We would like to acknowledge the staff and scientists at SBI for their support in the development and testing of these protocols.

1. Transfection of 293TN Cells with Packaging Plasmids and the Expression Construct
<ol>
 <li> Seed 7.0 - 8.0 x 106 293TN cells per 15 cm2 culture plate in 20 ml of culture medium containing DMEM medium supplemented with 4 mM L-glutamine, 4.5 g/l glucose, and fetal bovine serum (10%) without antibiotics. Grow for 18-24 hours at 37 &deg;C with 5% CO2 so that the cell density reaches 60-80% confluency at the time of transfection. In some cases, it may be necessary to seed several plates of cells to obtain a high enough titer for transduction of target cells.</li>
 <li> For each plate of cells, add 1.6 ml serum-free DMEM to an autoclaved 2 ml Eppendorff tube. Add 45 &mu;l pPACKH1 or pPACKF1 and 4.5 &mu;g of your lentivector construct to the DMEM and mix by pipetting up and down. </li>
 <li> Add 55 &mu;l of PureFection into the same tube. Vortex for 10 seconds. Incubate the DMEM-Plasmid-PureFection mixture at room temperature for 15 min. </li>
 <li> Add the DMEM-Plasmid-PureFection mixture to the tissue culture plate drop-wise and swirl gently to evenly disperse throughout the plate. Return the dish to the incubator and grow at 37 &deg;C with 5% CO2.</li>
 <li> There is no need to change the media after transfection. If your lentivector construct expresses a gene encoding a fluorescent protein like GFP, check your cells under a microscope 12-24 hours after transfection. You should be able to see >90% GFP-positive cells, indicating that the transfection was successful.</li>
 <li> Collect the cell culture supernatant after 48 and 72 hours into a 50 ml sterile centrifuge tube. This cell culture supernatant now contains infectious pseudoviral particles. (Follow the recommended guidelines for working with BSL-2 safety class.) Centrifuge at 1,500 x g for 15 min at room temperature to pellet the cellular debris. Transfer the viral supernatant to a new tube, ensuring not to disturb the pellet. </li>
 <li> 293TN cells that have been used for viral packaging should be disposed of in a biohazard container and are not to be used for further rounds of viral packaging. </li>
</ol>
2. Concentration of the Viral Supernatant
<ol>
 <li> Add 1 volume of cold (4 &deg;C) PEG-it Virus Precipitation Solution to every 4 volumes of lentiviral particle-containing supernatant. Precipitation of lentiviral particles from large volumes can be achieved with Corning 250 ml polypropylene centrifuge tubes. Mix the solution by inverting the tubes several times, but do not vortex the mixture.</li>
 <li> Refrigerate the solution at 4 &deg;C for at least 12 hours (up to 4 days is acceptable). Do not shake or rotate the tubes during the refrigeration step. </li>
 <li> Centrifuge the supernatant/ PEG-it mixture at 1,500 x g for 30 minutes at 4 &deg;C. After centrifugation, the lentiviral particles will appear as a beige or white pellet at the bottom of the tube. Pour out the supernatant.</li>
 <li> Remove all traces of fluid by aspiration, taking care not to disturb the precipitated lentiviral particles in the pellet. Traces of residual PEG-it will dilute the virus (thus reducing the titer) but will not affect the integrity of the target cells since PEG-it is inert and not toxic.</li>
 <li> Resuspend and combine the lentiviral particles in 1/100 of the original volume using cold (4 &deg;C), sterile phosphate buffered saline or DMEM containing 25 mM HEPES buffer.</li>
 <li> Aliquot into sterile cryogenic vials and store at -70 &deg;C until ready for use. </li>
</ol>
3. Viral Titering and Transduction of Target Cells
<ol>
 <li> We recommend titering the lentiviral particles before transducing the target cells-of-interest and calculating the appropriate multiplicity of infection (MOI) for the target cells for consistency between experiments. For viral titering, we recommend using HT1080 cells as an easy-to-infect positive control line. Please note that the titering protocol involves transducing HT1080 cells with a small aliquot of the lentiviral particles you have packaged. Once the titer is known, the target cells-of-interest can also be transduced with the produced lentiviral particles. A recommend titering protocol can be found at: 
 <a href="http://www.systembio.com/downloads/manual_titer_kit_web.pdf" target="_blank">http://www.systembio.com/downloads/manual_titer_kit_web.pdf</a> 7.</li>
 <li> Plate 50,000 target cells-of-interest per well in a 24-well plate in cell culture media. Use the media that the cells are normally cultured in. Grow the cells overnight at their specified culture conditions. Cells should be between 50 to 70% confluent on the day of transduction.</li>
 <li> On the day of transduction, aspirate the media from the cells. Combine culture medium with TransDux to a 1 x final concentration (for example, 2.5 &mu;l TransDux to 500 &mu;l of culture medium). Then transfer the TransDux-cell culture medium mixture to each well.</li>
 <li> Pipette the appropriate volume of virus which corresponds to the optimal MOI for the target cells into each well. If the optimum MOI is unknown, we recommend trying a range of different MOIs (such as an MOI of 1, 2 and 5 for easy-to-infect tissue culture cells, and an MOI of 1, 10 and 20 for more difficult-to-infect primary cells). If the concentration of the lentiviral particles is not known (as determined through a virus titering protocol), different volumes of virus can be tried, such as 1, 2 and 5 &mu;l of virus. Virus can remain in contact with the target cells for up to 72 hours. </li>
 <li> Further experiments with the transduced target cells-of-interest may be commenced 72 hours after transduction, once the viral construct has integrated into the host cell genome. Change the medium and passage the target cells-of-interest according to their specific needs. </li>
</ol>
4. Representative Results
Transfection of 293TN cells
The starting density of the 293TN cells is critical for successful viral packaging. 293TN cells must be between 60 -80% confluent the day of transfection (Figure 2). If the 293TN cells are less than 60% confluent, allow them to grow for a few more hours before attempting the transfection. If the 293TN cells are >80% confluent, they should be replated before transfection.
If using a lentivector expressing GFP, at least 90% of the 293TN cells should fluoresce green 24 hours after the transfection, indicating a successful transfection (Figure 3). If less than 90% of the cells are GFP-positive, do not proceed to the next step because this indicates that the transfection was not successful, and the viral titers will be low. Instead, plate new 293TN cells and start over, making sure that the 293TN cells are at the proper cell density before transfecting.
293TN cells may pull off the tissue culture plates 48-72 hours after transfection. This does not negatively affect the production of lentiviral particles.
Titering with HT1080 Cells
Viral titers can be calculated using SBI's Global UltraRapid Titering kit according to the manufacturer's instructions. This system utilizes qPCR to measure viral integration of the WPRE sequence from the lentiviral vector into HT1080 cells and compares this to a standard curve, based on a genomic reference sequence to accurately measure infectious units per ml (Figure 4).
Transduction of Target Cells
If using lentiviral particles expressing a constitutively active GFP marker, GFP positive cells should start to appear within 12-24 hours of transduction, if the transduction reaction is working. GFP expression should continue after the lentiviral construct stably integrates into the host cell genome. If transducing with different MOIs, the GFP fluorescence should approximately match the MOI, where low MOIs show less GFP fluorescence and higher MOIs show increased GFP fluorescence (Figure 5). 
<img src="/files/ftp_upload/3171/3171fig1.jpg" alt="Figure 1" /> 
Figure 1. Overview of the viral production protocol. The lentivector and pPACK packaging plasmids are mixed and co-transfected into 293TN cells. After 48 -72 hours, the viral supernatant is collected, and viral particles are precipitated with PEG-it. After titering the lentiviral particles, they can be used to transduce the target cells-of-interest. <a href="https://www.jove.com/files/ftp_upload/3171/3171fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/3171/3171fig2.jpg" alt="Figure 2" /> 
Figure 2. Phase contrast image of 293TN cells. Cell density should be between 60-80% confluent the day of transfection.
<img src="/files/ftp_upload/3171/3171fig3.jpg" alt="Figure 3" /> 
Figure 3. Fluorescent image of GFP-positive 293TN cells 24 hours after transfection with Purefection, pPACKH1, and a lentivector construct encoding GFP.
<img src="/files/ftp_upload/3171/3171fig4.jpg" alt="Figure 4" /> 
Figure 4. WPRE qPCR results and standard curve obtained using SBI's Global UltraRapid Titering kit to calculate MOI and corresponding titer of packaged viral particles.
<img src="/files/ftp_upload/3171/3171fig5.jpg" alt="Figure 5" /> 
Figure 5. Fluorescent images of HT1080 cells transduced with increasing amounts of lentivirus. Images are from 72 hours post-transduction.

<ol>
	<li>Cann, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA Viruses. A Practical Approach. , (2000).</li><li>Gould, D. J., Favorov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+for+the+treatment+of+autoimmune+diseases.">Vectors for the treatment of autoimmune diseases.</a> Gene Therapy. 10, 912-927 (2003).</li><li>Naldini, L., Blomer, U., Gage, F. H., Trono, D., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfer,+integration,+and+sustained+long-term+expression+of+the+transgene+in+adult+rat+brains+injected+with+a+lentiviral+vector.">Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 11382-11388 (1996).</li><li>Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+delivery+and+stable+transduction+of+nondividing+cells+by+a+lentiviral+vector.">In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.</a> Science. 272, 263-267 (1996).</li><li>Dull, T., Zufferey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72, 8463-8471 (1998).</li><li>Federico, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lentivirus Gene Engineering Protocols. , (2003).</li><li>Curran, M. A., Kaiser, S. M., Achacoso, P. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transduction+of+nondividing+cells+by+optimized+feline+immunodeficiency+virus+vectors.">Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.</a> Mol. Ther. 1, 31-38 (2000).</li><li>Kahlig, K. M., Saridey, S. K., Kaja, A., Daniels, M. A., George, A. L., Wilson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+transposon-mediated+stable+gene+transfer+in+human+cells.">Multiplexed transposon-mediated stable gene transfer in human cells.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (4), 1343-1348 (2010).</li><li>Kim, A., Pyykko, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+matters:+versatile+use+of+PiggyBac+transposons+as+a+genetic+manipulation+tool.">Size matters: versatile use of PiggyBac transposons as a genetic manipulation tool.</a> Mol. Cell Biochem. , (2011).</li></ol>

Production and Free Access to this video article is sponsored by System Biosciences.

This viral packaging and transduction of target cells protocol has been optimized for use with SBI's viral packaging reagents. Substitution of other reagents may affect the outcome of the protocol. This protocol has only been optimized for lentiviral production and may not be suitable for producing other types of viruses or pseudoviruses.
Successful viral packaging is dependent upon several key steps. The 293TN cells express the SV40 large T antigen, which is absolutely necessary for secretion of the lentiviral particles into the cell culture supernatant. The density at which the 293TN cells are at the time of transfection is especially important in order for the PureFection reagent to sufficiently transfer the lentivector and packaging plasmids into the cells. Adding the PureFection mixture to the tissue culture plate in a drop-wise manner followed by thoroughly swirling the plate is important for dispersal of the packaging plasmids and lentivector construct throughout all of the cells. Failure to appropriately distribute the vectors may result in insufficient lentiviral particles being produced. The viral packaging reaction can be scaled up for production of high titer virus, based on the surface area of the tissue culture plates. One can choose to plate several tissue culture dishes of 293TN cells per construct that needs to be packaged. Concentration of the viral supernatants is especially helpful in creating high-titer virus. Viral supernatant from multiple plates of cells can be combined and concentrated for use in in vivo experiments, or for cells that are difficult to transduce. 
Titering the produced lentiviral particles is important for calculating an accurate MOI to use for transduction of target cells. Knowing the MOI that was used in a given transduction enables troubleshooting in the event that the transduction is not successful. For example, using an MOI that is too low may result in very few target cells expressing the construct-of-interest. Using an MOI that is too high, however, may result in target cells that show signs of stress. The goal is to use an MOI that maximizes expression of the construct-of-interest while preserving the health of the cells. While we recommend a qPCR-based titering protocol that measures integrated copies of the lentiviral construct in HT1080 cells, other titering approaches can also be used, such as p24 ELISA or estimation of the percent of GFP-positive cells.
Successful transduction of target cells is also dependent upon several key factors. TransDux is a unique infection reagent that enables high transduction efficiencies, but that is not toxic to cells. TransDux can be used instead of polybrene, which is often toxic to the target cells. Inclusion of TransDux in the transduction of the target cells helps to neutralize charges on the viral particles and allows the virus into the cells. Since TransDux is not toxic to the cells, one can allow the lentiviral particles to remain in contact with the cells for up to 72 hours (the time at which the viral constructs have integrated into the host cell genome), thus increasing the probability of the viral particles entering the cell. For some difficult-to-transduce cells, transductions can be repeated on successive days to boost the transduction efficiency. For example, transduce the cells on day 1, allow the cells to rest on day 2, and then transduce the cells again on day 3. It is important to wait 72 hours after the last transduction before beginning selection, further experiments or characterization of the target cells.
Efficient viral packaging is also dependent upon the size of the lentiviral construct, between the 5' and 3'LTR regions. This section of the lentiviral construct should be approximately 9 kb or less, which corresponds to the size of the native HIV genome. Exceeding 9 kb may decrease the titer of the produced pseudoviral particles. New technology, such as piggyBac transposon vectors, allow for reliable, stable integration of transgenes up to 100 kb through a single transfection. This approach may be used for constructs that exceed the size limit for lentiviral vectors, in cases where it is possible to transfect target cells, and provides the additional advantage of not requiring a viral packaging step 9,10.
The pseudoviral particles produced using this protocol are infectious in that they can infect most mammalian cell types. The products used in this protocol, however are third-generation biosafe in that they produce non-replicative and self-inactivating pseudoviruses. Therefore, once transduced into target cells or animals, the target cells or animals are not infectious or contagious. Substitution of other lentivector plasmids that are not third-generation biosafe is possible although not recommended from a biosafety perspective. The viral production protocol and subsequent transduction of target cells should be carried out under Biosafety Level 2, according to the NIH guidelines 11, and researchers should always wear a lab coat and gloves when handling the viral particles. Used 293TN producer cells, tubes of viral particles, and disposable pipettes should be disposed of in an appropriate biohazard container. Reusable pipettes and glassware should be decontaminated with bleach and autoclaving to decrease exposure of personnel.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DMEM High Glucose Medium</td>
 <td>Gibco</td>
 <td>11995-073</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>293TN Cells</td>
 <td>SBI</td>
 <td>LV900A-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pPACKH1</td>
 <td>SBI</td>
 <td>LV500A-1</td>
 <td>For HIV-based lentivectors</td>
 </tr>
 <tr>
 <td>pPACKF1</td>
 <td>SBI</td>
 <td>LV100A-1</td>
 <td>For FIV-based lentivectors</td>
 </tr>
 <tr>
 <td>Any Lentiviral vector</td>
 <td>SBI</td>
 <td>Various</td>
 <td>cDNA, shRNA, microRNA, transcription reporters</td>
 </tr>
 <tr>
 <td>PureFection</td>
 <td>SBI</td>
 <td>LV750A-1 
 LV750A-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PEG-it</td>
 <td>SBI</td>
 <td>LV810A-1 
 LV825A-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Global UltraRapid Titering kit</td>
 <td>SBI</td>
 <td>LV961A-1</td>
 <td>Not described in this protocol, but needed to titer the viral particles</td>
 </tr>
 <tr>
 <td>TransDux</td>
 <td>SBI</td>
 <td>LV850A-1</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

packaging hiv- or fiv-based lentivector expression constructs &amp; transduction of vsv-g pseudotyped viral particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Watch this Scientific Journal Video about Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles at JoVE.com

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

packaging-hiv-or-fiv-based-lentivector-expression-constructs

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JoVE Journal

Immunology and Infection

35.0K Views.  System Biosciences. Vettori di espressione lentivirali sono i veicoli più efficaci per esprimere stabilmente diverse molecole effettrici o costrutti reporter nel dividere e non la divisione delle cellule di mammiferi e di interi organismi. Qui forniamo un protocollo su come confezionare costrutti di espressione lentivector particelle pseudoviral e di trasdurre cellule bersaglio utilizzando le particelle pseudoviral.

Video: Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

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 ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

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

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

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

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

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 ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...