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><tbody><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>DMEM</td><td>Corning Cellgro</td><td>10-017</td><td></td></tr><tr><td>ATPlite Luminescence ATP Detection Assay System</td><td>Perkin Elmer</td><td>6016941</td><td></td></tr><tr><td>Steady Lite Plus High Sensitivity Luminescence Reporter Gene Assay System</td><td>Perkin Elmer</td><td>6016751</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Gibco-Life Technologies-Invitrogen</td><td>14190-136</td><td></td></tr><tr><td>SpectraMax Gemini EM</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>KaleidaGraph</td><td>Synergy Software</td><td></td><td></td></tr><tr><td>Nunc F96 Microwell White Polystyrene Plate</td><td>Thomas Scientific</td><td>12-566-26</td><td></td></tr><tr><td>Eppendorf Thermomixer Compact</td><td>Sigma Aldrich</td><td>T1442-1EA</td><td></td></tr><tr><td>Turbo DNase</td><td>Ambion-Life Technologies-Invitrogen</td><td>AM2238</td><td></td></tr><tr><td>Millex HA Filter Unit, 0.45 &amp;micro;M</td><td>Millipore</td><td>SLAHA033SS</td><td></td></tr><tr><td>Alliance HIV-1 p24 Elisa Kit</td><td>Perkin Elmer</td><td>NEK050B001KT</td><td></td></tr><tr><td>HOS cells</td><td>ATCC</td><td>CRL-1543</td><td></td></tr><tr><td>TZM-bl cells</td><td>NIH AIDS Reagent Program</td><td>8129</td><td></td></tr><tr><td>pNL4.3&amp;Delta;Env.LUC</td><td>NIH-NCI HIV Drug Resistance Program- Hughes Lab</td><td></td><td></td></tr><tr><td>VSV-G</td><td>NIH-NCI HIV Drug Resistance Program- Hughes Lab</td><td></td><td></td></tr><tr><td>SoftMax Pro</td><td>Molecular Devices</td><td>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 Views.  National Cancer Institute. 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.

Video: Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Genetics

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

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 ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

Medicine

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 ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Assembly and Purification of Prototype Foamy Virus Intasomes

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

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

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

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.

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 ...

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 ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Summary

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Chapters in this video

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

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

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

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

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Assembly and Purification of Prototype Foamy Virus Intasomes

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

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