Name: identification of nucleolar factors during hiv-1 replication through rev immunoprecipitation and mass spectrometry
Uploaded: 2019-06-26T12:30:00
Description: Watch this Scientific Journal Video about Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry at JoVE.com

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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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.

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	<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

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

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

In vitro Uncoating of HIV-1 Cores

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

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

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

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

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

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

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

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 ...