The authors acknowledge Dr. Barbara K. Felber and Dr. George N. Pavlakis for the HLfB adherent culture provided by the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), NIH. The authors also acknowledge financial sources provided by the NIH, Grants AI042552 and AI029329.

1. Cell culture
<ol>
	<li>Maintain HLfB in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1 mM sodium pyruvate within tissue-culture-treated 100 mm plates. Keep the cell cultures at 37 &#176;C in a humidified incubator supplied with 5% CO2. Passage confluent cells to a cell density of 1 x 106 cells/mL.</li>
	<li>Discard the cell culture media. Gently rinse the cells with 10 mL of 1x phosphate-buffered saline (PBS). Remove...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+B23+is+an+important+human+factor+for+the+nucleolar+localization+of+the+human+immunodeficiency+virus+protein+Tat.">Protein B23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein Tat.</a> Journal of Virology. 71, 4098-4102 (1997).</li><li>Szebeni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleolar+protein+B23+stimulates+nuclear+import+of+the+HIV-1+Rev+protein+and+NLS-conjugated+albumin.">Nucleolar protein B23 stimulates nuclear import of the HIV-1 Rev protein and NLS-conjugated albumin.</a> Biochemistry. 36, 3941-3949 (1997).</li><li>Truant, R., Cullen, B. 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The authors have nothing to disclose.

Mass spectrometric analyses comparing Rev-NoLS mutations and WT Rev in the presence of HIV-1 were assessed to understand nucleolar factors involved in the viral replication cycle. This would identify nucleolar components required for viral infectivity. Nucleolar B23 has a high affinity to Rev-NoLS and functions in the nucleolar localization of Rev3 and nucleocytoplasmic transport of Rev-bound HIV mRNAs22. The affinity of B23 with Rev-NoLS mutations, which contained single o...

The nucleolus is postulated as the interaction ground of various cellular host and viral factors required for viral replication. The nucleolus is a complex structure subdivided into three different compartments: the fibrillar compartment, the dense fibrillar compartment, and the granular compartment. The HIV-1 Rev protein localizes specifically within granular compartments; however, the reason for this localization pattern is unknown. In the presence of single-point mutations within the NoLS sequence (Rev mutations 4, 5, and 6), Rev maintains a nucleolar pattern and has previously been shown to rescue HIV-1HXB2 replication, however, with reduced efficiency compared to WT Rev1. All single-point mutations are unable to sustain the HIV-1NL4-3 infectious cycle. In the presence of multiple single-point mutations within the NoLS sequence (Rev-NoLS mutations 2 and 9), Rev has been observed to disperse throughout the nucleus and cytoplasm and has not been able to rescue HIV-1HXB2 replication1. The goal of this proteomics study is to decipher nucleolar as well as nonnucleolar cellular factors involved in the Rev-mediated HIV-1 infectious pathway. Rev immunoprecipitation conditions are optimized through interaction with the nucleolar B23 phosphoprotein, which has previously been shown to lose interaction with Rev in the presence of nucleolar mutations.
Rev cellular factors have been extensively studied in the past; however, this has been done in the absence of viral pathogenesis. One protein, in particular, that is characterized in this study through Rev interaction during HIV-1 replication is the nucleolar phosphoprotein B23 - also called nucleophosmin (NPM), numatrin, or NO38 in amphibians2,3,4. B23 is expressed as three isoforms (NPM1, NPM2, and NPM3) - all members of the nucleophosmin/nucleoplasmin nuclear chaperone family5,6. The NPM1 molecular chaperone functions in the proper assembly of nucleosomes, in the formation of protein/nucleic acid complexes involved in chromatin higher-order structures7,8, and in the prevention of aggregation and misfolding of target proteins through an N-terminal core domain (residues 1-120)9. NPM1 functionality extends to ribosome genesis through the transport of preribosomal particles between the nucleus and cytoplasm10,11, the processing of preribosomal RNA in the internal transcribed spacer sequence12,13, and arresting the nucleolar aggregation of proteins during ribosomal assembly14,15. NPM1 is implicated in the inhibition of apoptosis16 and in the stabilization of tumor suppressors ARF17,18 and p5319, revealing its dual role as an oncogenic factor and tumor suppressor. NPM1 participates in the cellular activities of genome stability, centrosome replication, and transcription. NPM1 is found in nucleoli during cell cycle interphase, along the chromosomal periphery during mitosis, and in prenucleolar bodies (PNB) at the conclusion of mitosis. NPM2 and NPM3 are not as well-studied as NPM1, which undergoes altered expression levels during malignancy20.
NPM1 is documented in the nucleocytoplasmic shuttling of various nuclear/nucleolar proteins through an internal NES and NLS9,21 and was previously reported to drive the nuclear import of HIV-1 Tat and Rev proteins. In the presence of B23-binding-domain-&#946;-galactosidase fusion proteins, Tat mislocalizes within the cytoplasm and loses transactivation activity; this demonstrates a strong affinity of Tat for B232. Another study established a Rev/B23 stable complex in the absence of RRE-containing mRNAs. In the presence of RRE mRNA, Rev dissociates from B23 and binds preferably to the HIV RRE, leading to the displacement of B2322. It is unknown where, at the subnuclear level, Tat transactivation and the Rev exchange process of B23 for HIV mRNA take place. Both proteins are postulated to enter the nucleolus simultaneously through B23 interaction. The involvement of other host cellular proteins in the HIV nucleolar pathway is expected. The methods described in this proteomics investigation will help elucidate the interplay of the nucleolus with host cellular factors involved during HIV-1 pathogenesis.
The proteomics investigation was initiated through the expression of Rev NoLS single-point mutations (M4, M5, and M6) and multiple arginine substitutions (M2 and M9) for HIV-1HXB2 production. In this model, a HeLa cell line stably expressing Rev-deficient HIV-1HXB2 (HLfB) is transfected with WT Rev and Rev nucleolar mutations containing a flag tag at the 3&#39; end. The presence of WT Rev will allow viral replication to occur in HLfB culture, in comparison to Rev-NoLS mutations that do not rescue Rev deficiency (M2 and M9), or allow viral replication to occur but not as efficiently as WT Rev (M4, M5, and M6)1. The cell lysate is collected 48 h later after viral proliferation in the presence of Rev expression and subjected to immunoprecipitation with a lysis buffer optimized for Rev/B23 interaction. Lysis buffer optimization using varying salt concentrations is described, and protein elution methods for HIV-1 Rev are compared and analyzed in silver-stained or Coomassie-stained SDS-PAGE gels. The first proteomics approach involves the direct analysis of an eluted sample from expressed WT Rev, M2, M6, and M9 by tandem mass spectrometry. A second approach by which the eluates of WT Rev, M4, M5, and M6 underwent a gel extraction process is compared to the first approach. Peptide affinity to Rev-NoLS mutations in comparison to WT Rev is analyzed and the protein identification probability displayed. These approaches reveal potential factors (nucleolar and nonnucleolar) that participate in HIV-1 mRNA transport and splicing with Rev during HIV-1 replication. Overall, the cell lysis, IP, and elution conditions described are applicable to viral proteins of interest for the understanding of host cellular factors that activate and regulate infectious pathways. This is also applicable to the study of cellular host factors required for the persistence of various disease models. In this proteomics model, HIV-1 Rev IP is optimized for B23 interaction to elucidate nucleolar factors involved in nucleocytoplasmic shuttling activity and HIV-1 mRNA binding. Additionally, cell lines stably expressing infectious disease models that are deficient for key proteins of interest can be developed, similar to the HLfB cell line, to study infectious pathways of interest.

<table border="0" cellpadding="0" cellspacing="0" style="width:964px;" width="964">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Acetic acid</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A38S-212</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Acetonitrile</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A955-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Acrylamide:Bisacrylamide</td>
			<td style="width:321px;">BioRad</td>
			<td style="width:119px;">1610158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ammonium bicarbonate</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A643-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ammonium persulfate</td>
			<td style="width:321px;">Sigma-Aldrich</td>
			<td style="width:119px;">7727-54-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">ANTI-Flag M2 affinity gel</td>
			<td style="width:321px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag M2 mouse monoclonal IgG</td>
			<td>Sigma-Aldrich</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">BioMax MS film</td>
			<td style="width:321px;">Carestream</td>
			<td style="width:119px;">8294985</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Bio-Rad Protein Assay Dye Reagent Concentrate, 450 mL</td>
			<td style="width:321px;">Bio-Rad</td>
			<td style="width:119px;">5000006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">B23 mouse monoclonal IgG</td>
			<td style="width:321px;">Santa Cruz Biotechnologies</td>
			<td style="width:119px;">sc-47725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Bromophenol blue</td>
			<td style="width:321px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Carnation non-fat powdered milk</td>
			<td style="width:321px;">Nestle</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Cell scraper</td>
			<td style="width:321px;">ThermoFisher Scientific</td>
			<td style="width:119px;">179693PK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">C18IonKey nanoTile column</td>
			<td style="width:321px;">Waters</td>
			<td>186003763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 100-mm TC-treated culture dishes</td>
			<td>Fisher Scientific</td>
			<td>08-772-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol</td>
			<td>Thermo Scientific</td>
			<td>J1539714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">1 x DPBS</td>
			<td style="width:321px;">Corning</td>
			<td style="width:119px;">21-030-CVRS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">ECL Estern blotting substrate</td>
			<td style="width:321px;">Pierce</td>
			<td style="width:119px;">32106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ethanol, 200 proof</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A409-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">FBS</td>
			<td style="width:321px;">Gibco</td>
			<td style="width:119px;">16000044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Formic Acid</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A117-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">GelCode blue stain reagent</td>
			<td style="width:321px;">ThermoFisher</td>
			<td style="width:119px;">24590</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Glycerol</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">goat-anti-mouse IgG-HRP</td>
			<td style="width:321px;">Santa Cruz Biotechnologies</td>
			<td style="width:119px;">sc-2005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Iodoacetamide</td>
			<td style="width:321px;">ACROS Organics</td>
			<td style="width:119px;">122270050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">KimWipe delicate task wiper</td>
			<td style="width:321px;">Kimberly Clark Professional</td>
			<td style="width:119px;">34120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">L-glutamine</td>
			<td style="width:321px;">Gibco</td>
			<td style="width:119px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Methanol</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">NanoAcuity UPLC</td>
			<td style="width:321px;">Waters</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pierce Silver Stain Kit</td>
			<td style="width:321px;">Thermo Scientific</td>
			<td style="width:119px;">24600df</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">15-mL Polypropylene conical tube</td>
			<td style="width:321px;">Falcon</td>
			<td style="width:119px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Prestained Protein Ladder, 10 to 180 kDa</td>
			<td style="width:321px;">Thermo Scientific</td>
			<td style="width:119px;">26616</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Protease inhibitor cocktail</td>
			<td style="width:321px;">Roche</td>
			<td style="width:119px;">4693132001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Purified BSA</td>
			<td style="width:321px;">New England Biolabs</td>
			<td style="width:119px;">B9001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PVDF&#160; Western blotting membrane</td>
			<td style="width:321px;">Roche</td>
			<td style="width:119px;">3010040001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Sodium Pyruvate</td>
			<td style="width:321px;">Gibco</td>
			<td style="width:119px;">11360070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">10 x TBS</td>
			<td style="width:321px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP2471500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">TEMED</td>
			<td style="width:321px;">BioRad</td>
			<td style="width:119px;">1610880edu</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Triton X-100 detergent solution</td>
			<td style="width:321px;">BioRad</td>
			<td style="width:119px;">1610407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizaic source</td>
			<td style="width:321px;">Waters</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">trypsin-EDTA</td>
			<td style="width:321px;">Corning</td>
			<td style="width:119px;">25-051-CIS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Tween 20</td>
			<td style="width:321px;">BioRad</td>
			<td style="width:119px;">1706531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Synapt G2 mass spectrometer</td>
			<td style="width:321px;">Waters</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Whatman filter paper</td>
			<td style="width:321px;">Tisch Scientific</td>
			<td>10427813</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of nucleolar factors during hiv-1 replication through rev immunoprecipitation and mass spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Rev-NoLS single- and multiple-point arginine mutations, corresponding to a variety of subcellular localization patterns, were examined in their ability to interact with cellular host factors in comparison to WT Rev. WT Rev-3&#39;flag and pcDNA-flag vector were expressed in HLfB culture. Protein complexes were processed from total cell lysate and stained with silver stain reagent. Rev-NoLS-3&#39;flag is detectable (approximately 18 kDa) in three different lysis buffer conditions containing various concentrations ...

Watch this Scientific Journal Video about Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry at JoVE.com

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...

Our protocol provides alternative methods for the identification and characterization of viral, nucleolar, and non-nucleolar host factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways. Troubleshooting techniques are described for modification to other protein models.Demonstrating the procedure will be Haitang Li and Pritsana Chomchan, scientists in the Molecular and Cellular Biology Department at the Beckman Research Institute at the City of Hope, and Roger Moore, members of the City of Hope Proteomics Core and the Department of Molecular Immunology. Begin by growing an HLfB stably expressing HeLa cell line culture in three, 100-millimeter culture plates to a two-times-10-to-the-six-cells-per-milliliter density. When the cells reach the appropriate concentration, mix 20 micrograms of plasmid containing the Rev nucleolar localization signal three-prime flag mutation of interest with 1.76 milliliters of TE 79/10 in a 15-milliliter tube, followed by the addition of 240 microliters of two-molar calcium chloride, with thorough mixing.Next, transfer the contents of the tube dropwise into a new 15-milliliter tube containing two milliliters of 2x HBS, followed by mixing on a vortex machine. After 30 minutes at room temperature, vortex the precipitation. Add one milliliter of the suspension dropwise to each of the three, 100-millimeter HLfB culture plates while gently swirling the medium.After a six-hour incubation in the cell culture incubator, replace the transfection mixture with 10 milliliters of fresh culture medium, and return the cells to the incubator for another 42 hours. 48 hours after the transfection, place each plate on a bed of ice within a biohazard level two tissue culture hood. Remove the cell culture media, and gently rinse the cells with 10 milliliters of prechilled PBS, without disrupting the cell layers.Discard the PBS after rinsing the cells. Next, treat the cells with one milliliter of lysis buffer, supplemented with protease inhibitor cocktail, per plate. Tilting the plates to gather the cells into a pool, use a cell scraper to manually disrupt the layers.Using a 1, 000-microliter micropipette, pool the lysate from each plate into a prechilled, 15-milliliter tube for a 15-minute incubation on ice, with vortexing every five minutes. At the end of the incubation, aliquot the lysates into three, 1.5-milliliter microcentrifuge tubes, and collect the lysates by centrifugation. Transfer the supernatant into a new 15-milliliter tube, and obtain the viral protein lysate concentration, as described in the manuscript.For coimmunoprecipitation of the Rev nucleolar localization signal three-prime flag, rinse 25 microliters of M2 affinity gel beads three times with 500 microliters of fresh lysis buffer, supplemented with protease inhibitor cocktail, per wash. Perform these steps in a cold room or with the reagents on ice. After the last rinse, add a one-milligram-per-milliliter concentration of viral protein lysate to the rinsed beads in a final volume of five milliliters of lysis buffer.Incubate the reaction for three hours at four degrees Celsius with rotation. At the end of the incubation, pellet the viral protein lysate-conjugated beads by centrifugation. Collect the supernatant for measurement of the post-immunoprecipitation lysate protein concentration.See the manuscript for sample storage instructions. Add 750 microliters of lysis buffer to the viral protein lysate-conjugated bead pellet, transfer the beads into a 1.5-milliliter microcentrifuge tube, and wash the beads on a rotator for five minutes at four degrees Celsius. Collect the beads by centrifugation for an additional two washes on the rotator, as just demonstrated.After the last wash, use a pinched, long, gel-loading tip to remove any traces of lysis buffer from the bead-coimmununoprecipitation complex. Resuspend the beads in 55 microliters of 2x loading buffer. Then, boil the sample at 95 degrees Celsius for 10 minutes.Load 25 microliters of eluate onto one western blot gel and one Coomassie staining SDS-PAGE gel. After completion of SDS-PAGE gel electrophoresis, rinse the gel three times in 25 milliliters of ultrapure water for 15 minutes. After the last wash, add 100 milliliters of Coomassie stain reagent onto the gel.See the manuscript for the complete staining protocol. For digestion of the gel bands, first use a clean razor blade to cut the gel bands from the entire lane of the gel, representing each mutation, into approximately five-millimeter cubes. Place each band in a clean, 0.5-milliliter microcentrifuge tube.Cover the bands two times with 100-millimolar ammonium bicarbonate in a one-to-one acetonitrile-to-water solution at room temperature for 15 minutes per wash. Then, dry the gel pieces for five minutes in a vacuum centrifuge. To reduce the proteins, cover the dried gel pieces with 10-millimolar dithiothreitol in 100-millimolar ammonium bicarbonate for a one-hour incubation at 56 degrees Celsius, followed by alkylation in 100-millimolar iodoacetamide in water for one hour at room temperature in the dark.Next, shrink and reswell the gel pieces two times with acetonitrile, followed by 100-millimolar ammonium bicarbonate, both with gentle shaking at room temperature for 15 minutes. After the second reswelling, dry the gel pieces for five minutes in a vacuum centrifuge. Swell the gel pieces with 50 nanograms per microliter of sequencing-grade modified trypsin in 100-millimolar ammonium bicarbonate.After five minutes, cover the gel pieces with 100-millimolar ammonium bicarbonate, and allow them to reswell completely, adding additional 100-millimolar ammonium bicarbonate so the swollen gel pieces are completely covered. Then, incubate the gel pieces overnight at 37 degrees Celsius, stopping the reaction with 1/10 of the volume of the samples with 10%formic acid in water the next morning, and collect the supernatant from each tube. Rev nucleolar localization signal three-prime flag is detectable under three different lysis buffer conditions containing various concentrations of sodium chloride.B23 is also detectable in lysis buffer containing the lower salt concentration, barely detectable in lysis buffer containing the medium salt concentration, and undetectable in lysis buffer containing a high salt concentration. B23 detection is optimal in lysis buffer containing the low sodium chloride concentration with wild-type Rev three-prime flag and loses affinity with Rev nucleolar localization signal M1 three-prime flag. B23 affinity with wild-type Rev three-prime flag also decreases with a higher salt concentration, and the pcDNA negative control does not yield nonspecific immunodetection after immunoprecipitation under both lysis buffer conditions.Rev nucleolar localization signal three-prime flag is most detectable after elution through boiling in 2x sample loading buffer, while B23 is detectable under both the 37-and 95-degree Celsius sample buffer incubation conditions. After immunoprecipitation and silver staining, as demonstrated, wild-type Rev and Rev nucleolar localization signals M2, M6, and M9 are detectable at 18 kilodaltons. Bands corresponding to B23 protein are also observed at the 37-kilodalton marker.Staining with Coomassie reagent further demonstrates the detectability of Rev nucleolar localization signal three-prime flag in the presence and absence of mutations at 18 kilodaltons. Use as many controls, both positive and negative, as necessary for references that pertain to your disease model during the troubleshooting and screening process for potential binding factors. All deletion and single-point mutations can be examined for dominant-negative activity through multimerization with wild-type Rev and utilized in the arrest of HIV-1 replication.

identification-nucleolar-factors-during-hiv-1-replication-through-rev

Research

JoVE Journal

Immunology and Infection

7.9K Görüntüleme.  Beckman Research Institute at the City of Hope. Protokolümüz, HIV-1 enfeksiyöz döngüsünü koruyan viral, nükleolar ve nükleolar olmayan konak faktörlerinin tanımlanması ve karakterizasyonu için alternatif yöntemler sunmaktadır. Bu yaklaşımda kullanılan kavramlar, az çalışılan yolların karakterizasyonunu gerektiren diğer viral ve hastalık modelleri için de geçerlidir. Sorun giderme teknikleri diğer protein modellerinde değişiklik için açıklanmıştır.Prosedürü gösteren Haitang Li ve Pritsana Chomch...