We would like to thank Drs. Romas Geleziunas and Jeff Murry and the people at Gilead for providing the antiretroviral drugs used in this study. This work was funded by NCI 1R01CA239261-01 (to Kitchen), NIH Grants P30AI28697 (the UCLA CFAR Virology Core, Gene and Cell Therapy Core, and Humanized Mouse Core), U19AI149504 (PIs: Kitchen &#38; Chen), CIRM DISC2-10748, NIDA R01DA-52841 (to Zhen), NIAID R2120200174 (PIs: Xie &#38; Zhen), IRACDA K12 GM106996 (Carrillo). This work was also supported by the UCLA AIDS Institute, the James B. Pendleton Charitable Trust, and the McCarthy Family Foundation.

Anonymized human fetal tissue was acquired commercially. Animal research was carried out according to protocols approved by the University of California, Los Angeles, and (UCLA) Animal Research Committee (ARC) in accordance with all federal, state, and local guidelines. Specifically, all the experiments were carried out in accordance with the recommendations and guidelines for housing and care of laboratory animals of the National Institutes of Health (NIH) and the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC) International under UCLA ARC Protocol Number 2010-038-02B. All surgeries were performed under ketamine (100 mg/kg)/xylazine (5 mg/kg) and isoflurane anesthesia (2-3 vol%) and all efforts were made to minimize animal pain and discomfort.
1. Humanized mice infected with HIV-1
NOTE: Humanized mice were constructed as previously described in30,31,49. The protocol is briefly described below.
<ol>
	<li>Purify CD34+ hematopoietic progenitor cells from the human fetal liver via anti-CD34 microbeads according to the manufacturer&#39;s protocol.</li>
	<li>Anesthetize 6-8-week-old NOD/SCID/IL2R&#947;&#8722;/&#8722; (NSG) male and female mice and sub-lethally irradiate (2.7 Gy) before the surgery.</li>
	<li>Implant thymus, derived from the same donor as the fetal liver, under the kidney capsule along with the liver.</li>
	<li>Following implantation, inject mice with 0.5 million to one million CD34+ cells, intravenously.</li>
	<li>After 8-10 weeks, collect 100 &#181;L of mouse blood via retro-orbital bleed50 into microcentrifuge tubes containing 5 &#181;L of EDTA and centrifuge at 350 x g for 3 min.</li>
	<li>Store the plasma at -80 &#176;C to monitor viral load after the mouse has been infected with HIV-1. Add 2 mL of 83% NH4C solution and incubate for 5 min at room temperature to lyse red blood cells.</li>
	<li>Add 10 mL of RPMI with 10% Fetal Bovine Serum (FBS) to stop lysis. Spin at 300 x g for 5 min.</li>
	<li>Aspirate supernatant. Stain cells with antibody panel (see Table of Materials) and analyze by flow cytometry to check human immune cell engraftment.</li>
	<li>Infect mice exhibiting more than 50% of circulating CD45+ cells by retro-orbital vein injection51,52 with at least 200 ng of p24 of an HIV-1 strain (i.e., NFNSXSL930,53,54) using an insulin syringe. Collect blood biweekly for flow cytometry analysis and to measure the viral load.</li>
</ol>
2. Preparation of ART drugs
<ol>
	<li>Weigh individual drugs; for example, to make 10 food cups with cART, use sterile cell scrapers to weigh out 250 mg of FTC (Emtricitabine), 375 mg of TDF, and 500 mg of RAL or ELV into individual sterile 15 mL centrifuge tubes in a biosafety cabinet.</li>
	<li>Add 1 mL of DMSO into 250 mg FTC tube (final concentration of 250 mg/mL), add 1.5 mL of DMSO into 375 mg TDF tube (final concentration of 250 mg/mL), and add 1mL of DMSO into 500 mg RAL or ELV tube (final concentration of 500 mg/mL). Stir or pipet the drug mixture until fully dissolved and a clear solution is obtained.</li>
	<li>Use a 0.22 &#181;M pore size hydrophilic PVDF membrane filter to sterilize solutions with a sterile syringe. Individual drug solutions can be stored at -20 &#176;C for 12 weeks.</li>
	<li>When ready to use, freshly thaw one aliquot of each drug solution at 37 &#176;C until the solution becomes clear. Mix well using a pipet.</li>
	<li>Combine drugs and mix well to make up master mix: 1 mL of FTC in DMSO, 1.5 mL of TDF in DMSO, and 1 mL of ELV or RAL in DMSO. 
	NOTE: This amount will make 10 food cups.</li>
	<li>Add 350 &#181;L of cART master mix solution into one cup to make one DietGel Boost cART cup.</li>
	<li>Add 0.75 mL of Trimethoprim-Sulfamethoxazole (0.48 mg/mL final concentration) into the cup.</li>
	<li>Stir thoroughly using 1 mL sterile pipette tips.</li>
	<li>Aliquot the food cup containing cART from the original cup with a micro spatula onto a 60 mm Petri dish as needed. Weigh the food on a scale to calculate the amount of food cup containing cART for each cage according to the number of mice.</li>
</ol>
3. Administration of ART drugs to HIV-1 infected mice
<ol>
	<li>Remove regular chow from the cage and replace it with a food cup containing cART. 
	NOTE: On average, a mouse will eat up to 5 g of food per day. Approximately one food cup can be administrated to five mice for 2 days.</li>
	<li>Refresh cART food three times per week.</li>
	<li>Weigh used cups to monitor intake. Weigh mice weekly to confirm consumption.</li>
</ol>
4. Monitor viral load by real-time PCR
<ol>
	<li>Assess human immune cells (CD4 and CD8 T cell levels) and HIV-1 replication in BLT mice every 2 weeks by retro-orbital bleeding. Harvest plasma by following instructions in steps 1.5-1.8.</li>
	<li>Monitor plasma viral loads of mice infected with HIV-1 before and during oral cART administration for 8 weeks. Extract plasma viral RNA from plasma using a viral RNA extraction kit and quantify it by real-time PCR using the primers and probes (see Table of Materials) as previously described27,30,31. Use the following cycling protocol: 48 &#176;C (15 min), 95 &#176;C (10 min), then cycling 95 &#176;C (15 s), 60 &#176;C (1 min) for 45 cycles.</li>
</ol>
5. Assess CD4/CD8 ratios by flow cytometry
<ol>
	<li>Prepare single-cell suspensions from peripheral blood of biweekly bleeds following steps 1.5-1.8.</li>
	<li>Stain cells with surface markers and analyze by flow cytometry. Use the following surface marker antibodies27,30,43,49 in flow cytometry: CD45 (clone HI30), CD8 (clone SK1), CD3 (clone OKT3), CD4 (clone RPA-T4)27,30,42,49.</li>
</ol>

SK is a founder of CDR3 Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

An oral cART administration method is developed here for HIV-1 infected humanized mice by combining three antiretroviral drugs within high nutrient food. Compared to administration by daily injections, oral delivery is easier to use, limits administration frequency, reduces animal handling, minimizes stress, and improves safety55. Up to this point, only a few studies in humanized mice24,37,41 have used fo...

The life expectancy of chronic human immunodeficiency virus (HIV)-infected individuals has significantly improved with combinational antiretroviral treatment (cART)1,2. cART successfully reduces the HIV-1 replication and increases CD4+ T cell counts to normalcy in the majority of HIV-1 chronically infected participants3, resulting in improved overall health and dramatically reduced disease progression4. However, the latent HIV-1 reservoir is established even when ART is initiated during acute infection5,6,7. Reservoirs persist over years during ART and rapid viral rebound after ART interruption is well-documented8,9. People living with HIV on ART are also predisposed to a higher risk of comorbidities such as cardiovascular disease, cancer, and neuro disorders10,11,12. Therefore, a functional cure for HIV is needed. Animal models for HIV-1 infection offer obvious advantages in developing and validating novel HIV cure strategies13,14,15. Humanized mice, as a small animal model, can provide multilineage human immune cell reconstitution in different tissues, which allows for the close study of HIV infection16,17,18,19. Among humanized models, the humanized bone marrow-liver-thymus (BLT) model successfully recapitulates chronic HIV-1 infection as well as functional human immune responses to HIV-1 infection20,21,22,23,24. Therefore, the humanized BLT mouse model has been widely used to investigate various aspects in the HIV research field. Humanized BLT mice are not only well-established models for the recapitulation of persistent HIV-1 infection and pathogenesis, but also consequential tools for the evaluation of cell therapy-based intervention strategies. The current authors and others have demonstrated that the humanized BLT mice model recapitulates persistent HIV-1 infection and pathogenesis25,26,27 and provides tools to evaluate cell therapy-based intervention strategies 28,29,30,31,32,33.
cART regimens consisting of combinations of antiretroviral drugs that are taken daily suppress HIV-1 replication to the point that the viral load in successfully treated individuals remains undetectable over long term34. The outcomes of treating HIV-infected humanized mice with clinically relevant cART regimens resemble those observed in HIV-1 infected ART-treated individuals22: HIV-1 levels are suppressed below the limits of detection and interruption of cART results in a rebound of HIV replication from the latent reservoir35. Subcutaneous (SC)27,36,37 or intraperitoneal (IP)37,38,39 injection is the route commonly used for cART treatment in humanized mice. However, intensive daily injection induces stress to animals by physical restraint40. It is also labor-intensive and potentially unsafe for researchers due to increased exposure to HIV while using sharps. Oral administration is ideal to mimic the absorption, distribution, and excretion of cART drugs that are taken by HIV-1-infected individuals. Oral administration typically involves customized and often laborious procedures to put the antiretroviral drugs in sterilized (necessary due to the immunodeficiency of the mice) food24,37,41 or water42,43,44,45,46, which may or may not be chemically compatible with many antiretroviral drugs, or result in something the mice would not readily eat or drink (which would affect dose and drug levels in the body). The novel peroral cART administration method proposed here surpasses previous delivery attempts due to its compatibility with different types of antiretroviral drugs, safety and ease of preparation and administration, and reduction of animal stress and anxiety resulting from the daily injection.
Tenofovir disoproxil fumarate (TDF), Elvitegravir (ELV), and Raltegravir (RAL) are poorly water-soluble drugs. Interestingly, increased bioavailability of TDF is observed with fatty foods, suggesting that competitive inhibition of lipases by fatty food may provide certain protection for TDF47. Therefore, DietGel Boost cups were selected to replace regular rodent chow as the method of delivery based on their modest fat content (20.3 g per 100 g) as compared to regular rodent chow (10 g per 100 g) and a typical mouse high-fat diet (40-60 g per 100 g)48. The total weight of one cup is 75 g; thus, each cup will contain the amount of food, and therefore drug, sufficient for five mice over 3 days.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>60 mm petri dish</td>
			<td>Thermo Scientific Nunc</td>
			<td>150288</td>
			<td>For aliquoting ART food</td>
		</tr>
		<tr>
			<td>APC anti-human CD8 Antibody</td>
			<td>Biolegend</td>
			<td>344722</td>
			<td>For flow cytometry</td>
		</tr>
		<tr>
			<td>BD LSRFortessa</td>
			<td>BD biosciences</td>
			<td></td>
			<td>For flow data collection</td>
		</tr>
		<tr>
			<td>CD34 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-046-702</td>
			<td>For NSG-BLT mice generation</td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Falcon</td>
			<td>14-432-22</td>
			<td>For dissolving ART</td>
		</tr>
		<tr>
			<td>DietGel Boost</td>
			<td>ClearH2O</td>
			<td>72-04-5022</td>
			<td>For making ART food</td>
		</tr>
		<tr>
			<td>Elvitegravir</td>
			<td>Gilead</td>
			<td></td>
			<td>Gifted from Gilead</td>
		</tr>
		<tr>
			<td>Emtricitabine</td>
			<td>Gilead</td>
			<td></td>
			<td>Gifted from Gilead</td>
		</tr>
		<tr>
			<td>FITC anti-human CD3 Antibody</td>
			<td>Biolegend</td>
			<td>317306</td>
			<td>For flow cytometry</td>
		</tr>
		<tr>
			<td>Flowjo software</td>
			<td>FlowJo</td>
			<td></td>
			<td>For flow cytometry data analysis</td>
		</tr>
		<tr>
			<td>HIV-1 forward primer: 5&#8242;-CAATGGCAGCAATTTCACCA-3&#8242;;</td>
			<td>IDT</td>
			<td>Customized</td>
			<td>For viral load RT-PCR</td>
		</tr>
		<tr>
			<td>HIV-1 probe: 5&#8242;-[6-FAM]CCCACCAACAGGCGGCCT 
			TAACTG [Tamra-Q]-3&#8242;;</td>
			<td>IDT</td>
			<td>Customized</td>
			<td>For viral load RT-PCR</td>
		</tr>
		<tr>
			<td>HIV-1 reverse primer: 5&#8242;-GAATGCCAAATTCCTGCTTGA-3&#8242;;</td>
			<td>IDT</td>
			<td>Customized</td>
			<td>For viral load RT-PCR</td>
		</tr>
		<tr>
			<td>Human fetal tissue</td>
			<td>Advanced Bioscience Resources, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td>
			<td>The Jackson Laboratory</td>
			<td>5557</td>
			<td>For constructing the humanized mice</td>
		</tr>
		<tr>
			<td>Pacific Blue anti-human CD45</td>
			<td>Biolegend</td>
			<td>304022</td>
			<td>For flow cytometry</td>
		</tr>
		<tr>
			<td>PerCP anti-human CD4 Antibody</td>
			<td>Biolegend</td>
			<td>300528</td>
			<td>For flow cytometry</td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Kits</td>
			<td>Qiagen</td>
			<td>&#160;52904</td>
			<td>For measuring viral load</td>
		</tr>
		<tr>
			<td>Raltegravir</td>
			<td>Merck</td>
			<td></td>
			<td>Gifted from Merck</td>
		</tr>
		<tr>
			<td>Sterile cell scrapers</td>
			<td>Thermo Scientific</td>
			<td>179693</td>
			<td>For aliquoting ART food</td>
		</tr>
		<tr>
			<td>TaqMan RNA-To-Ct 1-Step Kit</td>
			<td>Applied Biosystems</td>
			<td>4392653</td>
			<td>For plasma viral load detection</td>
		</tr>
		<tr>
			<td>Tenofovir disoproxil fumarate</td>
			<td>Gilead</td>
			<td></td>
			<td>Gifted from Gilead</td>
		</tr>
		<tr>
			<td>Trimethoprim-Sulfamethoxazole</td>
			<td>Pharmaceutical Associates</td>
			<td>NDC 0121-0854-16</td>
			<td>For keeping ART food sterile. Each 5mL teaspoon contains 
			200 mg Sulfamethoxazole, USP 
			40 mg Trimethoprim, USP 
			NMT 0.5% Alcohol</td>
		</tr>
	</tbody>
</table>

oral combinational antiretroviral treatment in hiv-1 infected humanized mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

Assuming an average mouse weighing 25 g consumes 4 g of food per day, the daily drug dose through oral intake corresponds to 2.88 mg/kg TFV, 83 mg/kg FTC, and 768 mg/kg RAL. To test whether the optimized food regimen is toxic and influences overall health compared to daily injection of cART, mice weight was monitored weekly before and during cART through oral or subcutaneous injection. There were no significant weight differences before cART administration in each group (Figure 1). However, ...

Watch this Scientific Journal Video about Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice at JoVE.com

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...

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Immunology and Infection

2.2K Views.  University of California, Los Angeles. This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

Video: Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

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 ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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 ...

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 ...

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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.

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

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 ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.