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...

<ol>
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Invest. 116 (6), 1514-1524 (2006).</li><li>Mueller, Y. M., Do, D. H., Altork, S. R., Artlett, C. M., Gracely, E. J., Katsetos, C. D., Legido, A., Villinger, F., Altman, J. D., Brown, C. R., Lewis, M. G., Katsikis, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+treatment+during+acute+simian+immunodeficiency+virus+(SIV)+infection+increases+viral+set+point+and+accelerates+disease+progression+despite+the+induction+of+stronger+SIV-specific+CD8++T+cell+responses.">IL-15 treatment during acute simian immunodeficiency virus (SIV) infection increases viral set point and accelerates disease progression despite the induction of stronger SIV-specific CD8+ T cell responses.</a> J. Immunol. 180 (1), 350-360 (2008).</li><li>Ali, S. A., Huang, M. B., Campbell, P. E., Roth, W. W., Campbell, T., Khan, M., Newman, G., Powell, F., Powell, M. D., Bond, V. 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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...

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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.