter cells by placing it into a 37 &#176;C water bath. Pipette the cells from the vial into 10 mL of RPMI complete medium and then centrifuge the mixture at 800 x g for 5 min at 23 &#176;C. Resuspend the pellet in 20 mL of RPMI complete medium in a T-75 flask. Incubate the flask overnight (37 &#176;C and 5% CO2). 
		NOTE: RPMI complete medium contains RPMI 1640, 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin.</li>
		<li>The next day, add 0.5 &#181;g/mL of puromycin (1 &#181;L of a 2 mg/mL stock per 8 mL of media). Culture the cells, maintaining a density of 200,000&#8211;800,000 cells/mL (counted via hemocytometer).</li>
		<li>The day before setting up the transfer assay, split the cells down to 200,000&#8211;400,000 cells/mL in fresh RPMI medium containing 0.5 &#181;g/mL of puromycin.</li>
	</ol>
	</li>
	<li>Preparation of donor cells
	<ol>
		<li>Thaw untransduced Jurkat cells and culture them in a T-75 flask with 20 mL of RPMI complete medium (as described in step 2.1), keeping a density of 200,000&#8211;800,000 cells/mL.</li>
		<li>Centrifuge 7,500,000 cells (800 x g for 5 min) and resuspend them in 120 &#181;L of nucleofection solution V with supplement (see Table of Materials). Transfer the cells to an electroporation cuvette and add 4.5 &#181;g of Gag-iCre1 DNA. Transfect the cells (via an electroporation-based approach, see the Table of Materials) using an appropriate program (e.g., S-18) and then immediately transfer them to 3 mL of RPMI medium (with 10% FBS).</li>
		<li>Allow the cells to recover by incubating them overnight at 37 &#176;C with 5% CO2.</li>
		<li>The next morning, centrifuge the cells at 800 x g for 5 min at 23 &#176;C and resuspend them in 3 mL of RPMI complete medium, allowing them 2 h to recover at 37 &#176;C (5% CO2) before proceeding with the assay set-up.</li>
	</ol>
	</li>
	<li>Co-culture
	<ol>
		<li>Count the cells using a hemocytometer then spin down 50,000 cells (800 x g for 5 min at 23 &#176;C) per well to be assayed (of both donor and target cells). Resuspending 1 x 106 cell/mL in RPMI complete without puromycin.</li>
		<li>In one plate, add 25 &#181;L of donor cell suspension to each well; in another plate, add 25 &#181;L of target cells.</li>
		<li>To each well, add 25 &#181;L of the test compound diluted in RPMI complete medium at the appropriate concentration. Incubate the donor and target cells with the compound for 30 min. 
		NOTE: The concentration will vary per drug. If it has a known IC50, use this as a starting point, titrating above and below. If the IC50 is unknown, prepare a 200 &#181;M stock for a final concentration of 10 &#181;M.</li>
		<li>After the pretreatment, combine the donor cells with the target cells by pipetting the contents of one plate into the other and incubate the cells for 40 h in a tissue culture incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Add paraformaldehyde (PFA) to each well to a final concentration of 2%. 
		NOTE: For a 16% stock solution, this is 14 &#181;L per 100 &#181;L well.</li>
		<li>CAUTION: PFA is harmful to exposed skin and eyes. Wear a lab coat, gloves, and safety goggles, and handle the stock solution in a safety hood.</li>
		<li>Read the cells on a flow cytometer with mCherry and FITC channels. 
		NOTE: It is possible to see 5&#8211;20% of the cells expressing GFP above background in the infected cells vs. uninfected cells.</li>
		<li>Visualize the GFP signal via fluorescence microscopy (40X) on GFP-specific channels (with an excitation filter of 400 nm and an emission filter of 508 nm).</li>
	</ol>
	</li>
</ol>
3. Alternative Protocol for a Cell-free Virus Infection
<ol>
	<li>Preparation of target cells
	<ol>
		<li>Thaw one 500 &#181;L vial of Jurkat RG reporter cells by placing into a 37 &#176;C water bath. Pipette the cells from the vial into 10 mL of RPMI complete medium and then centrifuge the RPMI complete medium at 800 x g for 5 min at 23 &#176;C. Resuspend the pellet in 20 mL of RPMI complete medium in a T-75 flask. Incubate the flask overnight (37 &#176;C and 5% CO2).</li>
		<li>The next day, add 0.5 &#181;g/mL of puromycin (1 &#181;L of a 2 mg/mL stock per 8 mL of media). Culture the cells, maintaining a density of 200,000&#8211;800,000 cells/mL (counted via hemocytometer).</li>
		<li>The day before setting up the transfer assay, split the cells down to 200,000&#8211;400,000 cells/mL in fresh RPMI medium containing 0.5 &#181;g/mL of puromycin.</li>
	</ol>
	</li>
	<li>Production of a cell-free virus
	<ol>
		<li>Transfect a 70% confluent 10 cm plate of 293T cells11 (in 10 mL of DMEM with 10% FBS) with 5 &#181;g of Gag-iCre1 plasmid using calcium-based transfection13. 
		NOTE: This scale will produce 10 mL of virus, which is enough for approximately two to four 96-well plates, depending on the efficiency of the transfection.</li>
		<li>Harvest the entire viral supernatant at 48 h by pipetting the media from the plate into a 15 mL conical tube and centrifuging the tube at 3,000 x g for 5 min to pellet all cell debris.</li>
		<li>Attach a 0.45 &#181;m filter to a 10 mL syringe. After centrifugation, take the entire supernatant and load the syringe. Run the sample through into a clean 15 mL tube.</li>
		<li>Use the virus to infect target cells immediately (step 3.3) or freeze and store the virus at -80 &#176;C (thaw the sample as needed before use).</li>
	</ol>
	</li>
	<li>Infection
	<ol>
		<li>Count the RG Jurkat target cells (from step 3.1.3) using a hemocytometer and spin down 50,000 target cells per well at 800 x g for 5 min, resuspending the cells at 1 x 106 cells/mL in RPMI complete medium without puromycin.</li>
		<li>In one plate, add 25 &#181;L of RG Jurkat target cells to each well; in a separate plate, add 25 &#181;L (2 ng) virus to each well.</li>
		<li>To each well, add 25 &#181;L of the test compound, diluted in media at the appropriate concentration. Incubate the cells and the virus with the compound for 30 min. 
		NOTE: The test concentration will vary per drug. If it has a known IC50, use this as a starting point, titrating above and below. If unknown, prepare a 200 &#181;M stock medium for a final concentration of 10 &#181;M.</li>
		<li>After the pretreatment, add the entire content (50 &#181;L) of the virus/compound mix from the wells to the target cells and incubate everything for 40 h in a tissue culture incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Add PFA to each well to a final concentration of 2%. 
		NOTE: For a 16% stock solution, this is 14 &#181;L per 100 &#181;L well.</li>
		<li>Read the cells on a flow cytometer with mCherry and FITC channels. The GFP signal can also be visualized via fluorescence microscopy on GFP channels. 
		NOTE: Once can expect to see 5&#8211;20% of the cells expressing GFP above background in the infected cells vs. the uninfected cells.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Esposito, A. M., 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=A+high+throughput+Cre-lox+activated+viral+membrane+fusion+assay+identifies+pharmacological+inhibitors+of+HIV+entry.">A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.</a> Virology. 490, 6-16 (2016).</li><li>Cavrois, M., De Noronha, C., Greene, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+specific+enzyme-based+assay+detecting+HIV-1+virion+fusion+in+primary+T+lymphocytes.">A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes.</a> Nature Biotechnology. 20, 1151-1154 (2002).</li><li>Huerta, L., Lamoyi, E., Baez-Saldana, A., Larralde, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+envelope-dependent+cell-cell+fusion:+a+quantitative+fluorescence+cytometric+assay.">Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.</a> Cytometry. 47, 100-106 (2002).</li><li>Sakamoto, T., 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=Establishment+of+an+HIV+cell-cell+fusion+assay+by+using+two+genetically+modified+HeLa+cell+lines+and+reporter+gene.">Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene.</a> Journal of Virological Methods. 114, 159-166 (2003).</li><li>Herschhorn, A., 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=An+inducible+cell-cell+fusion+system+with+integrated+ability+to+measure+the+efficiency+and+specificity+of+HIV-1+entry+inhibitors.">An inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of HIV-1 entry inhibitors.</a> PLoS One. 6, e26731 (2011).</li><li>Swartz, T. H., Esposito, A. M., Durham, N. D., Hartmann, B. M., Chen, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X-selective+purinergic+antagonists+are+strong+inhibitors+of+HIV-1+fusion+during+both+cell-to-cell+and+cell-free+infection.">P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.</a> Journal of Virology. 88, 11504-11515 (2014).</li><li>Marin, M., 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=High-Throughput+HIV-Cell+Fusion+Assay+for+Discovery+of+Virus+Entry+Inhibitors.">High-Throughput HIV-Cell Fusion Assay for Discovery of Virus Entry Inhibitors.</a> Assay and Drug Development Technologies. 13, 155-166 (2015).</li><li>Giroud, 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=Screening+and+Functional+Profiling+of+Small-Molecule+HIV-1+Entry+and+Fusion+Inhibitors.">Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors.</a> Assay and Drug Development Technologies. 15, 53-63 (2017).</li><li>Hubner, W., 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=Sequence+of+human+immunodeficiency+virus+type+1+(HIV-1)+Gag+localization+and+oligomerization+monitored+with+live+confocal+imaging+of+a+replication-competent,+fluorescently+tagged+HIV-1.">Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1.</a> Journal of Virology. 81, 12596-12607 (2007).</li><li>Koo, B. K., 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=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nature Methods. 9, 81-83 (2011).</li><li>Pear, W. S., 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=Production+of+high-titer+helper-free+retroviruses+by+transient+transfection.">Production of high-titer helper-free retroviruses by transient transfection.</a> Proceedings of the National Academy of Sciences. 90, 8392-8396 (1993).</li><li>Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pCL+vector+system:+Rapid+production+of+helper-free,+high+titer,+recombinant+retroviruses.">The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses.</a> Journal of Virology. 70, 5701-5705 (1996).</li><li>Kingston, R. E., Chen, C. A., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Phosphate+Transfection.">Calcium Phosphate Transfection.</a> Current Protocols in Immunology. 10, 10-13 (2001).</li></ol>

The authors have nothing to disclose.

The Gag-iCre assay has proven to be very useful for screening drug candidates which may inhibit the fusion step of viral replication. When performing this assay, the most important steps to get a good signal are similar to most viral infection assays. The first critical step is producing high titers of good-quality virus. This step requires that the 293T cells are passaged frequently (at least 1x every 48 h) so they do not become overconfluent and clump together. Additionally, it may be worth experimenting with different...

The need for novel antiretroviral therapies has prompted the developement of&#160;high-throughput screens for&#160;inhibitors of HIV-1 entry. The Gag-iCre reporter assay is developed to identify inhibitors of viral entry at the fusion step in a cell-to-cell infection system by specifically measuring viral membrane fusion with the host cell membrane1. An assay was developed to&#160;screen for novel inhibitors that acted specifically at the early stages of HIV-1 infection up to the point of viral membrane fusion. One challenge to measuring cell-cell infection is that the initial inoculum contains infected donor cells and ininfected&#160;target cells, so measuring new infection is difficult to discriminate from the input cells. The ideal system would involve a heterologous gene marker that can be induced by the initiation of a target cell infection and is not present in the donor cell. A popular viral contents mixing assay based on a viral protein-enzyme fusion,&#160;BlaM-Vpr, can be effective, but has&#160;limitations for high throughput, cell-cell screening2. This assay involves a beta-lactamase (BlaM) reporter gene that is fused to HIV-1 Vpr, packaged into virions, and delivered into target cells upon viral membrane fusion. The substrate CCF2-AM is loaded into the cytoplasm of target cells and undergoes a fluorescence shift upon cleavage. The CCF2-AM substrate is expensive and can be prohibitive for high-throughput screens. In order to distinguish donor and target cells, it is necessary to dye-label the target populations. Finally, the protocol requires several wash and incubation steps which can be burdensome and costly when testing large numbers of compounds.
The Gag-iCre assay was developed as a solution to these issues, to develop a high-throughput screening for inhibitors of fusion in cell-to-cell transmission. This system does not require&#160;substrate to be loaded into cells. The assay can also be used for cell-free studies and is adaptable to other viral fusion studies using pseudo-typed HIV-1 Gag-iCre virus particles. HIV Env mediated cell-cell fusion assays can measure virus Env protein-mediated cell fusion, however these do not use actual virus particles as the HIV-1 Gag-iCre assay does3,4,5. This assay can be read with&#160;flow cytometry or fluorescence microscopy. It has been successfully used to screen the FDA library as well as a small library of purinergic inhibitors1,6. Other investigators have also identified purinergic inhibitors in HIV-1 fusion using an adapted and optimized high-throughput fusion assay that reports the transfer of virus-encapsulated beta-lactamase into the cytoplasm7,8.
The Gag-iCre virus was designed with a similar approach to the Gag-iGFP virus, inserting Cre recombinase between matrix and capsid, flanked by HIV-1 protease sites9. The Cre enzyme is made as a precursor inserted within the Gag polyprotein that matures when HIV-1 protease is activated within the nascent virus particle. The Cre delivery is, thus, dependent upon the delivery of the contents of a protease-activated HIV-1 particle into a target cell via a viral membrane fusion process. Two versions of the protocol are provided here. The first uses an HIV-1 infected population to infect target cells, to study direct cell-to-cell transmission of HIV. The second version uses a cell-free virus to study a cell-free viral infection. The cell-to-cell transmission assay takes 7 days to complete from the day the cells are thawed, or 5 days if the cells are already thawed and passaged. The cell-free infection assay can be performed&#160;in 5 days if the cells need to be thawed and 3 days if the cells are thawed and passaged. Instructions are also given to generate an RG (red-to-green) target cell line in the desired cell type (if a preexisting RG target cell line is not being used) using a plasmid created by the Clevers Lab10. It is recommended that appropriate biosafety precautions are taken with the virus and the virus-expressing cells in this assay. We conduct the infectious portion of this assay in a BSL2+ tissue culture facility. After the cells are fixed, they can be analyzed in standard flow cytometry and microscopy facilities.
Here we describe the application of this assay to screen for novel compounds that inhibit HIV-1 viral membrane fusion (Figure 1). Purinergic receptors are pro-inflammatory mediators. Our laboratory has demonstrated that non-selective inhibitors of purinergic receptors act as inhibitors of HIV-1 viral membrane fusion6. We report that the utilization of this assay in high throughput can identify novel HIV-1 viral membrane fusion inhibitors. We demonstrate that an inhibitor of the purinergic class of receptors represents a novel class of HIV-1 viral membrane fusion inhibitors.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Sigma Aldrich</td><td>D5546</td><td>Media for 293 Cells</td></tr><tr><td>RPMI-1640</td><td>Sigma Aldrich</td><td>R0883</td><td>Media for Jurkats</td></tr><tr><td>Fetal Bovine Serum Albumin</td><td>Gibco</td><td>16140-071</td><td>Serum for 293 Cells</td></tr><tr><td>Cosmic Calf Serum</td><td>Hyclone</td><td>SH30087.03</td><td>Serum for Jurkat Cells</td></tr><tr><td>Hyclone Pennecillin Streptomycin solution</td><td>GE Healthcare Life sciences</td><td>SV30010</td><td>Penn/Strep used in both media</td></tr><tr><td>T75 Flasks</td><td>Corning</td><td>3073</td><td>Used for Culture of Jurkat Cells</td></tr><tr><td>10 cm Tissue Culture Plates</td><td>Corning</td><td>430167</td><td>Used for Culture of 293 Cells</td></tr><tr><td>96 Well Plates (tissue culture Treated)</td><td>Corning</td><td>3595</td><td>Used for fusion assay</td></tr><tr><td>Polyjet Transfection Reagent</td><td>Signagen</td><td>SL100688</td><td>Used to transfect 293 Cells</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma Aldrich</td><td>D8537-100ML</td><td>Used in wash steps</td></tr><tr><td>Hyclone Trypsin Protease</td><td>GE Healthcare Life sciences</td><td>SH30042.01</td><td>For Trypsinization of 293 cells</td></tr><tr><td>Amaxa Cell Line Nucleofector Kit V</td><td>Lonza</td><td>VACA-1003</td><td>For Nucleofection of Jurkats</td></tr><tr><td>Ficoll-Paque plus</td><td>GE Healthcare Life sciences</td><td>17144002</td><td>For Nucleofection of Jurkats</td></tr><tr><td>Serological Pipettes</td><td>Fisher Brand</td><td>13-678-11E</td><td>For all tissue culture</td></tr><tr><td>Pipettor Tips</td><td>Denville Scientific</td><td>P3020-CPS</td><td>For all tissue culture and liquid handling steps</td></tr><tr><td>Millex Syringe Filter (0.45 &amp;mu;m)</td><td>Millipore</td><td>SLHA033</td><td>For filtration of virus</td></tr><tr><td>BD Slip Tip Sterile syringes</td><td>BD Diagnostics</td><td>309656</td><td>For filtration of virus</td></tr><tr><td>Amaxa Nucleofector</td><td>Lonza</td><td>2b</td><td>for Nucleofection of Jurkats (various models available)</td></tr><tr><td>BD Fortessa Flow Cytometer</td><td>BD Biosciences</td><td></td><td>for flow cytometry analyss of samples</td></tr><tr><td>Tissue Culture Hood</td><td>Various models</td><td>Fortessa 2</td><td></td></tr><tr><td>pMSCV-loxp-dsRed-loxp-eGFP-Puro-W PRE</td><td>Addgene</td><td>32702</td><td>&lt;strong&gt;Koo BK&lt;em&gt; et al.&lt;/em&gt;&lt;/strong&gt; Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods &lt;strong&gt;9:&lt;/strong&gt;81-83 (2011).</td></tr><tr><td>pCL10a1</td><td>Novus Bio</td><td>NBP2-29542</td><td>&lt;strong&gt;Naviaux, RK, Costanzi, E, Haas, M and Verma, I&lt;/strong&gt;. The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses. Journal of Virology &lt;strong&gt;70&lt;/strong&gt;: 5701-5705 (1996)</td></tr><tr><td>Gag-iCre</td><td>Benjamin Chen Lab</td><td></td><td>&lt;strong&gt;Esposito AM&lt;em&gt; et al.&lt;/em&gt; &lt;/strong&gt;A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry. Virology &lt;strong&gt;490:&lt;/strong&gt;6-16 (2016).</td></tr></tbody></table>

a high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of hiv-1 viral membrane fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

Uninfected RG Jurkat Cells exhibit a low level of background GFP signal (0.3%) with a very strong RFP signal (Figure 2A, uninfected column). The infection with Gag-iCre causes an increase in GFP signal (24.9%,) while the presence of the HIV-1 fusion inhibitor AMD3100 (20 &#181;M) inhibits the development of this signal, bringing it down to uninfected background levels (0.34%). When an inhibitor of a post-fusion event such as the reverse transcription inhibito...

Watch this Scientific Journal Video about A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion at JoVE.com

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

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Immunology and Infection

6.3K Views.  Icahn School of Medicine at Mount Sinai, Immunology Institute. We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

Video: A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

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 ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

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

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

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

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

Biology

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

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

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

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

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

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

Test przyłączania wirusa do badań przesiewowych związków przeciwwirusowych

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

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Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

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

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

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

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

Test przyłączania wirusa do badań przesiewowych związków przeciwwirusowych