This research was supported by the Intramural Research Program of the NCI.

1. Preparation of Master Stocks
<ol>
	<li>Make master stocks of the compounds to be tested in DMSO. Prepare stocks at a standard concentration of 20 mM. 
	Note: any concentration above 10 mM can be used. Compounds that can be used as positive controls to validate this assay include RAL, EVG, and DTG.</li>
	<li>Ensure the compounds are dissolved in DMSO by vortexing the solutions multiple times for 15 sec and incubating at RT for 1 hr. Store the 20 mM stock solutions in the dark at -20 &#176;C until use.</li>
</ol>
2. Preparation of the 96-well Plates for Compound Screening
<ol>
	<li>Choose the cell line to be tested (e.g. HOS or TZM-bl), and seed 100 &#181;l of those cells at a density of 4 x 104 cells/ml (4,000 cells/well) in media (e.g. Dulbecco&#8217;s modified Eagle&#8217;s or DMEM medium supplemented with 5% (v/v) fetal bovine serum, 5% newborn calf serum, and penicillin (50 units/ml) plus streptomycin).</li>
</ol>
3. Generation of Virus Stocks
Produce VSV-g-pseudotyped HIV by transfecting 293 cells (as shown in Figure 1, step 1)32-34.
<ol>
	<li>On the day prior to transfection, plate 293 cells on 100 mm diameter dishes at a density of 1.5 x 106 cells.</li>
	<li>On the day of transfection, transfect 293 cells with 16 &#181;g of wild type or mutant HIV (pNLNgoMIVR-&#916;LUC) and 4 &#181;g of VSV (pHCMV-g) using the calcium phosphate method35.</li>
	<li>Approximately 6 hr&#160; after the calcium phosphate precipitate is added, wash 293 cells twice with phosphate buffered saline (PBS) and incubate with fresh media for 48 hr. [DMEM &#160;supplemented with 5% (v/v) fetal bovine serum, 5% newborn calf serum, and penicillin (50 units/ml) plus streptomycin (50 &#181;g/ml)].</li>
	<li>Harvest the virus containing supernatants by removing the media from the 100 mm diameter dishes, clarify the supernatants by low speed centrifugation at 3,000 rpm for 10 min , filter the supernatants through a 45 &#181;m&#160;pore&#160;size syringe filter, treat the supernatants with Turbo DNase for 30 min at RT and dilute the supernatants in media for preparation in infection assays. Store the viral supernatants frozen, in aliquots, at -80 &#176;C. 
	Note: the amount of p24 in the supernatant is determined by using a HIV-1 p24 enzyme-linked immunosorbent assay kit. The p24 concentration is used to control the amount of virus in the sample. Approximately 500 ng of virus is added to HOS cells plated on 60 mm diameter dishes at a density of 1.5 x&#160;105 cells/dish on the day prior to infection. After a 48 hr&#160; of incubation, the cells are harvested, collected by centrifugation, washed, and resuspended in 100 &#181;l of PBS. Add an equal amount of Luminescence reporter gene assay reagent and measure luciferase activity as described in sections 5.4.1 and 5.4.2. From this, an appropriate dilution of the virus can be made as discussed in step 4.6.</li>
</ol>
4. Compound Screening in 96-well Plates
Screen each compound in triplicate and average the results.
Note: the effect of each compound on viral replication is corrected by normalizing to the level of replication obtained in the absence of any compound.
<ol>
	<li>Determine the empirical concentration range to be screened. 
	Note: typically, screens with 11 serial dilutions are made by adding the compound to the plate column by column and screens with 7 serial dilutions are made by adding the compound by row. One triplicate set of wells must be reserved for the no compound control. In addition, one column or one row must remain blank to act as the negative/background control.&#160;Lastly, whether it is a cellular cytotoxicity or infectivity assay will dictate if virus is added.&#160;</li>
	<li>Prepare serial dilutions from the 20 mM stock solution. The concentrations are chosen depending on the empirically determined range of concentrations to be tested (as shown in Table 1). Prepare the dilutions in media at 10x the final concentration desired, i.e. if the final concentration is going to be 100 &#181;M, make a 1 mM working stock. 
	Note: compounds with IC50s above 5-10 &#956;M are not usually good candidates for drug development. In the initial assays we test the compounds only against the WT vector. Promising compounds that effectively inhibit the WT vector are then tested against a panel of drug resistant mutants.&#160;</li>
	<li>Remove the 96-well plates from the incubator and add the serial dilutions of the compounds to be tested to the wells in triplicate (as shown in Figure 1, step 2). 
	Note: the volume added to the well should be 1/10 volume of the final concentration. Thus, add 22 &#181;l/well (final volume post virus addition is 220 &#181;l) for infectivity assays. For cytotoxicity assays, add 11 &#181;l/well (final volume is 110 &#181;l/well).</li>
	<li>Return the 96-well plates to the incubator. For a cellular cytotoxicity screen, incubate the 96-well plates 48 hr&#160; at 37 &#176;C and no further manipulations are needed in Protocol 4: proceed to Protocol 5. For infectivity assays, continue following the instructions in Protocol 4.</li>
	<li>Infectivity assays only. Remove the plates from the incubator after a&#160;minimum of 3 hr&#160;incubation at 37 &#176;C with the compounds to be analyzed. 
	Note: this allows the compound to be taken up by the cells prior to infection with the HIV vector.</li>
	<li>Prepare a stock dilution of the virus33,34 (usually approximately 1:3) that will produce a luciferase signal between 0.2-1.5 relative luciferase units (RLUs) in untreated cells. An entire plate will take approximately 10 ml of diluted virus. Add 100 &#181;l of virus to each of the wells, using either an 8 or 12 multichannel pipettor. Do not add virus to the negative/background control wells. Return the plates to the 37 &#176;C incubator for 48 hr. 
	Note: a 1:3 stock dilution of the virus is typical dilution that will produce a luciferase signal between 0.2 -1.5 RLUs based on a p24 assay showing that the viral concentration in the supernatant is approximately 500 ng in 1.0 ml.</li>
</ol>
5. Preparation and Measurement of Cytotoxicity and Infectivity in 96-well Plates
<ol>
	<li>Aspirate the media from the wells (phenol red in the media can interfere with the luciferase signal). Use a glass pipette with a 200 &#181;l pipette tip attached to the end. Start at the top of the media and slowly work down toward the bottom corner of the well. Do not spend too much time at the bottom of the well, or cells can be removed from the well.</li>
	<li>Add 100 &#181;l of PBS supplemented with 0.5 mM MgCl2 to each well. This needs to be done immediately after the media is removed so that the cells do not dry out.</li>
	<li>Only for cytotoxicity assays, add 5 ml of substrate buffer from the Luminescence ATP detection assay to each vial of supplied lyophilized reagent. One vial is sufficient for a 96-well plate.
	<ol>
		<li>Add 50 &#181;l of cell lysis buffer from the Luminescence ATP detection assay to each well. Shake the 96-well plate at 700 rpm at RT for 5 min using a compact thermomixer.</li>
		<li>Add 50 &#181;l of reconstituted Luminescence ATP detection assay reagent to all wells except for the negative control/background wells. Shake at 700 rpm at room temperature for 5 min using a compact thermomixer. Incubate the plates at RT for 20 min&#160; to allow time for signal development.</li>
		<li>Read the 96-well plate using the microplate luminometer. 
		Note: open the SoftMax Pro microplate luminometer program. Make sure the luminometer is set to measure luminescence at a sensitivity of 5 readings/well.</li>
	</ol>
	</li>
	<li>Only for infectivity assays, add 10 ml of substrate buffer from the Luminescence reporter gene assay to each vial of supplied lyophilized reagent. One vial is sufficient for each 96-well plate.
	<ol>
		<li>Add 100 &#181;l of reconstituted Luminescence reporter gene assay reagent to each well. Incubate at room temperature for 20 min&#160; to allow time for signal development.</li>
		<li>Read the 96-well plate using a microplate luminometer as performed in section 5.3.3.</li>
	</ol>
	</li>
</ol>
6.&#160;Determination of CC50 and IC50 Values for Compounds
<ol>
	<li>Transfer the luciferase data from the microplate luminometer into an excel spreadsheet.
	<ol>
		<li>Average both the luciferase data in triplicate and the background/control signal data. Subtract the average background/control signal from the average triplicate signal for the entire concentration range.</li>
		<li>Normalize the background corrected average signal for the concentration ranges against the activity, whether it is cytotoxicity or infectivity, in the absence of any compound to determine the percent inhibition. 
		Note: percent inhibition is defined as the luciferase activity in the presence of drug divided by the luciferase activity in the absence of drug multiplied by 100.&#160;</li>
	</ol>
	</li>
	<li>Use Kaleidagraph software program to obtain CC50 and IC50 values
	<ol>
		<li>Transfer both the empirically determined concentration range and percent inhibition of luciferase activity into Kaleidagraph.</li>
		<li>Plot the data with the concentration range on the x&#160;axis and the percent inhibition of luciferase activity on the y&#160;axis.</li>
	</ol>
	</li>
</ol>

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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=A+novel+polymorphism+at+codon+333+of+human+immunodeficiency+virus+type+1+reverse+transcriptase+can+facilitate+dual+resistance+to+zidovudine+and+L-2',3'-dideoxy-3'-thiacytidine.">A novel polymorphism at codon 333 of human immunodeficiency virus type 1 reverse transcriptase can facilitate dual resistance to zidovudine and L-2',3'-dideoxy-3'-thiacytidine.</a> J. Virol. 72, 5093-5098 (1998).</li><li>Butler, S. L., Hansen, M. S., Bushman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+HIV+DNA+integration+in+vivo.">A quantitative assay for HIV DNA integration in vivo.</a> Nat. Med. 7, 631-634 (2001).</li><li>Brussel, 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=Longitudinal+monitoring+of+2-long+terminal+repeat+circles+in+peripheral+blood+mononuclear+cells+from+patients+with+chronic+HIV-1+infection.">Longitudinal monitoring of 2-long terminal repeat circles in peripheral blood mononuclear cells from patients with chronic HIV-1 infection.</a> AIDS. 17, 645-652 (2003).</li><li>Daelemans, D., Pauwels, R., De Clercq, E., Pannecouque, 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+time-of-drug+addition+approach+to+target+identification+of+antiviral+compounds.">A time-of-drug addition approach to target identification of antiviral compounds.</a> Nat. Protoc. 6, 925-933 (2011).</li></ol>

The authors have nothing to disclose.

We describe a quick, efficient, and reproducible assay that can be used to screen compounds for cytotoxicity and for their ability to inhibit the replication of both WT and drug resistant HIV-1. The ability to quickly identify compounds and test their efficacy and cytotoxicity is crucial in the development of new and improved drugs against HIV-1. Once lead compounds are identified, analogues of the lead compound can be produced and tested using the same assay. The assay is relatively simple. There are both positive and negative controls that allow the user to diagnose the most common problems (toxic compounds, problems with the vector stock). The use of a triplicate set of wells with no added compound shows that viral infection has occurred. The fact that cytotoxicity is measured in an independent assay avoids misinterpreting a reduction in luciferase caused by cytotoxicity as a specific effect on viral replication.
The critical steps in the protocol are preparing the plates so that the cells are uniformly distributed in the wells, that the wells have the proper concentrations of the compounds to be tested, adding the same amount of virus to each of the wells, and measuring the luciferase activity to determine the CC50 and IC50 values.
The assay is safe, quantitative, and reproducible. The assay is safe because the vector is replication defective. The assay is quantitative and reproducible because it is based on a single round vector that expresses luciferase, which can be assayed accurately and conveniently. In a multi round virus replication assay, the measured IC50 depends on the number of viral life cycles; this is a particular problem when the assays involve both WT and drug resistant viruses that may have significantly different replication capacities.
There have been several enzymatic assays reported previously that can be used to screen for IN inhibitors. Assays that involve real time PCR technology to measure integrated DNA require purified recombinant proteins(s), and are, in general, both more labor intensive and expensive36,37. Although it is possible to use enzymatic assays to measure the impact of compounds on the other viral enzymes (RT and protease), each enzyme requires its own assay system. The one round vector assay, as described, can be used, without modification, to screen for RT inhibitors. A similar assay can be used to screen for protease inhibitors; however, in a protease inhibitor assay, the compounds must be added to the cells used to produce the vectors. A related assay, using different cells and vectors, can also be used to screen for envelope (env) and HIV entry fusion inhibitors. Lastly, the assay can be used, on a larger scale, with automated robotic dispensers. Thus, the assay can be used to screen large libraries of compounds against WT and mutant HIV. However, the fact the assay can detect an inhibitor of HIV replication that acts at different stages of the viral life cycle points to a limitation in interpreting the data. By itself the assay does not define which step in the life cycle is blocked by a compound. If this question arises, it can be resolved by using time of addition assays38, and by testing the compound against purified recombinant viral proteins.
Unfortunately, despite the success of anti HIV drugs, there are still problems with both resistance and toxicity. In the absence of an effective anti HIV vaccine, there is a need not only to develop new therapeutic drugs that will be effective against the existing drug resistant mutants, but also to develop prophylactic drugs that can reduce the spread of the virus. If the prophylactic use of anti HIV drugs incudes the treatment of uninfected people, this approach will place a special burden on developing drugs that are have little or no long term toxicity.

The availability of drugs targeting several essential steps in the HIV-1 viral life cycle has led to combination drug therapy (called highly active anti retroviral therapy, or HAART) that has greatly improved the treatment of HIV-1 infections, and the long term survival of patients.&#160;HAART, which typically uses combinations of two nucleoside reverse transcriptase (RT) inhibitors and either a protease inhibitor or a non nucleoside RT inhibitor, has now converted a deadly disease into a life long condition1-5. However, despite the many successes of HAART in sustained suppression of viral replication, it has limitations. HAART does not eradicate HIV, so the patients are not cured and therapy is life long. There are problems with drug toxicity and with the emergence of drug resistant strains. Resistance can arise to all of the approved anti HIV drugs, including the newly approved drugs that target HIV integrase (IN). Drug resistance likely arises because of spontaneous mutations that occur during viral replication (error rate of 3 x 10-5 mutations/base/replication cycle)6. When mutations arise in the genes coding for the targets of anti retroviral drugs, a small subset of these mutations will lead to a reduction in susceptibility of the viral strain to the drugs. To avoid the development of drug resistance, drug concentrations must be maintained at levels that fully suppress HIV replication. Poor adherence to the therapeutic regimen exacerbates the problem, and can lead to the rapid development of resistance7-9. Although drug concentrations can vary in patients due to differing absorption, metabolism, distribution, and excretion levels, high drug concentrations can lead to toxicity10. Since therapy continues for the life of the patient, there are serious safety concerns about the long term toxicity of anti retrovirals. The anti retrovirals commonly used in HAART can have adverse effects and there have been incidences where the side effects have been life threatening11-15. The problems patients encounter with the development of resistance and toxicity underlie the need to develop new drugs that effectively block the replication of the common drug resistant strains of the virus with little or no long term toxicity.
Thus, there is a need for an assay that can screen for compounds that block essential steps in the viral life cycle quickly. Here we describe an efficient assay that can be used to evaluate the cytotoxicity of a compounds and their ability to block the replication of both WT and drug-resistant HIV strains rapidly and efficiently. The assay we use is similar to an assay that was developed to screen for drug resistance in virus isolated from patients16-18.
Although the assay can be used without modification to screen for compounds that can block HIV reverse transcription, we will describe using the assay to evaluate IN inhibitors. IN is an essential viral enzyme that inserts the viral DNA into the cellular genome19. Although a number of promising IN inhibitors are being developed, some of which are currently undergoing clinical trials, only Isentress20,21 (also known as Raltegravir or RAL) and more recently, Elvitegravir (EVG)22 and Dolutegravir (DTG)23 have been approved by the FDA.&#160;These compounds are active against both HIV B and non B subtypes in cell culture and in patients24,25. However, treatment with RAL selects for drug-resistant HIV-1 IN mutants, including Y143R, N155H, and G140S/Q148H24,26-31. N155H and G140S/Q148H also reduce the efficacy of EVG, which emphasizes the need to design and develop second generation IN strand transfer inhibitors (INSTIs) that are effective against these resistance mutations.

<table border="0" cellpadding="0" cellspacing="0" width="659">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:157px;">Corning Cellgro</td>
			<td style="width:119px;">10-017</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ATPlite Luminescence ATP Detection Assay System</td>
			<td style="width:157px;">Perkin Elmer</td>
			<td style="width:119px;">6016941</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Steady Lite Plus High Sensitivity Luminescence Reporter Gene Assay System</td>
			<td style="width:157px;">Perkin Elmer</td>
			<td style="width:119px;">6016751</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s PBS</td>
			<td style="width:157px;">Gibco-Life Technologies-Invitrogen</td>
			<td style="width:119px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SpectraMax Gemini EM</td>
			<td style="width:157px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KaleidaGraph</td>
			<td style="width:157px;">Synergy Software</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc F96 Microwell White Polystyrene Plate</td>
			<td style="width:157px;">Thomas Scientific</td>
			<td style="width:119px;">12-566-26</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Thermomixer Compact</td>
			<td style="width:157px;">Sigma Alrich</td>
			<td style="width:119px;">T1442-1EA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Turbo Dnase</td>
			<td style="width:157px;">Ambion-Life Technologies-Invitrogen</td>
			<td style="width:119px;">AM2238</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Millex HA Filter Unit, 0.45 &#181;M</td>
			<td style="width:157px;">Millipore</td>
			<td style="width:119px;">SLAHA033SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alliance HIV-1 p24 Elisa Kit</td>
			<td style="width:157px;">Perkin Elmer</td>
			<td style="width:119px;">NEK050B001KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HOS cells</td>
			<td style="width:157px;">ATCC</td>
			<td style="width:119px;">CRL-1543</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TZM-bl cells</td>
			<td style="width:157px;">NIH AIDS Reagent Program</td>
			<td style="width:119px;">8129</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">pNL4.3&#916;Env.LUC</td>
			<td style="width:157px;">NIH-NCI HIV Drug Resistance Program- Hughes Lab</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">VSV-G</td>
			<td style="width:157px;">NIH-NCI HIV Drug Resistance Program- Hughes Lab</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SoftMax Pro</td>
			<td style="width:157px;">Molecular Devices</td>
			<td style="width:119px;">0200-310</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

If the assay (Figure 1, steps 1 and 2) was successfully performed, then the luciferase values should resemble the data presented in Table 2. Scan across the concentration range; a potentially potent compound will reveal increasing luciferase activity from left to right, and the control should have the highest luciferase activity. If the luciferase activity does not exceed 0.1 Relative Luciferase Units (RLUs) across the concentration range, this usually indicates that the compound killed the cells. If the luciferase data is greater than or equal to 2.0 RLUs across all the serial dilutions, then the compounds were not able to inhibit HIV-1 infections at the tested concentrations.
Plotting the concentration of the compounds versus the percent inhibition of luciferase activity in Kaleidagraph (Table 3, part A), after performing linear regression analysis, will produce results similar to those shown in Table 3, part B.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51400/51400fig1highres.jpg" width="500" /> 
Figure 1.&#160;Preparation of HIV-1 Viral Stocks and the Setup of Cellular Cytotoxicity and Single-round Infectivity Assays. In step 1, 293T cells are transfected with pNL4.3&#916;Env.LUC and VSV-G and incubated for 48 hr&#160; to produce virus34. The virus is harvested and stored (frozen at -80 &#176;C in aliquots) until it is used in the infectivity assays. For step 2, HOS cells are seeded in a 96-well plate and incubated for 24 hr. The cells are then preincubated with serial dilutions of the compounds to be tested for 3 hr&#160; and then infected with virus (either WT or drug resistant). After a 48 hr incubation, luciferase activity is measured.
<img alt="Table 1" fo:content-width="5in" src="/files/ftp_upload/51400/51400table1.jpg" width="300" /> 
Table 1. Drug Screening Serial Dilution Prototype. A more stringent screening involves 11 serial dilutions which typically start at 10 &#181;M and end at 0.0005 &#181;M. The serial dilutions are prepared 10x; there are 100 &#181;l&#160;of cells and 100 &#181;l&#160;of virus in each well). At this volume and following these calculations, the serial dilutions will be enough for 3 rows of an entire 96-well plate. The cytotoxicity assays are prepared similarly; however the 11 serial dilutions start at 250 &#956;M and end at 0.05 &#956;M. Only 11 &#956;l&#160;of the dilutions are added to the wells in the plate that contain 100 &#956;l&#160;of cells. <a href="https://www.jove.com/files/ftp_upload/51400/51400table1.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 2" fo:content-width="6in" src="/files/ftp_upload/51400/51400table2.jpg" width="600" /> 
Table 2. Luciferase Signal data Readout and Determination of Percent Inhibition of Luciferase Activity. The data table shows a typical set of luciferase data for a successful compound. The table also shows the additional calculations needed to determine the percent inhibition of luciferase activity and the CC50 and IC50 values. <a href="https://www.jove.com/files/ftp_upload/51400/51400table2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 3" fo:content-width="6in" src="/files/ftp_upload/51400/51400table3.jpg" width="600" /> 
Table 3. Linear Regression Analysis Data Table. Part A. Graphing the concentration range used versus the percent inhibition of luciferase activity in Kaleidagraph will produce the appropriate inhibition curves. Part B, The inhibition curves are defined by the 3 parametric sigmoidal function &#160;and fit to the data by linear regression analyses18. This data table is then used in conjunction with Microsoft Excel to calculate the drug concentrations required to inhibit virus integration and cellular cytotoxicity by 50%, e.g. IC50 and CC50. <a href="https://www.jove.com/files/ftp_upload/51400/51400table3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors at JoVE.com

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

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 ...

rapid-screening-of-hiv-reverse-transcriptase-and-integrase-inhibitors

Research

JoVE Journal

Immunology and Infection

17.7K vistas.  National Cancer Institute.  Aquí se describe la citotoxicidad celular y ensayos de infectividad redondas individuales que permiten la detección rápida y precisa de los compuestos para determinar su citotoxicidad celular (CC 50) y los valores de IC50 contra WT y VIH-1 resistente drogas. 

Video: Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.