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

Summary

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.

Abstract

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.

Introduction

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-1_HXB2 replication, however, with reduced efficiency compared to WT Rev¹. All single-point mutations are unable to sustain the HIV-1_NL4-3 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-1_HXB2 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 amphibians^2,3,4. B23 is expressed as three isoforms (NPM1, NPM2, and NPM3) - all members of the nucleophosmin/nucleoplasmin nuclear chaperone family^5,6. The NPM1 molecular chaperone functions in the proper assembly of nucleosomes, in the formation of protein/nucleic acid complexes involved in chromatin higher-order structures^7,8, 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 cytoplasm^10,11, the processing of preribosomal RNA in the internal transcribed spacer sequence^12,13, and arresting the nucleolar aggregation of proteins during ribosomal assembly^14,15. NPM1 is implicated in the inhibition of apoptosis¹⁶ and in the stabilization of tumor suppressors ARF^17,18 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 NLS^9,21 and was previously reported to drive the nuclear import of HIV-1 Tat and Rev proteins. In the presence of B23-binding-domain-β-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-1_HXB2 production. In this model, a HeLa cell line stably expressing Rev-deficient HIV-1_HXB2 (HLfB) is transfected with WT Rev and Rev nucleolar mutations containing a flag tag at the 3' 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.

Protocol

1. Cell culture

1. Maintain HLfB in Dulbecco’s modified Eagle’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 °C in a humidified incubator supplied with 5% CO₂. Passage confluent cells to a cell density of 1 x 10⁶ cells/mL.
2. Discard the cell culture media. Gently rinse the cells with 10 mL of 1x phosphate-buffered saline (PBS). Remove and discard the 1x PBS without disrupting the cell layer.
3. Add 2 mL of 1x trypsin-EDTA solution to the cells. Rock the dish to coat the monolayer and incubate at 37 °C within a humidified chamber for 5 min.
4. Firmly tap the side of the dish with the palm of the hand to detach the cells. Resuspend the detached cells in 8 mL of fresh culture media. Spin the cells at 400 x g for 5 min.
5. Discard the culture media without disrupting the cell pellet. Resuspend the cell pellet in 10 mL of fresh culture media. Subculture 1 mL of concentrated cells with 9 mL of fresh culture media within tissue-culture-treated 100 mm plates.
   NOTE: For each Rev-NoLS mutation, 3x 100 mm HLfB or HeLa culture plates will yield enough protein lysate for western blot analysis and mass spectrometry. Add extra plates for a WT Rev positive control and negative control. The subculture cell volume will require optimization with the use of different cell types.

2. Expression of Rev-NoLS-3'flag mutations during HIV-1 replication

1. Grow the HLfB cell culture to a cell density of 2 x 10⁶ cells/mL. Prepare 4 mL of calcium phosphate-DNA suspension for each 100 mm plate as follows.
   1. Label two 15 mL tubes as 1 and 2. Add 2 mL of 2x HBS (0.05 M HEPES, 0.28 M NaCl, and 1.5 mM Na₂HPO₄ [pH 7.12]) to tube 1. Add TE 79/10 (1 mM Tris-HCl and 0.1 mM EDTA [pH 7.9]) to tube 2. The volume of TE 79/10 is 1.760 mL - the volume of DNA.
   2. Add 20 µg of plasmid containing the Rev-NoLs-3'flag mutation of interest to tube 2 and mix its contents through resuspension. Add 240 µL of 2 M CaCl₂ to tube 2 and mix again through resuspension.
   3. Transfer the mixture of tube 2 to tube 1 dropwise, gently mixing. Allow the suspension to sit at room temperature for 30 min. Vortex the precipitation.
2. Add 1 mL of the suspension dropwise to each of the 3-cell culture, 100 mm plates while gently swirling the media. Return the plates to the incubator and leave the transfection mixture for 6 h. Replace the transfection mixture with 10 mL of fresh culture media and incubate the cells for 42 h.

3. Collection of viral protein lysate

1. Discard the cell media 48 h posttransfection. Place each 100 mm plate on a bed of ice. Label 15 mL tubes for each Rev-NoLS mutation sample and place the tubes on ice.
2. Gently rinse the cells with 10 mL of prechilled 1x PBS without disrupting the cell layer. Discard the 1x PBS. Add 3 mL of lysis buffer (50 mM Tris-HCl [pH 8.0], 137 mM NaCl, and 1% X-100 detergent [see the Table of Materials]), treated with protease inhibitor cocktail, to each of the 100 mm plates.
3. Use a cell scraper to disrupt the cell layer. Tilt the plate and gently scrape and gather the cells into a pool. Collect the cell lysate using a 1,000 µL micropipette and mix the cell lysates from each of the three 100 mm plates in the prelabeled 15 mL tube.
4. Incubate the cell lysate on ice for 15 min, vortexing every 5 min. Centrifuge the cell lysate at 15,000 x g for 5 min.
5. Collect the protein supernatant without disrupting the cell debris pellet and transfer it to another sterile 15 mL tube. Obtain the viral protein lysate concentration using the Bradford method (see section 4).
6. Save an aliquot of the input sample (20 µg) for western immunoblot analysis. Add 2x sample buffer (20% glycerol, 0.02% bromophenol blue, 125 mM Tris-Cl [pH 6.8], 5% SDS, and 10% 2-mercaptoethanol) to the final volume. Boil it at 95 °C for 10 min, and store the input sample at -20 °C.

4. Bradford assay

NOTE: Prepare 10x bovine serum albumin (BSA) from 100x BSA stock before generating protein standard curves.

1. Aliquot water into microcentrifuge tubes for the following blank and standards: Blank = 800 µL; Standard 1 = 2 mg/mL, 798 µL; Standard 2 = 4 mg/mL, 796 µL; Standard 3 = 6 mg/mL, 794 µL; Standard 4 = 8 mg/mL, 792 µL; Standard 5 = 10 mg/mL, 790 µL. Aliquot 10x BSA into the following designated standards: Standard 1 = 2 µL; Standard 2 = 4 µL; Standard 3 = 6 µL; Standard 4 = 8 µL; Standard 5 = 10 µL.
2. Prepare a mixture of protein samples by mixing 795 µL of water with 5 µL of protein samples. Add 200 µL of protein assay dye reagent (see the Table of Materials) to each blank, standard, and protein sample. Vortex the samples briefly for an even mixture and incubate at room temperature (18-20 °C) for 5 min.
3. Transfer the blank, standards, and protein samples to cuvettes. Measure the protein concentrations at an OD of 595 nm.

5. Coimmunoprecipitation of Rev-NoLS-3'flag

1. Rinse 25 µL of M2 affinity gel beads (see the Table of Materials) with 500 µL of lysis buffer, treated with protease inhibitor cocktail. Rinse more affinity gel beads for every mutation sample and controls.
   NOTE: Prepare enough M2 affinity gel beads for two gels (50 µL) - one for western immunoblot analysis and the other for protein staining and mass spectrometry.
2. Spin at 820 x g for 2 min at 4 °C. Remove the supernatant. Rinse 2x more.
3. Add viral protein lysate (1 mg/mL in 5 mL of total volume) to the prerinsed M2 affinity beads. Adjust the total volume using lysis buffer.
4. Incubate the reaction for 3 h, rotating at 4 °C. Centrifuge the M2 affinity beads/viral protein lysate at 820 x g for 1 min.
5. Collect the supernatant and save an aliquot of post-IP sample (20 µg) for western immunoblot analysis. Measure the protein concentration of the post-IP lysate. Collect 20 µg for western immunoblotting.
6. Add 2x sample buffer to the final volume. Boil it at 95 °C for 10 min, and store the post-IP sample at -20 °C.
7. Rinse the M2 beads with 750 µL of lysis buffer and wash the beads on a rotator at 4 °C for 5 min. Centrifuge the M2 beads at 820 x g and discard the supernatant.
8. Repeat steps 5.7 for two more washes on a rotator at 4 °C for 5 min. After the third wash, remove any traces of lysis buffer from the M2 beads/co-IP complex using a long gel-loading tip.
   NOTE: Pinch the end of the gel-loading tip with flat tweezers before removing any trace amounts of the lysis buffer. This will prevent the disruption and uptake of the M2 beads.
9. Resuspend the M2 beads in 55 µL of 2x loading buffer. Boil the sample at 95 °C for 10 min.
10. Load 25 µL of eluate onto two separate SDS-PAGE gels (one gel for western immunoblotting and the other for Coomassie staining).

6. Preparation of SDS-PAGE gels

1. Cast two 15% SDS-acrylamide resolving gels by mixing the following reagents in a 50 mL tube (at a final volume of 40 mL, enough for four gels): 4.16 mL of ultrapure water, 15 mL of 40% acrylamide:bisacrylamide (29:1), 10 mL of 1.5 M Tris-HCl (pH 8.8), 400 µL of 10% SDS, 400 µL of 10% ammonium persulfate, and 40 µL of TEMED.
2. Mix the resolving gel by inverting the 50 mL tube several times. Pipette the resolving gel mixture into a precleaned western gel apparatus (four gels - three for western immunoblotting and one for Coomassie/silver staining).
3. Gently pipette enough water to cover the top layer of the gel mixture. Allow the resolving gel to polymerize.
4. Pour the water layer from the resolving gel, using a delicate task wiper (see the Table of Materials) to absorb any excess water.
5. Cast two 5% SDS-acrylamide stacking gels by mixing the following reagents in a 50 mL tube (at a final volume of 20 mL, enough for four gels): 11.88 mL of ultrapure water, 2.5 mL of 40% acrylamide:bisacrylamide (29:1), 5.2 mL of 1.5 M Tris-HCl (pH 8.8), 200 µL of 10% SDS, 200 µL of 10% ammonium persulfate, and 20 µL of TEMED.
6. Mix the stacking gel by inverting the 50 mL tube several times. Pipette the stacking gel mixture above the resolving gel to the top of the apparatus.
7. Place a gel cassette comb containing the appropriate number of lanes into the stacking gel. Absorb any overflow of the gel mixture using a delicate task wiper (see the Table of Materials). Allow the stacking gel to polymerize completely.
8. Flood the Western gel apparatus with 1x western running buffer (5x concentration: 250 mM Tris-Cl [pH 8.3], 1.92 M glycine, 0.5% SDS, and 10 mM EDTA).
9. Gently pull the gel cassette comb from the stacking gel. Allow the 1x western running buffer to fill the loading wells. Flush each well with 1x western running buffer using a syringe prior to loading the samples.
10. Load the western immunoblot samples into each corresponding gel (input samples, coimmunoprecipitated samples, and post-IP samples). Load the western gel protein markers.
11. Load the coimmunoprecipitated samples for the Coomassie/silver staining into another gel. Load the western gel protein markers.
12. Connect the running gel apparatus to a power source and run the gels at 100 V until the loading dye reaches the resolving gel. Increase the voltage to 140 V until the loading dye reaches the bottom of the resolving gel.

7. Western blot transfer

1. Disassemble the western gel apparatus. Slice and discard the stacking gel, leaving the resolving gel intact.
2. Gently transfer the resolving gels to a clean tray filled with western transfer buffer (25 mM Tris, 194 mM glycine, 0.005% SDS, 20% methanol) and soak them for 15 min.
3. Assemble the gel transfer apparatus as follows.
   1. Cut three PVDF transfer membranes and six pieces of filter paper (see the Table of Materials) to the size of the resolving gel.
   2. Soak the PVDF membrane in methanol for 5 min. Hydrate it in water for 5 min. Place the PVDF membrane in the western transfer buffer until ready to use.
   3. Place the gel holder cassette in a glass baking tray filled partially with western transfer buffer, with the black side at the bottom.
   4. Place a foam pad soaked with western transfer buffer against the black side of the gel holder cassette.
   5. Wet a piece of filter paper in western transfer buffer and place it on top of the foam pad. Place the resolving gel on top of the filter paper.
      NOTE: Place the resolving gel in the correct loading orientation to be transferred to the PVDF membrane.
   6. Place one PVDF transfer membrane on top of the resolving gel. Wet a piece of filter paper with western transfer buffer and place it on top of the PVDF transfer membrane.
   7. Place another foam pad soaked with western transfer buffer on top of the filter paper. Carefully fold the white side of the gel holder cassette on top of the soaked foam pad. Lock the cassette tightly.
   8. Place the gel holder cassette into the transfer apparatus electrode assembly. Repeat steps 7.3.4-7.3.8 for each remaining resolving gel.
   9. Fill the transfer apparatus tank with western transfer buffer. Place a stirring rod into the apparatus tank.
   10. Place the apparatus tank on top of a stir plate. Adjust the stir setting to 5-6, making sure that the stir bar is not stuck or hitting the gel holder cassettes.
   11. Connect the gel transfer apparatus to a power source and transfer the gel at 100 V for 1 h at 4 °C.

8. Immunoblotting

1. Remove the gel holder cassette and place the black side down against a clean glass baking tray. Open the cassette and carefully discard the foam pad and filter paper. Mark a corner of the PVDF membrane to identify the correct loading orientation. Keep the membrane wet.
   NOTE: The PVDF membrane can be air-dried and stored in a clean, sealed container. Rehydrate the membrane by repeating steps 7.3.2.
2. Place the membrane in 100 mL of blocking solution (5% milk, 1x TBS, and 0.1% Tween 20). Block the membrane by gentle rocking at room temperature (18-20 °C) for 1 h.
3. Cut across the membrane above the 25 kDa protein marker. Place the top portion of the membrane, containing protein bands larger than 25 kDa, in blocking solution containing B23 mouse monoclonal IgG₁ (1:500 dilution). Block overnight, rocking at 4 °C.
4. Place the bottom portion of the membrane, containing protein bands smaller than 25 kDa, in blocking solution containing M2 mouse monoclonal IgG₁ (1:1,000 dilution, see the Table of Materials). Block overnight, rocking at 4 °C.
5. Wash the membrane 3x for 10 min in 25 mL of western wash solution (1x TBS, 0.1% Tween 20) on a rocking platform.
6. Incubate the membranes in goat-anti-mouse IgG₁-HRP (1:5,000 dilution) diluted in blocking solution for 1 h at room temperature. Wash the membrane 3x for 10 min in 25 mL of western wash solution on a rocking platform.
7. Prepare chemiluminescence western blotting substrate. Use a p1000 micropipette to add the substrate to the membrane.
8. Develop each membrane in chemiluminescence western blotting substrate for 5 min. Remove the membrane from the substrate. Absorb excess substrate using a delicate task wiper (see the Table of Materials).
9. Place the membrane into a clean sheet protector taped to the inside of a cassette. Take the cassette into a dark room and place one sheet of film into the cassette. Lock the cassette in place and incubate for 5–15 min. Remove the film from the cassette and develop it.

9. Coomassie staining

1. Disassemble the western gel apparatus. Slice and discard the stacking gel, leaving the resolving gel intact. Gently transfer the resolving gel to a clean tray filled with 25 mL of ultrapure water.
2. Incubate the gel on a rocking platform for 15 min. Use gentle rocking to prevent the resolving gel from breaking. Discard the ultrapure water and repeat the washing step 2x more.
   NOTE: If SDS bubbles remain after the washing steps, the gel can be washed in ultrapure water overnight. Residual SDS can cause high background staining of the gel.
3. Mix the Coomassie stain reagent by inverting the bottle (see the Table of Materials). Place 100 mL of Coomassie stain reagent to cover the resolving gel and incubate the gel on a rocking platform for 1 h. Discard the Coomassie stain reagent and wash the gel in deionized water on a rocking platform for 15 min.
4. Discard the deionized water. Repeat the washing step 2x more. Continue to wash the gel until the desired resolution of protein bands is observed.

10. Silver staining

1. Disassemble the western gel apparatus. Slice and discard the stacking gel, leaving the resolving gel intact. Gently transfer the resolving gel to a clean tray filled with 25 mL of ultrapure water.
2. Incubate the gel on a rocking platform for 15 min. Use gentle rocking to prevent the resolving gel from breaking. Discard the ultrapure water and repeat the washing step 2x more.
   NOTE: If SDS bubbles remain after the washing steps, the gel can be washed in ultrapure water overnight. Residual SDS can cause high background staining of the gel.
3. Fix the gel in 30% ethanol:10% acetic acid solution (6:3:1 water:ethanol:acetic acid) overnight at room temperature. Wash the gel in a 10% ethanol solution for 5 min at room temperature. Replace the ethanol solution and wash for another 5 min.
4. Prepare sensitizer working solution from the Pierce Silver Stain Kit by mixing one-part silver stain sensitizer with 500 parts ultrapure water (50 µL of sensitizer with 25 mL of ultrapure water). Incubate the resolving gel in the sensitizer working solution for 1 min. Wash the gel in ultrapure water for 1 min, replace the water, and wash the gel again for 1 min.
5. Prepare stain working solution by mixing one-part silver stain enhancer with 50 parts silver stain (500 µL of enhancer with 25 mL of silver stain). Incubate the gel in stain working solution for 30 min.
6. Prepare developer working solution by mixing 1 part silver stain enhancer with 50 parts silver stain developer (500 µL of enhancer with 25 mL of developer). Prepare 5% acetic acid solution as stop solution. Wash the gel with ultrapure water for 1 min, replace the water, and wash the gel for an additional 1 min.
7. Replace the water with developer working solution and incubate until the desired protein band intensity is resolved (5 min). Replace the developer working solution with stop solution and incubate for 10 min.

11. In-gel reduction, alkylation, and digestion of Coomassie-stained gel bands

1. Cut the gel bands from the gel using a clean razor blade. Cut each gel band into approximately 5 mm cubes and place them in a clean 0.5 mL microcentrifuge tube.
2. Destain the gel pieces by covering them with 100 mM ammonium bicarbonate in 1:1 acetonitrile:water at room temperature for 15 min. Discard the supernatant. Repeat this step.
3. Dry the gel pieces for 5 min in a vacuum centrifuge. Reduce the proteins by covering the dried gel pieces with 10 mM dithiothreitol in 100 mM ammonium bicarbonate and incubating them for 1 h at 56 °C.
4. Pipette off any supernatant. Alkylate the proteins by covering the gel pieces with 100 mM iodoacetamide in water and incubating them for 1 h at room temperature in the dark.
5. Pipette off the supernatant and shrink the gel pieces by covering them with acetonitrile and shaking them gently at room temperature for 15 min. Pipette off the supernatant and reswell the gel pieces by covering them with 100 mM ammonium bicarbonate and shaking them gently at room temperature for 15 min.
6. Repeat step 11.5. Dry the gel pieces for 5 min in a vacuum centrifuge.
7. Cover the gel pieces with 50 ng/µL sequencing grade modified trypsin (see the Table of Materials) in 100 mM ammonium bicarbonate. Allow the gel to swell for 5 min; then, pipette off any remaining solution. Cover the gel pieces with 100 mM ammonium bicarbonate and allow them to reswell completely, adding additional 100 mM ammonium bicarbonate so the gel pieces are completely covered.
8. Incubate the gel pieces overnight at 37 °C. Stop the reaction by adding 1/10 of the volume of 10% formic acid in water. Collect the supernatant from each tube.
9. Extract the gel pieces by covering them with 1% formic acid in 60% acetonitrile and incubating them for 15 min with gentle shaking.
10. Reduce the volume of the combined supernatants to less than 20 µL in a vacuum centrifuge, while taking care to avoid drying the supernatants completely. Add 1% formic acid to bring the total volume back to 20 µL.

12. Liquid chromatography/mass spectrometry

NOTE: The samples were analyzed using a mass spectrometer equipped with ultra HPLC, a nanospray source, and a column (see the Table of Materials). Solvents A and B are 0.1% formic acid in water and acetonitrile, respectively.

1. Load the digested proteins into high recovery polypropylene autosampler vials. Load the vials into the sample manager of a UPLC system.
2. Inject 6 µL of each sample. Load each sample onto the trapping column of the nanotile for 1.5 min at 8 µL/min, using 99% solvent A/1% solvent B.
3. Elute the peptides into the mass spectrometer with a linear gradient from 3% to 35% of solvent B over 30 min, followed by a gradient from 35% to 50% of solvent B over 4 min and 50% to 90% of solvent B over 1 min. Maintain 90% acetonitrile for 3 min; then reduce the %B back to 3% over 5 min.
4. Acquire positive ion profile mass spec data in resolution (20,000 resolution) mode. Acquire data from 100 to 2,000 Da at a rate of one scan every 0.6 s. Acquire data in MS^E mode by alternating scans with no collision energy and scans with elevated collision energy.
5. For the elevated collision energy, ramp the collision energy in the Trap cell from 15 V to 40 V. Acquire a lock mass scan every 30 s, using the +2 ion of [Glu₁]-Fibrinopeptide B as the lock mass. Acquire a data file using a blank injection of solvent A, using the same acquisition method between each pair of samples to control the carryover.

13. Data analysis for mass spectrometry

1. Copy the mass spectrometry results files to the computer running a quantitative and qualitative proteomics research platform (e.g., ProteinLynx Global Server). Data analysis is highly CPU-intensive and should be performed on a separate, high-performance data analysis computer.
2. Create a new project for the data. Create a new microtiter plate representing the autosampler plate. Assign the samples to the same position in the microtiter plate as their position in the autosampler.
3. Assign each sample processing parameters. Parameters to use are automatic chromatographic peak width and MSTOF resolution; low-energy threshold, 100 counts; elevated-energy threshold, 5 counts; intensity threshold, 500 counts.
4. Assign each sample workflow parameters. Parameters to use are database, concatenated human SwissProt and HIV, with reversed sequences; automatic peptide and fragment tolerance; min fragment ion matches per peptide, 3; min fragment ion matches per protein, 7; min peptide matches per protein, 1; primary digest reagent, trypsin; missed cleavages, 1; fixed modifier reagents, carbamidomethyl C; variable modifier reagents, oxidation M; false discovery rate, 100.
5. Select the samples and choose Process Latest Raw Data. When the search completes, select the samples and choose Export Data to Scaffold (version 3). Open Scaffold, create a new file, and import each file exported from the proteomics platform as a new biosample using precursor ion quantitation.
6. When all files have been imported, proceed to the Load and Analyze Data screen. Select the same database used for the search and import data using LFDR scoring and standard experiment-wide protein grouping. Set display options to Protein Identification Probability, the protein threshold to 20%, the minimum number of peptides to 1, and the peptide threshold to 0% during the analysis.

Results

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'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'flag is detectable (approximately 18 kDa) in three different lysis buffer conditions containing various concentrations of NaCl ...

Discussion

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...

Materials

List of materials used in this article

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