We thank Britta Weseloh and Bettina Abel for technical assistance. We also thank Arne D&#252;sedau and Jana Hennesen (flow cytometry technology platform, Heinrich Pette Institut) for technical support.

<ol>
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R., 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=HIV-1-mediated+insertional+activation+of+STAT5B+and+BACH2+trigger+viral+reservoir+in+T+regulatory+cells.">HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells.</a> Nature Communications. 8 (1), 498 (2017).</li><li>Cohn, L. B., Silva, I. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Integration+Landscape+during+Latent+and+Active+Infection.">HIV-1 Integration Landscape during Latent and Active Infection.</a> Cell. 160 (3), 420-432 (2015).</li><li>Han, Y., Lassen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+CD4++T+cells+from+human+immunodeficiency+virus+type+1+(HIV-1)-infected+individuals+carry+integrated+HIV-1+genomes+within+actively+transcribed+host+genes.">Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes.</a> Journal of Virology. 78 (12), 6122-6133 (2004).</li><li>Ikeda, T., Shibata, J., Yoshimura, K., Koito, A., Matsushita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+HIV-1+integration+at+the+BACH2+locus+in+resting+CD4++T+cell+populations+during+effective+highly+active+antiretroviral+therapy.">Recurrent HIV-1 integration at the BACH2 locus in resting CD4+ T cell populations during effective highly active antiretroviral therapy.</a> The Journal of Infectious Diseases. 195 (5), 716-725 (2007).</li><li>Wagner, T. A., Mclaughlin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+of+cells+with+HIV+integrated+into+cancer+genes+contributes+to+persistent+infection.">Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection.</a> Science. 345 (6196), 570-573 (2014).</li><li>Maldarelli, F., Wu, X., 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=Specific+HIV+integration+sites+are+linked+to+clonal+expansion+and+persistence+of+infected+cells.">Specific HIV integration sites are linked to clonal expansion and persistence of infected cells.</a> Science. 345 (6193), 179-183 (2014).</li><li>Jordan, A., Bisgrove, D., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+reproducibly+establishes+a+latent+infection+after+acute+infection+of+T+cells+in+vitro.">HIV reproducibly establishes a latent infection after acute infection of T cells in vitro.</a> The EMBO Journal. 22 (8), 1868-1877 (2003).</li><li>Mack, K. D., Jin, X., 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=HIV+insertions+within+and+proximal+to+host+cell+genes+are+a+common+finding+in+tissues+containing+high+levels+of+HIV+DNA+and+macrophage-associated+p24+antigen+expression.">HIV insertions within and proximal to host cell genes are a common finding in tissues containing high levels of HIV DNA and macrophage-associated p24 antigen expression.</a> Journal of Acquired Immune Deficiency Syndromes. 33 (3), 308-320 (2003).</li><li>Byrne, S. M., Ortiz, L., Mali, P., Aach, J., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-kilobase+homozygous+targeted+gene+replacement+in+human+induced+pluripotent+stem+cells.">Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells.</a> Nucleic Acids Research. 43 (3), 1-12 (2014).</li><li>CRISPR Genome Engineering Toolbox: Target Sequence Cloning Protocol. Addgene website Available from: <a target="_blank" href="https://www.addgene.org/static/cms/filer_public/e6/5a/e65a9ef8-c8ac-4f88-98da-3b7d7960394c/zhang-lab-general-cloning-protocol.pdf">https://www.addgene.org/static/cms/filer_public/e6/5a/e65a9ef8-c8ac-4f88-98da-3b7d7960394c/zhang-lab-general-cloning-protocol.pdf</a> (2013)</li><li>Gibson Assembly Protocol. Addgene website Available from: <a target="_blank" href="https://www.addgene.org/protocols/gibson-assembly/">https://www.addgene.org/protocols/gibson-assembly/</a> (2009)</li><li>Addgene Plasmid Cloning by PCR. Addgene website Available from: <a target="_blank" href="https://www.addgene.org/protocols/pcr-cloning/">https://www.addgene.org/protocols/pcr-cloning/</a> (2014)</li><li>Addgene Plasmid Cloning by Restriction Enzyme Digest (aka Subcloning). Addgene website Available from: <a target="_blank" href="https://www.addgene.org/protocols/subcloning/">https://www.addgene.org/protocols/subcloning/</a> (2013)</li><li>Stemmer, M., Thumberger, T., Del Sol Keyer, M., Wittbrodt, J., Mateo, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCTop:+An+intuitive,+flexible+and+reliable+CRISPR-Cas9+target+prediction+tool.">CCTop: An intuitive, flexible and reliable CRISPR-Cas9 target prediction tool.</a> Public Library of Science (PLoS) ONE. 10 (4), (2015).</li><li>Lange, U. C., Bialek, J. K., Walther, T., Hauber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pinpointing+recurrent+proviral+integration+sites+in+new+models+for+latent+HIV-1+infection.">Pinpointing recurrent proviral integration sites in new models for latent HIV-1 infection.</a> Virus Research. 249, (2018).</li><li>Bialek, J. K., Dunay, G. 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=Targeted+HIV-1+Latency+Reversal+Using+CRISPR-Cas9-Derived+Transcriptional+Activator+Systems.">Targeted HIV-1 Latency Reversal Using CRISPR-Cas9-Derived Transcriptional Activator Systems.</a> PloS ONE. 11 (6), e0158294 (2016).</li><li>Lee, C. M., Davis, T. H., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+CRISPR-Cas9+design+tools+and+the+effect+of+target+site+accessibility+on+Cas9+activity.">Examination of CRISPR-Cas9 design tools and the effect of target site accessibility on Cas9 activity.</a> Experimental Physiology. 103 (4), 456-460 (2018).</li><li>Jensen, K. T., Fl&#248;e, L., 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=Chromatin+accessibility+and+guide+sequence+secondary+structure+affect+CRISPR-Cas9+gene+editing+efficiency.">Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency.</a> FEBS Letters. 591 (13), 1892-1901 (2017).</li><li>Simonetti, F. R., Sobolewski, M. 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=Clonally+expanded+CD4+++T+cells+can+produce+infectious+HIV-1+in+vivo.">Clonally expanded CD4 + T cells can produce infectious HIV-1 in vivo.</a> Proceedings of the National Academy of Sciences. 113 (7), 1883-1888 (2016).</li><li>Chen, H. C., Martinez, J. P., Zorita, E., Meyerhans, A., Filion, G. 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The authors have nothing to disclose.

Here, we describe a protocol to generate HIV-1-derived Jurkat reporter models with chosen proviral integration sites applying CRISPR-Cas9-based genome engineering.
Several points of the protocol require careful attention during the planning stage. First, the locus to be targeted should be chosen carefully, as some loci might be easier to target than others (e.g., depending on the chromatin status of the region and the target sequence itself). Repetitive sequences are hard to clone int...

Integration of proviral DNA into the host genome upon infection is a critical step in the life cycle of human immunodeficiency virus (HIV). Following integration, HIV persists by establishing latency in long-lived CD4+ T cell subsets such as memory CD4+ T cells. HIV integration appears to be non-random1,2. A number of genomic hotspots with recurrently integrated proviral DNA has been detected in several studies through the sequencing of integration sites in acutely and chronically infected individuals2,3,4,5,6,7,8. Interestingly, at some of these integration sites, the same locus was detected in a large fraction of infected cells, leading to the idea that integration at recurrent sites might positively affect clonal expansion1.
To advance our understanding of the significance of recurrent integration sites, proviral integration site choice must be explored. However, several technical aspects hamper studying HIV integration site choice and the consequences. Broadly used cell culture models for HIV latency like JLat cell lines do not reflect clinically relevant recurrent integration sites9. Studies on primary patient-derived cells, on the one hand, enable description of integration site landscape by sequencing but do not allow for functional analyses. To our knowledge, no adequate experimental model is available to functionally analyze selected clinically relevant integration sites.
Here we present a detailed workflow to generate novel models for HIV infection using CRISPR-Cas9-based genome engineering technology. The workflow described herein can be used to generate T cell-derived reporter cell lines that model HIV infection, carrying a genomically integrated proviral reporter at a chosen integration site. They are thus serving as new tools to explore how the proviral integration site can impact HIV biology and how the provirus responds to different treatment strategies (e.g., inducibility by latency reversing agents). Our method uses the advantages of CRISPR-Cas9-based genome engineering, in which integration of the reporter sequence by homologous recombination is facilitated by a Cas9 nuclease-induced double-strand break at the target site. Target sites for integration are chosen according to proximity to the described recurrent integration sites from studies on HIV-infected individuals and the presence of suitable PAM motifs for Cas9-mediated genome engineering.
In our exemplary results, we have focused on the BACH2 gene locus, which codes for the BTB And CNC Homology transcriptional regulator 2. In chronically HIV-infected individuals on antiretroviral therapy, BACH2 is one of the loci showing enrichment of integrated HIV-1 sequences3,6,7,8,10. We have chosen a minimal HIV-derived reporter consisting of HIV-1-derived long terminal repeat (LTR), tdTomato coding sequence, and bovine growth hormone (BGH) polyadenylation signal (PA), which we have targeted to two specific sites in BACH2 intron 5. The presented protocol is optimized for Jurkat cells, a human CD4+ T cell-derived suspension cell line, but other cell lines may be used and the protocol adapted accordingly. We present a detailed workflow for selection of target site, construction of target vector with homology arms, CRISPR-Cas9-mediated targeting of the reporter into the chosen genomic site, generation and selection of clonal lines, and comprehensive verification of newly generated, targeted reporter cell lines.

<table>
	<tbody>
		<tr>
			<td>pX330-U6-Chimeric_BB-cBh-hSpCas9</td>
			<td>Addgene</td>
			<td>42230</td>
			<td>vector for expression of SpCas9 and gRNA</td>
		</tr>
		<tr>
			<td>pMK</td>
			<td>GeneArt</td>
			<td></td>
			<td>mammalian expression vector for cloning</td>
		</tr>
		<tr>
			<td>cDNA3.1</td>
			<td>Invitrogen</td>
			<td>V79020</td>
			<td>mammalian expression vector for cloning</td>
		</tr>
		<tr>
			<td>BbsI</td>
			<td>New England Biolabs</td>
			<td>R0539S</td>
			<td>restriction enzyme</td>
		</tr>
		<tr>
			<td>NEBuilder Hifi DNA Assembly Cloning Kit</td>
			<td>New England Biolabs</td>
			<td>E5520S</td>
			<td>Assembly cloning kit used for target vector generation</td>
		</tr>
		<tr>
			<td>TaqPlus Precision PCR System</td>
			<td>Agilent Technologies</td>
			<td>600210</td>
			<td>DNA polymerase with proofreading activity used for amplification of homology arms (step 1.2.2.2), verification of integration site and reporter sequence (step 3.3.3 and 3.3.5), generation of genomic probe for Southern blot (step 3.4.1.5) and analysis of off-target events (step 3.5.4)</td>
		</tr>
		<tr>
			<td>96-well tissue culture plate (round-bottom)</td>
			<td>TPP</td>
			<td>92097</td>
			<td>tissue culture plates for dilution plating</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530 L</td>
			<td>DNA polymerase used for detection of targeting events (step 2.4.2) and generation ofreporter-specific probe for Southern blot (step 3.4.1.4)</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D9170</td>
			<td>dimethyl sulfoxide as PCR additive</td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2) Solution</td>
			<td>New England Biolabs</td>
			<td>B9021S</td>
			<td>MgCl2 solution as PCR additive</td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Mix</td>
			<td>New England Biolabs</td>
			<td>N0447S</td>
			<td>dNTP mixture with 10 mM of each nt for PCR reactions</td>
		</tr>
		<tr>
			<td>5PRIME HotMasterMix</td>
			<td>5PRIME</td>
			<td>2200400</td>
			<td>ready-to-use PCR mix used for screening PCR (step 3.2.11)</td>
		</tr>
		<tr>
			<td>QIAamp DNA blood mini kit</td>
			<td>Qiagen</td>
			<td>51106</td>
			<td>DNA isolation and purification kit</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>PCR Purification Kit</td>
		</tr>
		<tr>
			<td>RPMI 1640 without glutamine</td>
			<td>Lonza</td>
			<td>BE12-167F</td>
			<td>cell culture medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum South Africa Charge</td>
			<td>PAN Biotech</td>
			<td>P123002</td>
			<td>cell culture medium supplement</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Biochrom</td>
			<td>K 0282</td>
			<td>cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10.000 U/ml / 10.000 &#181;g/ml</td>
			<td>Biochrom</td>
			<td>A 2212</td>
			<td>cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Gibco Opti-MEM Reduced Serum Media</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985062</td>
			<td>cell culture medium with reduced serum concentration optimized for transfection</td>
		</tr>
		<tr>
			<td>TransIT-Jurkat</td>
			<td>Mirus Bio</td>
			<td>MIR2125</td>
			<td>transfection reagent</td>
		</tr>
		<tr>
			<td>phorbol 12-myristate 13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P8139-1MG</td>
			<td>cell culture reagent</td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I0634-1MG</td>
			<td>cell culture reagent</td>
		</tr>
		<tr>
			<td>Syringe-driven filter unit, PES membrane, 0,22 &#181;m</td>
			<td>Millex</td>
			<td>SLGP033RB</td>
			<td>filter unit for sterile filtration</td>
		</tr>
		<tr>
			<td>Heracell 150i incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>51026280</td>
			<td>tissue culture incubator</td>
		</tr>
		<tr>
			<td>Amershan Hybond-N+</td>
			<td>GE Healthcare</td>
			<td>RPN1520B</td>
			<td>positively charged nylon membrane for DNA and RNA blotting</td>
		</tr>
		<tr>
			<td>Stratalinker 1800</td>
			<td>Stratagene</td>
			<td>400072</td>
			<td>UV crosslinker</td>
		</tr>
		<tr>
			<td>High Prime</td>
			<td>Roche</td>
			<td>11585592001</td>
			<td>kit for labeling of DNA with radioactive dCTP using random oligonucleotides as primers</td>
		</tr>
		<tr>
			<td>illustra ProbeQuant G-50 Micro Columns</td>
			<td>GE Healthcare</td>
			<td>28-9034-08</td>
			<td>chromatography spin-columns for purification of labeled DNA</td>
		</tr>
	</tbody>
</table>

crispr-cas9-based genome engineering to generate jurkat reporter models for hiv-1 infection with selected proviral integration sites

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites.
In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed.
By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.

In this representative experiment we have chosen to target a minimal HIV-1-derived reporter consisting of a LTR, tdTomato-coding sequence, and polyA-signal sequence to two loci in intron 5 of the BACH2 gene17. The loci for targeting were chosen according to proximity to published recurrent integration sites found in different studies on primary T cells from HIV-infected patients2,4,<sup class="xr...

Watch this Scientific Journal Video about CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites at JoVE.com

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we ...

crispr-cas9-based-genome-engineering-to-generate-jurkat-reporter

Research

JoVE Journal

Immunology and Infection

9.7K Views.  Leibniz Institute for Experimental Virology. We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

Video: CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

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 ...

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 ...

In vitro Uncoating of HIV-1 Cores

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

In vitro Uncoating of HIV-1 Cores

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

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

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

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

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

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

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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

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

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

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

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.