This work was supported by NIH/NIGMS/MBRS (Grant 58268), NIH/NCRR/RCMI (Grant G12-RR03034), Georgia Research Alliance funding grant GRA.VAC08.W, NIH/NIAID/NRSA grant F31AI091484, Emory CFAR grant P30 A1050409. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR18386 from NIH/NCRR. The Jurkat cells, the set of 20 HIV-1 Nef peptides, as well as the rabbit anti-HIV-1 Nef antiserum were obtained from the NIH AIDS Research &amp; Reference Reagent Program, (Rockville, MD).

I. Use of Short Peptides in Biological Analysis
I.1. Mapping Biologically Functional Motifs Using Peptides
<ol>
 <li>Treating cells with Nef scanning peptides
 <ol>
 <li>Procure a set of peptides scanning the region of interest. For our experiments, 20mer peptides, with 10 amino acid overlap, spanning the entire HIV-1 Nef protein (205 aa), were obtained from the AIDS Reagent Program. The peptides are assayed individually as follows:</li>
 <li>Add 10 ng/ml of peptide to the cell culture medium.</li>
 <li>Add 10 ml of culture medium per plate to Jurkat cell cultures.</li>
 <li>Incubate cultures for 24 hr at 37 &deg;C.</li>
 </ol>
 </li>
 <li>Terminal deoxynucleotidyltransferase-mediated dUTP biotin Nick End Labeling (TUNEL) assay of apoptosis
 <ol>
 <li>Wash cells with 1X PBS, and fix for 30 min at room temperature with 4% paraformaldehyde in 1X PBS, pH 7.4.</li>
 <li>Wash with 1X PBS, and permeabilize with 0.1% Triton X-100 for 10 min at room temperature.</li>
 <li>Rinse the cells twice with 1X PBS.</li>
 <li>Count stained cells by epifluorescence on a computer-controlled microscopy system.</li>
 </ol>
 </li>
</ol>
I.2. Competitive Inhibition Analysis using Peptides
<ol>
 <li>Co-transfection of Peptides using the Chariot Protein Delivery Reagent Kit
 <ol>
 <li>Per transfection reaction mixture, dilute 1 &mu;g of wtNef-GFP plasmid DNA and 100 ng of either wildtype peptides derived from the SMR domain, or peptides containing alanine-replacement of individual amino acids in the SMR motif, to a final volume of 100 &mu;l with 1X PBS.</li>
 <li>Dilute 6 &mu;l of a 1/10 dilution of Chariot Reagent to a final volume of 100 &mu;l with sterile water, and combine with the diluted DNA-peptide mix.</li>
 <li>Incubate at room temperature for 30 min to allow formation of the Chariot/DNA-peptide complex.</li>
 <li>Pellet 3 x 105 Jurkat cells by centrifugation at 600 x g for 5 min.</li>
 <li>Wash cells twice with 1X PBS, and resuspend in the Chariot/DNA-peptide complex.</li>
 <li>Add 400 &mu;l of serum-free RPMI 1640 medium to the suspension and incubate the cells in culture plates at 37 &deg;C for 2 hr.</li>
 <li>Add 1 ml of fresh RPMI 1640 medium containing 10% FBS to each plate, and incubate the cells for 48 hr at 37 &deg;C.</li>
 <li>Harvest cells and determine transfection efficiency by fluorescent microscopy.</li>
 </ol>
 </li>
 <li>Nef-GFP secretion fluorescent plate reader assay of peptide inhibition
 <ol>
 <li>Collect the cell-free conditioned media from the harvested cells, and transfer 100 &mu;l to individual wells of a 96-well black microtiter plate.</li>
 <li>Measure GFP fluorescence in the media on a Tecan GENEios fluorimeter with excitation wavelength 485 nm and emission wavelength 515 nm in relative fluorescent units.</li>
 <li>Set the level of GFP fluorescence in the control (media from cells co-transfected with Nef-GFP and a scrambled peptide) at 100%.</li>
 <li>Compare this to the level of GFP fluorescence in the media from cells co-transfected with various SMR peptides to determine changes in Nef-GFP secretion.</li>
 </ol>
 </li>
</ol>
I.3. Isolating Motif Binding Partners Using Peptides as Bait
<ol>
 <li>Co-immunoprecipitation with SMR peptides
 <ol>
 <li>Grow Jurkat cells in RPMI 1640 medium containing 10% FBS at 37 &deg;C in a T-75 tissue culture flask, and pellet cells by centrifugation at 1,000 x g for 20 min at 4 &deg;C. Determine the transfection efficiency, by placing the plate under a fluorescent microscope, capture images, and subsequently count the total and labeled cells and determine the percent labeled cells.</li>
 <li>Resuspend the cell pellet in 1 ml 1X PBS, and transfer it to a 1.5-ml Eppendorf tube. Microcentrifuge at 1,000 x g for 1 min. Resuspend the pellet in 1 ml &alpha;-FLAG Lysis buffer by gentle pipetting. Lysis buffer: Mix 50 mM Tris&bull;HCl pH 7.4, 150 mM sodium chloride [NaCl], 1 mM EDTA, and 1% Triton X-100, with 1 mM phenylmethylsulfonyl fluoride [PMSF], and 10 &mu;l/ml Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts [PIC; Sigma]. Make immediately before using.</li>
 <li>Differentially microcentrifuge the suspensions at 2,000 x g for 5 min, and at 13,000 x g for 10 min to pellet the cell debris and DNA, respectively. Collect the supernatant (hereafter referred to as the "lysate").</li>
 <li>Add 1 mg of lysate protein and 1 &mu;g of either the SMRwt or SMRmut peptide to 20 &mu;l of anti-FLAG M2 Affinity Gel (hereafter called "resin"; Sigma). Note that both SMRwt and SMRmut used here have a FLAG tag sequence embedded in the peptides. Bring the mixture to a final volume of 1 ml in Co-IP buffer (&alpha;-FLAG Lysis buffer minus PMSF and PIC). Incubate the mixture at 4 &deg;C overnight with end-over-end rotation. As a negative control, incubate the lysate with the resin in the absence of either peptide.</li>
 <li>Pellet the resin by microcentrifugation at 5,000 - 8,000 x g for 30 sec. Allow resin to settle for 2 min before handling the sample and then aspirate the supernatant. Remove the supernatant with a narrow-end pipette tip, or a Hamilton syringe, being careful not to transfer any resin. Narrow-end pipette tips can be made using forceps to pinch the opening of a plastic pipette tip until it is partially closed. Wash the resin four times with 500 &mu;l of wash buffer (50 mM Tris&bull;HCl pH 7.4, and 150 mM NaCl). Elute the bound cellular proteins with 15 &mu;g of 3X FLAG peptide (Sigma) in 50 &mu;l of wash buffer and incubate on rocker/shaker for 30 min at room temperature.</li>
 <li>Concentrate the eluates by acetone precipitation, followed by boiling in Laemmli sample buffer (Bio-Rad). Separate the proteins by SDS-PAGE on a 4-20% Tris-HCl Criterion precast gel (Bio-Rad). Stain the gel with Coomassie Brilliant Blue R-250 (Bio-Rad). Note: for mass spectrometry identification experiments (Figure 4A), each condition (Co-IP with SMRwt, SMRmut, or no peptide) was performed in quadruplicate, and the eluates from all four replicates were pooled prior to acetone precipitation for a total of 4 mg of total protein input/condition).</li>
 </ol>
 </li>
 <li>Co-immunoprecipitation with Nef-GFP protein
 <ol>
 <li>Mix 50 &mu;l of Dynabeads Protein G magnetic beads (Invitrogen) with 2 &mu;g of murine monoclonal anti-GFP in 1 ml of 1X PBS with 0.02% Tween 20 (PBS-T). Incubate the mixture with end-over-end rotation in a 1.5-ml Eppendorf tube for 1 hr at 4 &deg;C.</li>
 <li>Separate the beads from the buffer by placing the tube on a 1.5-ml MagnaSphere Technology Magnetic Separation Stand (Promega Corp., Madison, WI) allowing magnetic pulldown followed by removal of the supernatant. Wash the anti-GFP coated beads with 1 ml of PBS-T using gentle pipetting.</li>
 <li>Lyse Nef-GFP transfected Jurkat cells with 1 ml of 1X RIPA+ buffer by gentle pipetting and incubation for 15 min on ice. RIPA+ buffer: 10X RIPA Lysis Buffer, pH 7.4 [Millipore, Bedford, MA] diluted 1:10 in sterile water, 0.02% sodium azide [Sigma], and 0.1% sodium dodecyl sulfate [SDS], with 1 mM PMSF and 10 &mu;l/ml PIC added immediately before using.</li>
 <li>Pellet the cell debris by microcentrifugation at 2,000 x g for 10 min. Collect the supernatant (hereafter referred to as the "lysate").</li>
 <li>Add 1 mg of lysate protein to the anti-GFP coated beads, bring the final volume to 1 ml with 1X RIPA+ buffer, and incubate the mixture with end-over-end rotation for 1 hr at 4 &deg;C. Note: For peptide competition studies, 1 mg of Lysate protein may be pre-incubated with 2 &mu;g of SMRwt or SMRmut peptide in 1 ml of 1X RIPA+ buffer, prior to being added to the anti-GFP coated beads.</li>
 <li>Wash the beads through resuspension in 1 ml of wash buffer (50 mM Tris&bull;HCl pH 7.4, 150 mM NaCl, and 0.1% Tween 20). Incubate with end-over-end rotation for 5 min at 4 &deg;C. Separate the beads from the wash using the Magnetic Separation Stand. Wash the beads a second and third time with wash buffers containing 250 mM and 300 mM NaCl, respectively.</li>
 <li>Resuspend the beads in 200 &mu;l of PBS, transfer the mixture to a clean tube, and separate the beads from the wash on the magnetic stand. Boil the beads in 50 &mu;l of Laemmli sample buffer 5 min at 95 &deg;C, allow the mixture to cool for 10 min at room temperature, and then place the mixture on the magnetic stand for separation, and collect the supernatants ("eluates").</li>
 <li>Separate the proteins of the eluate by SDS-PAGE on an 8-16% Tris-HCl Criterion precast gel.</li>
 </ol>
 </li>
 <li>Mass spectrometry
 <ol>
 <li>Excise the protein bands of interest from Coomassie-stained gels.</li>
 <li>Reduce the samples in 10 mM 1,4-dithiothreitol (DTT) for 45 min at 37 &deg;C.</li>
 <li>Alkylate the samples in 55 mM iodoacetamide for 45 min at 25 &deg;C.</li>
 <li>Digest the samples overnight with sequencing grade trypsin (Promega) at 37 &deg;C.</li>
 <li>Desalt the sample peptides with C-18 ZipTip (Millipore). Mix the sample peptides with alpha-CHC Matrix (Agilent Technologies, Santa Clara, CA), and spot them onto a MTP target frame III (Bruker Daltonics Inc., Billerica, MA).</li>
 <li>Perform tandem matrix-assisted laser desorption/ionization time of-flight (MALDI-TOF/TOF) Mass Spectrometry analysis using a Bruker Daltonics ultraflex III TOF/TOF.</li>
 <li>Compare the MS/MS data to the NCBI non-redundant and Swiss-Prot databases using the Mascot algorithm (<a href="http://www.matrixscience.com" target="_blank">www.matrixscience.com</a>).</li>
 </ol>
 </li>
 <li>Immunoblot Analysis
 <ol>
 <li>Separate the protein samples by SDS-PAGE on 4-20% or 8-16% Tris-HCl Criterion precast gels (Bio-Rad). Transfer the bands electrophoretically to a nitrocellulose membrane.</li>
 <li>Wash the membrane in Tris Buffered Saline (TBS; Bio-Rad) for 5 min. Block with 5% nonfat milk in TTBS (TBS with 0.1% Tween 20) for 1 hr by shaking at room temperature.</li>
 <li>Process the membrane for immunoblotting using a specific primary antibody in 5% nonfat milk with shaking at 4 &deg;C overnight. Wash in TTBS for 20 min followed by a secondary HRP conjugated IgG (H+L) antibody in 5% nonfat milk for 1 hr at room temperature. Wash in TTBS for 30-60 min.</li>
 <li>Detect the protein bands by Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Expose the filter to photographic film. Note: In some experiments, a stripping reagent was used to strip the membrane (Pierce, Rockford, IL) with subsequent hybridization with a different primary and secondary antibody.</li>
 <li>Scan the X-ray films into Adobe Photoshop 5.0.2. Perform densitometry analysis using ImageJ software (National Institutes of Health, Bethesda, MD).</li>
 </ol>
 </li>
</ol>
II. Use of Antibody Inhibition in Functional Analysis
II.1. Co-transfection of Antibodies using the Chariot Protein Delivery Reagent Kit
<ol>
 <li>Per transfection reaction mixture, dilute 1 &mu;g of wtNef-GFP plasmid DNA and 1 &mu;g of either anti-mortalin antibody or anti-&alpha;-tubulin antibody, to a final volume of 100 &mu;l with 1X PBS. Note: It was previously found that co-transfection of the anti-&alpha;-tubulin antibody has no detectable effect on exNef secretion, and thus, it serves as an effective negative control in these experiments.</li>
 <li>Dilute 6 &mu;l of undiluted Chariot Reagent to a final volume of 100 &mu;l with sterile water, and combine with the diluted DNA-antibody mix.</li>
 <li>Incubate at room temperature for 30 min to allow formation of the Chariot/DNA-antibody complex.</li>
 <li>Pellet 3 x 105 Jurkat cells by centrifugation at 600 x g for 5 min.</li>
 <li>Wash cells twice with 1X PBS, and resuspend in the Chariot/DNA-antibody complex.</li>
 <li>Add 400 &mu;l of serum-free RPMI 1640 medium to the suspension and incubate the cells in culture plates at 37 &deg;C for 2 hr.</li>
 <li>Add 1 ml of fresh RPMI 1640 medium containing 10% FBS to each plate, and incubate the cells for 48 hr at 37 &deg;C.</li>
</ol>
II.2. Nef-GFP Secretion Fluorescent Plate Reader Assay of Antibody Inhibition
<ol>
 <li>Collect the cell-free conditioned media from the harvested cells, and transfer 100 &mu;l to individual wells of a 96-well black microtiter plate.</li>
 <li>Measure GFP fluorescence in the media on a Tecan GENEios fluorimeter with excitation wavelength 485 nm and emission wavelength 515 nm in relative fluorescent units.</li>
 <li>Set the level of GFP fluorescence in the control (media from cells co-transfected with Nef-GFP and anti-&alpha;-tubulin antibody) at 100%.</li>
 <li>Compare this to the level of GFP fluorescence in the media from cells co-transfected with anti-mortalin antibody to determine changes in Nef-GFP secretion.</li>
</ol>

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The authors declare that they have no competing financial interests.

Understanding the mechanisms underlying Nef's ability to manipulate the exosomal trafficking pathway will be useful in engineering novel inhibitors of exNef secretion. Inhibition of exNef secretion should diminish the CD4 T-cell depletion and CIA that drive HIV/AIDS pathogenesis. Towards this goal, we developed a number of novel reagents and methodologies that allowed us to analyze the genetics of exNef secretion, and begin to determine the cellular proteins involved. This approach also led to the development of the firs...

With the advent of anti-retroviral therapy, the AIDS epidemic in the western world has been slowed, but not curtailed, and the spread of HIV continues to be a major health burden throughout the world. With the exception of the marginally effective RV144 Thai trial, HIV vaccines have so far shown failure to protect from infection. Thus, research into additional, potential therapeutic targets is still warranted.
Along with CD4 T-cell depletion, persistent generalized immune activation is a hallmark of HIV infection. This Chronic Immune Activation (CIA) leads to increases in cell turnover, activated and differentiated lymphocytic subpopulations, cellular exhaustion and senescence, and the killing of T cells and B cells via Activation-Induced Cell Death (AICD)1,2,3, and it is well established as one of the strongest predictors of disease progression4,5,6,7,8,9,10,11,12. However, the mechanisms underlying CIA and CD4 T-cell depletion in HIV infection remain to be fully elucidated.
Evidence from our lab and others led us to a model for disease progression (Figure 1) wherein the HIV protein Nef (Negative Regulatory Factor) induces its secretion in exosomes from HIV-1 infected cells13,14. These Nef-containing exosomes (exNef) induce apoptosis in a number of cell lineages including uninfected CD4 T-cells15,16. Alternatively, in monocytes/macrophages exNef alters gene expression patterns, e.g. cytokine expression, and appears to induce a state of unscheduled immune activation. This body of evidence suggests an important role for exNef in CIA and CD4 T-cell depletion.
Understanding the mechanisms underlying Nef's ability to manipulate the exosomal trafficking pathway will be useful in engineering novel inhibitors of exNef secretion. Inhibition of exNef secretion should diminish the CD4 T-cell depletion and CIA that drive HIV/AIDS pathogenesis.
To gather the evidence that led to our model for HIV/AIDS disease progression and the subsequent data that built onto this model, we developed a number of novel reagents and methodologies that allowed us to analyze the genetics of exNef secretion, and begin to determine the cellular proteins involved. In the initial work, we found that Nef protein induces apoptosis in bystander cells and is released extracellularly from Nef-transfected and HIV-infected cells15. Peptides derived from SDF-1&alpha; (Stromal cell Derived Factor-1, with alternative splicing alpha) had been previously shown to retain much of the binding and signaling activity of the full-length molecule17. We speculated that peptides of Nef might retain some of the apoptotic activity of the full protein, and that these peptides would necessarily contain the Nef apoptotic domain(s). To identify these peptides, and consequently the Nef apoptotic domain(s), we obtained a set of 20 HIV-1 Nef scanning peptides from the NIH AIDS Research and Reference Reagent Program. These 20-aa peptides, each overlapping 10 amino acids of its neighbor, are named by the number of their last amino acid, i.e. N20 spans Nef amino acids 1-20, N30 spans Nef amino acids 11-30, etc16. We found that exposing T-cells extracellularly to specific peptides overlapping two distinct 10-aa domains in the full-length Nef protein induced apoptosis in these cells. Subsequent analysis of these Nef-derived apoptotic peptides revealed their ability to physically interact with the chemokine receptor CXCR4 on the surface of T-cells, with binding kinetics that allowed these peptides to competitively inhibit binding between CXCR4 and its natural ligand SDF-1&alpha;. Finally, the Nef-derived apoptotic peptides' interaction with CXCR4 was found to induce a stress response in these T-cells leading to apoptosis. This evidence allowed us to quickly map Nef's functional domains; a process that would have taken much longer using standard DNA mutagenic techniques such as alanine scanning mutagenesis. It also showed that these short Nef-derived peptides retained the biological function of the apoptotic domains in the full-length protein.
Having identified a role for extracellular Nef, i.e. apoptosis of T-cells, we sought a better understanding of how Nef was secreted from cells. Using a series of mutated Nef constructs, we mapped highly conserved Nef protein motifs in the N-terminal regions of both HIV Nef, and its Rhesus macaque equivalent SIVmac (Simian Immunodeficiency Virus) Nef, that are critical for exNef secretion13 . One of these motifs, the Secretion Modification Region (SMR; 66VGFPV70), was particularly critical, as alanine replacement of any of its five amino acids either greatly reduced or abolished exNef secretion. When delivered to cells via Active Motif's Chariot Protein Delivery Reagent, a peptide containing the SMR attached to a FLAG peptide sequence (SMRwt) was found to inhibit exNef secretion from both Nef-transfected and HIV-infected cells 18. Based on our previous experience with peptides, we decided to use this peptide to elucidate the molecules and mechanisms underlying the Nef SMR's role in exNef secretion.
Using the SMRwt peptide as our "bait protein", we co-immunoprecipitated cellular binding partners of the SMR from uninfected T-cell lysates18. The FLAG peptide sequence provided a convenient handle for capturing the SMRwt peptide using anti-FLAG affinity resin. Our previous finding that a single valine to alanine mutation in the SMRwt peptide was sufficient for abolishing its inhibition of exNef secretion identified a convenient, highly specific control peptide (SMRmut) that we used to rule out co-immunoprecipitated proteins not specific for the SMR. Using a peptide with the specific domain of interest rather than the full-length protein allowed us to bypass the screening of dozens of cellular factors that bind to other domains in Nef19.
Once we identified the SMR-binding partners, a logical next step was to show that the identified cellular binding partners are important for the biological function18. The standard procedure to accomplish this is to knockdown protein levels using target protein-specific miRNA or siRNA, and subsequently assay the effect on the biological function. We performed miRNA knockdown, which inhibits translation of the target mRNA, reducing production of that target, in this case a SMR-binding protein. This reduction of the target protein is an indirect effect, and possibly has a delayed effect on the biological function the targeted protein plays a role in. Consequently, we also employed the less common antibody inhibition technique to determine if directly disrupting the activity of the target protein reduces or eliminates the biological function. In this procedure, antibodies raised against the targeted protein are transfected into the cell using Chariot Reagent, and interact directly with the target protein either sequestering it from its site of function, or blocking its relevant binding domain. Inhibition of the target protein through this procedure directly disrupts its function, and can complement RNA knockdown procedures by further confirming the importance of the target protein to the biological function.
While Chariot Reagent is effective at delivering peptides and proteins into cells; this process is time-consuming and limits the types of experiments, e.g. prolonged or repeated exposures and in vivo animal studies that can be performed. Consequently, we added a cell-penetrating peptide (CPP) sequence to the SMRwt peptide (SMRwt-CPP)18 to generate a peptide that could be taken up by cells passively from the culture media. This version was as effective as the former at inhibiting exNef secretion.
The evidence from these published experiments demonstrates the ability of small peptides containing specific functional motifs to antagonize the function of the full-length protein through competitive inhibition, and to isolate the proteins that bind these motifs. One would expect that these techniques should be useful in many experimental protocols. They should also be effective in engineering novel peptide inhibitors of many cellular processes; a function that can be further enhanced by linkage to CPP sequences.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent </td>
 <td>Company </td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>20mer peptide set with 10 amino acid overlap</td>
 <td>NIH AIDS Research and Reference Reagent Program</td>
 <td>4641</td>
 <td></td>
 </tr>
 <tr>
 <td>TUNEL Assay</td>
 <td>Roche</td>
 <td>11 684 809 910</td>
 <td></td>
 </tr>
 <tr>
 <td>Chariot Protein Delivery Reagent</td>
 <td>Active Motif</td>
 <td>30100</td>
 <td></td>
 </tr>
 <tr>
 <td>Tecan GENEios fluorimeter</td>
 <td> (Tecan Group, Switzerland)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>96-well black microtiter plate</td>
 <td>Corning</td>
 <td>3792</td>
 <td></td>
 </tr>
 <tr>
 <td>anti-FLAG M2 Affinity Gel</td>
 <td>Sigma</td>
 <td>A2220</td>
 <td></td>
 </tr>
 <tr>
 <td>Dynabeads Protein G magnetic beads </td>
 <td>Invitrogen</td>
 <td>100.03D</td>
 <td></td>
 </tr>
 <tr>
 <td>MagnaSphere Technology Magnetic Separation Stand (two position)</td>
 <td>Promega Corp., Madison, WI</td>
 <td>Z5332</td>
 <td></td>
 </tr>
 <tr>
 <td>C-18 ZipTip </td>
 <td>Millipore</td>
 <td>ZTC18S096</td>
 <td>C18 Resin (0.6 &mu;l or 0.2 &mu;l bed volumes). Oligonucleotides or small (&lt;50 kDa) proteins/ peptides in aqueous solution</td>
 </tr>
 <tr>
 <td>MALDI TOF/TOF</td>
 <td>Bruker Daltonics ultraflex III TOF/TOF</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

peptide-based identification of functional motifs and their binding partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Mapping Biologically Functional Motifs using Peptides. Two regions were identified on Nef proteins that induce apoptosis. Peptide-driven apoptosis was observed (Figure 2) beginning with peptide N50 (aa30-50), peaking at N60 (aa40-60) and N70 (aa50-70), and diminishing to background levels at peptide N100 (aa80-100). The major Motif 1 (M1) peak, centering on aa50-60, induces &gt;80% of the apoptotic levels of the full-length Nef protein. A second, smaller apoptotic peak was observed spann...

Watch this Scientific Journal Video about Peptide-based Identification of Functional Motifs and their Binding Partners at JoVE.com

Peptide-based Identification of Functional Motifs and their Binding Partners

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

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12.3K Views.  Morehouse School of Medicine. Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef’s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Video: Peptide-based Identification of Functional Motifs and their Binding Partners

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 &#176;C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.

Here we present two protocols, one for measuring the specific growth rate and the other for the cell-binding ability of rotavirus using the plaque assay and RT-qPCR. These protocols are available for confirming the differences in phenotypes between rotavirus strains.

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known ...