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

Jerlisa Ann C. Arizala; Pritsana Chomchan; Haitang Li; Roger Moore; Helen Ge; Dominique  L. Ouellet; John  J. Rossi

doi:10.3791/59329

JoVE Journal > Immunology and Infection

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

Published: June 26, 2019

doi:

10.3791/59329

Jerlisa Ann C. Arizala^1,2, Pritsana Chomchan¹, Haitang Li¹, Roger Moore³, Helen Ge³, Dominique L. Ouellet¹, John J. Rossi^1,2

¹Molecular and Cellular Biology Department,Beckman Research Institute at the City of Hope, ²Irell & Manella Graduate School of Biological Sciences, ³Department of Molecular Immunology,Beckman Research Institute and the City of Hope

概要

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_NL4-3infectious 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 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-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

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.
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.
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.
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.
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

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 CaCl₂to 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.
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

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.
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.
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.
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.
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).
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.

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.
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.
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

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.
Spin at 820 x g for 2 min at 4 °C. Remove the supernatant. Rinse 2x more.
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.
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.
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.
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.
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.
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.
Resuspend the M2 beads in 55 µL of 2x loading buffer. Boil the sample at 95 °C for 10 min.
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

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.
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).
Gently pipette enough water to cover the top layer of the gel mixture. Allow the resolving gel to polymerize.
Pour the water layer from the resolving gel, using a delicate task wiper (see the Table of Materials) to absorb any excess water.
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.
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.
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.
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).
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.
Load the western immunoblot samples into each corresponding gel (input samples, coimmunoprecipitated samples, and post-IP samples). Load the western gel protein markers.
Load the coimmunoprecipitated samples for the Coomassie/silver staining into another gel. Load the western gel protein markers.
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

Disassemble the western gel apparatus. Slice and discard the stacking gel, leaving the resolving gel intact.
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.
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

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.
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.
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.
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.
Wash the membrane 3x for 10 min in 25 mL of western wash solution (1x TBS, 0.1% Tween 20) on a rocking platform.
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.
Prepare chemiluminescence western blotting substrate. Use a p1000 micropipette to add the substrate to the membrane.
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).
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

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.
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.
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.
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

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.
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.
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.
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.
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.
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.
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

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.
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.
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.
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.
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.
Repeat step 11.5. Dry the gel pieces for 5 min in a vacuum centrifuge.
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.
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.
Extract the gel pieces by covering them with 1% formic acid in 60% acetonitrile and incubating them for 15 min with gentle shaking.
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.

Load the digested proteins into high recovery polypropylene autosampler vials. Load the vials into the sample manager of a UPLC system.
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.
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.
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.
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

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.
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.
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.
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.
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.
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.

Representative 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 (137 mM, 200 mM, and 300 mM) in Figure 1. B23 was detectable (37 kDa) in lysis buffer containing lower salt concentration (137 mM, lanes 2–4), barely detectable in lysis buffer containing 200 mM NaCl, and undetectable in lysis buffer containing a high salt concentration (300 mM NaCl). In Figure 2, WT Rev-3'flag, Rev-NoLS M1-3'flag, and pcDNA-flag vector were expressed in HLfB culture. Protein complexes were processed from total cell lysates prepared from two lysis buffer conditions (137 mM and 200 mM NaCl). M2 mouse monoclonal IgG₁ was used in the detection of Rev-3'flag expression from cell lysates. B23 detection was optimal in lysis buffer containing 137 mM NaCl with WT Rev-3'flag (input and α-flag-Rev IP) but lost affinity with Rev-NoLS M1-3'flag. B23 affinity with WT Rev-3'flag decreased with a higher salt concentration in lysis buffer containing 200 mM NaCl. pcDNA negative control did not yield nonspecific immunodetection in input and α-flag-Rev IP of all lysis buffer conditions.

Elution conditions were optimized for Rev-NoLS-3'flag IP in Figure 3. WT Rev-3'flag and pcDNA-flag vector were expressed in HLfB culture. Protein complexes were processed from total cell lysates and eluted using three different conditions to eradicate light and heavy chain background (25 and 50 kDa) – 2x sample loading buffer at 37 °C for 15 min, 2x sample loading buffer at 95 °C for 3 min, and 3x flag peptide at 4 °C for 30 min. Rev NoLS-3'flag (~18 kDa) was most detectable after elution through boiling in 2x sample loading buffer. B23 (~37 kDa) was detectable under two conditions – 37 °C incubation in 2x sample loading buffer for 15 min and 95 °C incubation in 2x sample loading buffer for 3 min.

M2 (nuclear/nucleolar in localization, nonfunctional in HIV-1_HXB2 production), M6 (nucleolar in pattern, functional in HIV-1_HXB2 production), and M9 (dispersed in the cytoplasm/nucleus, nonfunctional in viral production) were expressed in HLfB culture. Protein complexes were processed from total cell lysate, eluted through 95 °C incubation in 2x sample loading buffer for 3 min, and resolved in silver stain reagent (Figure 4). WT Rev was detectable after IP flag reaction. Rev-NoLS M2, M6, and M9 were also detectable at 18 kDa. Bands corresponding to B23 protein were observed at the 37 kDa marker. Protein complexes were further observed in each lane corresponding to IP reactions of WT Rev, M2, M6, M9, and pcDNA negative background control. Protein lysates were analyzed by immunodetection for α-flag-Rev and B23. Abundant WT Rev and moderate levels of M6 were expressed (α-flag input) and detectable after flag IP (α-flag-Rev IP, Figure 5). M2 and M9 were not highly expressed from 20 μg of protein lysate input but detectable at low intensity after flag IP from 5 mg of protein lysate. pcDNA negative control did not yield nonspecific immunodetection in input and α-flag-Rev IP. B23 affinity with WT Rev was observed after IP flag reaction (B23 co-IP). B23 affinity was slightly observed with M2 (two single-point mutations R48,50G) and M6 (single-point mutation R50G). B23 affinity was lost in the presence of three single-point mutations of M9 (R46,48,50G) within Rev-NoLS.

Total lysates from immunoprecipitated WT Rev and Rev-NoLS-3'flag mutations (4, 5, 6, and 8) were processed, stained with Coomassie reagent, and visualized in comparison to BSA serial dilutions (right panel). Rev-NoLS-3'flag is detectable (12.5-25 µg) in the presence and absence of mutations at 18 kDa (Figure 6). Protein complexes processed from IP reactions of WT Rev-3'flag, nucleolar-localizing Rev-NoLS-3'flag mutations (M4, M5, and M6), and negative control pcDNA-flag were visualized in SDS-PAGE gels stained with Coomassie reagent (Figure 7). WT Rev (WT1 and WT2) was detectable after IP flag reaction at 18 kDa. Rev-NoLS mutations were slightly detectable after expression in HLfB and IP flag reaction. pcDNA negative control did not yield nonspecific background at 18 kDa.

Immunoprecipitated lysates prepared from WT Rev-3'flag, M2, M6, M9, and negative control pcDNA-flag (shown in Figure 4) were analyzed by tandem mass spectrometry. Protein identification probability (in percentages) is displayed for comparison of protein interactions occurring with each nucleolar-localizing Rev-NoLS mutation versus WT Rev (Table 1). Cellular proteins, some of which are nucleolar in localization pattern (ribosomal isoforms, eukaryotic translation initiation factor 48, snoRNA C/D box 58B, and nucleophosmin B23), were identified as direct/indirect binding partners of WT Rev. These nucleolar factors lost binding affinity to M2 (two single-point mutations R48,50G), M6 (single-point mutation R50G), and M9 (three single-point mutation R46,48,50G), similar to pcDNA negative control. Each lane of the Coomassie-stained gel in Figure 6 was processed for tandem mass spectrometry (Table 2). Peptide affinity to Rev-NoLS mutations was analyzed and displayed using protein identification probability (in percentages). A variety of cellular proteins, some of which are nucleolar in localization pattern (nucleolin C23, nucleophosmin B23, and nucleosome assembly protein), were identified as direct/indirect protein binding factors of WT Rev. These nucleolar factors were not identified to bind with M4, M5, and M6. Transport factors ARHGEF1 (rho guanine nucleotide exchange factor 1) and TBC1D24 (TCB1 domain family, member 24) were lost in affinity in the presence of Rev-NoLS mutations. Splicing factors hnRNPC (heterogeneous ribonuclear protein C) and PNN (pinin, desmosome-associated protein) were additionally observed to bind to WT Rev, which lost interaction with Rev single-point nucleolar mutations.

Figure 1: Optimization of cell lysis conditions for Rev-3'flag co-IP during HIV-1 production. WT Rev-NoLS-3'flag and negative control pcDNA-flag IP conditions were optimized in three different NaCl concentrations (137 mM, 200 mM, and 300 mM) in lysis buffer. As observed through silver staining, 137 mM NaCl was the optimal salt concentration for Rev immunoprecipitation and B23 binding. Please click here to view a larger version of this figure.

Figure 2: Optimization of lysis conditions for Rev-3'flag co-IP and B23 immunodetection during HIV-1 production. WT Rev-NoLS-3'flag, M1-3'flag, and negative control pcDNA-flag IP conditions were optimized in two different NaCl concentrations (137 mM and 200 mM) in lysis buffer. As observed through immunodetection, 137 mM NaCl was the optimal salt concentration for Rev immunoprecipitation and B23 binding. Please click here to view a larger version of this figure.

Figure 3: Optimization of Rev-3'flag co-IP elution during HIV-1 production, visualized by silver staining. Protein complexes were processed from total cell lysates prepared during WT Rev-3'flag and pcDNA-flag IP. Three different elution conditions were tested – 2x sample loading buffer at 37 °C for 15 min, 2x sample loading buffer at 95 °C for 3 min, and 3x flag peptide at 4 °C for 30 min. The optimal elution conditions of WT Rev occurred after the 15 min incubation period in 2x sample loading buffer at 37 °C and after the 3 min incubation period in 2x sample loading buffer at 95 °C. Please click here to view a larger version of this figure.

Figure 4: Immunoprecipitation of Rev-3'flag mutations 2, 6, and 9 during HIV-1 production. Protein complexes that bound directly/indirectly with WT Rev, M2 (R48,50G), M6 (R50G), M9 (R46,48,50G), and negative control pcDNA-flag in the presence of HIV-1 replication are shown in a silver-stained SDS-PAGE gel. Please click here to view a larger version of this figure.

Figure 5: Rev-3'flag co-IP of mutations 2, 6, and 9 for B23 immunodetection during HIV-1 production. The protein lysates and IP reactions prepared from WT Rev, M2, M6, M9, and negative control pcDNA-flag in Figure 4 were further analyzed by immunodetection for α-flag-Rev and B23. WT Rev and mutant expression, as well as interaction with B23 nucleocytoplasmic protein, are observed. Please click here to view a larger version of this figure.

Figure 6: Quantification of Rev-NoLS mutations 4, 5, 6, and 8 after flag IP reaction. IP reactions from HLfB expressing WT Rev and nucleolar-localizing M4 (R46G), M5 (R48G), M6 (R50G), M8 (ΔRQ), and negative control pcDNA-flag were measured for protein concentration using BSA serial dilutions. A protein concentration of 12.5-25 µg was observed for each sample. Please click here to view a larger version of this figure.

Figure 7: Immunoprecipitation of nucleolar-localizing Rev-NoLS mutations 4, 5, and 6 during HIV-1 production. Coomassie-stained SDS-PAGE gel containing immunoprecipitated protein complexes of WT Rev (1 and 2), M4, M5, and M6 are shown. Please click here to view a larger version of this figure.

Identified Proteins	Molecular Weight	WT Rev	M2	M6	M9
signal recognition particle 14kDa (homologous Alu RNA binding protein)	15 kDa	95%	0	0	0
ribosomal protein S3A	30 kDa	95%	0	0	0
eukaryotic translation initiation factor 4B	69 kDa	95%	0	0	0
ribosomal protein L31	14 kDa	91%	0	0	0
ribosomal protein L12	18 kDa	93%	0	0	0
ribosomal protein L22	15 kDa	91%	0	0	0
small nucleolar RNA, C/D box 58B	15 kDa	87%	0	0	0
zinc finger, CCHC domain containing 11	185 kDa	87%	0	0	0
actin binding LIM protein 1	79 kDa	79%	0	0	0
ribosomal protein S13	17 kDa	78%	0	0	0
nucleophosmin (nucleolar phosphoprotein B23, numatrin)	33 kDa	73%	0	0	0

Table 1: Identification of cellular host factors that interact with WT Rev during HIV-1 production. Protein eluates prepared from WT Rev, M2, M6, and M9 were directly analyzed by tandem mass spectrometry. Protein interactions that are directly/indirectly bound to WT Rev versus M2, M6, and M9 are summarized by protein identification probability (in percentages).

Identified Proteins	Molecular Weight	M4	M5	M6	WT	pCDNA
poly(A) binding protein, cytoplasmic 1	61 kDa	98%	0	100%	100%	0
heat shock 70kDa protein 1B	70 kDa	100%	100%	100%	100%	0
ribosomal protein L7	29 kDa	91%	0	100%	100%	0
histone cluster 1, H1e	22 kDa	0	0	100%	100%	0
ribosomal protein L4	48 kDa	95%	0	100%	100%	0
ribosomal protein L13	24 kDa	99%	0	100%	100%	0
complement component 1, q subcomponent binding protein	31 kDa	100%	100%	100%	100%	0
Y box binding protein 1	36 kDa	0	0	100%	100%	0
nucleosome assembly protein 1-like 1	45 kDa	0	0	0	100%	0
ribosomal protein L8	28 kDa	89%	36%	100%	100%	0
ribosomal protein L18	22 kDa	27%	0	100%	100%	0
heat shock 70kDa protein 8	71 kDa	5%	99%	100%	100%	0
ribosomal protein L19	23 kDa	10%	0	81%	100%	0
ribosomal protein L27	16 kDa	92%	0	100%	100%	0
ribosomal protein L7a	30 kDa	0	0	100%	100%	0
ribosomal protein L24	18 kDa	0	0	100%	99%	0
tubulin, beta class I	50 kDa	92%	69%	100%	100%	0
ribosomal protein L6	33 kDa	0	0	100%	100%	0
heat shock 70kDa protein 9 (mortalin)	74 kDa	33%	99%	100%	100%	0
ribosomal protein S2	31 kDa	87%	0	100%	100%	0
casein kinase 2, alpha 1 polypeptide	45 kDa	0	0	50%	100%	0
ribosomal protein L14	23 kDa	0	0	81%	100%	0
ribosomal protein, large, P0	34 kDa	0	0	100%	100%	0
histone cluster 1, H1b	23 kDa	0	0	73%	100%	0
heat shock 70kDa protein 5 (glucose-regulated protein)	72 kDa	99%	0	100%	100%	0
ribosomal protein S4, X-linked	30 kDa	0	0	100%	100%	0
ribosomal protein S20	13 kDa	98%	94%	99%	99%	0
ribosomal protein L28	16 kDa	0	0	100%	99%	0
ribosomal protein S14	16 kDa	0	0	100%	100%	0
ribosomal protein S3A	30 kDa	0	0	100%	100%	0
ribosomal protein L21	19 kDa	0	0	100%	100%	0
zinc finger CCCH-type, antiviral 1	101 kDa	0	0	97%	100%	0
histone cluster 1, H1c	21 kDa	0	0	0	98%	0
ribosomal protein L36	12 kDa	0	0	100%	100%	0
ribosomal protein S18	18 kDa	90%	0	100%	100%	0
modulator of apoptosis 1	40 kDa	42%	88%	34%	0	0
ribosomal protein L17	21 kDa	0	0	100%	100%	0
ribosomal protein S26	13 kDa	91%	0	99%	98%	0
ribosomal protein L32	18 kDa	0	0	48%	100%	0
ribosomal protein L35	15 kDa	0	0	97%	100%	0
ribosomal protein L29	18 kDa	0	0	57%	94%	0
ribosomal protein S9	23 kDa	0	0	100%	100%	0
heterogeneous nuclear ribonucleoprotein H1 (H)	51 kDa	98%	0	100%	100%	0
ribosomal protein S8	22 kDa	0	0	78%	100%	0
ribosomal protein L10	25 kDa	0	0	100%	100%	0
ribosomal protein L23a	18 kDa	0	0	68%	100%	0
ribosomal protein S6	29 kDa	0	0	100%	100%	0
ribosomal protein L31	14 kDa	0	0	95%	100%	0
ribosomal protein S13	17 kDa	0	0	100%	99%	0
ribosomal protein L10a	25 kDa	45%	0	81%	100%	0
poly(A) binding protein, cytoplasmic 4 (inducible form)	70 kDa	0	0	100%	100%	0
transmembrane emp24 protein transport domain containing 1	25 kDa	0	0	0	100%	0
EBNA1 binding protein 2	35 kDa	0	0	69%	100%	0
conserved helix-loop-helix ubiquitous kinase	85 kDa	74%	92%	65%	83%	0
ribosomal protein L12	18 kDa	0	0	57%	100%	0
ribosomal protein S24	15 kDa	0	0	100%	100%	0
cold shock domain protein A	40 kDa	0	0	0	100%	0
ribosomal protein L27a	17 kDa	0	0	89%	99%	0
heterogeneous nuclear ribonucleoprotein F	46 kDa	0	0	99%	99%	0
ribosomal protein L11	20 kDa	0	0	99%	100%	0
ribosomal protein L26-like 1	17 kDa	0	0	100%	100%	0
casein kinase 2, alpha prime polypeptide	41 kDa	0	0	0	100%	0
tubulin, alpha 1a	50 kDa	33%	0	100%	0	0
ribosomal protein L3	46 kDa	0	0	86%	94%	0
mitochondrial ribosomal protein L15	33 kDa	0	0	100%	99%	0
nucleolin	77 kDa	0	0	25%	100%	0
mitochondrial ribosomal protein S21	11 kDa	0	0	93%	94%	0
KRR1, small subunit (SSU) processome component, homolog (yeast)	44 kDa	89%	0	76%	99%	0
mitochondrial ribosomal protein S7	28 kDa	0	0	0	100%	0
chloride channel, nucleotide-sensitive, 1A	22 kDa	0	0	97%	86%	0
cyclin B3	158 kDa	0	0	67%	49%	0
guanine nucleotide binding protein-like 3 (nucleolar)	61 kDa	0	0	99%	98%	0
mitochondrial ribosomal protein S23	22 kDa	0	0	100%	50%	0
mitochondrial ribosomal protein S22	41 kDa	0	0	0	99%	0
nucleophosmin (nucleolar phosphoprotein B23, numatrin)	33 kDa	0	0	52%	97%	0
ribosomal protein S19	16 kDa	0	0	0	99%	0
glyceraldehyde-3-phosphate dehydrogenase	36 kDa	0	0	100%	0	0
histone cluster 1, H2bg	14 kDa	0	0	53%	72%	0
ribosomal protein S23	16 kDa	0	0	68%	0	0
Rho guanine nucleotide exchange factor (GEF) 1	104 kDa	0	0	0	94%	0
death associated protein 3	46 kDa	0	0	87%	87%	0
mitochondrial ribosomal protein S6	14 kDa	0	0	52%	84%	0
ribosomal protein L39	6 kDa	0	0	0	87%	0
mitochondrial ribosomal protein S28	21 kDa	0	0	85%	99%	0
histone cluster 1, H4h	11 kDa	0	0	0	93%	0
mitochondrial ribosomal protein L43	23 kDa	0	0	0	97%	0
ribosomal protein S15	17 kDa	0	0	0	85%	0
ribosomal protein S12	15 kDa	0	0	68%	39%	0
ribosomal protein L35a	13 kDa	0	0	0	96%	0
mitochondrial ribosomal protein L38	45 kDa	0	0	42%	98%	0
mitochondrial ribosomal protein S14	15 kDa	0	0	45%	76%	0
phosphodiesterase 5A, cGMP-specific	95 kDa	14%	0	0	72%	0
ribosomal protein L15	24 kDa	0	0	0	56%	0
heterogeneous nuclear ribonucleoprotein C (C1/C2)	22 kDa	0	0	0	100%	0
mitochondrial ribosomal protein S16	11 kDa	0	0	0	92%	0
ribosomal protein S15a	15 kDa	0	0	0	91%	0
neurexin 2	185 kDa	0	50%	0	0	0
autism susceptibility candidate 2	139 kDa	0	0	46%	0	0
mitochondrial ribosomal protein L17	20 kDa	0	0	0	92%	0
splicing factor 3a, subunit 1, 120kDa	89 kDa	0	0	56%	89%	0
La ribonucleoprotein domain family, member 1	116 kDa	0	0	65%	66%	0
leprecan-like 2	62 kDa	0	0	0	68%	0
ribosomal protein L18a	21 kDa	0	0	0	86%	0
ribosomal protein L23	15 kDa	0	0	60%	47%	0
transmembrane emp24 protein transport domain containing 9	27 kDa	0	0	0	78%	0
signal recognition particle 72kDa	75 kDa	0	0	0	100%	0
lectin, galactoside-binding, soluble, 3	26 kDa	0	71%	28%	0	0
mitochondrial ribosomal protein L1	37 kDa	0	0	0	69%	0
H1 histone family, member X	22 kDa	0	0	0	96%	0
casein kinase 2, beta polypeptide	25 kDa	0	0	0	89%	0
solute carrier family 4, sodium bicarbonate cotransporter, member 7	118 kDa	82%	0	0	0	0
WD repeat domain 13	54 kDa	0	81%	0	0	0
transcription elongation factor A (SII), 3	17 kDa	0	0	0	38%	0
ribosomal protein S16	16 kDa	0	0	75%	0	0
SP140 nuclear body protein	92 kDa	0	0	0	64%	0
otoferlin	227 kDa	62%	0	0	0	0
golgi transport 1B	15 kDa	0	0	0	33%	0
mitochondrial ribosomal protein S34	26 kDa	0	0	0	33%	0
ribosomal protein L30	13 kDa	0	0	0	49%	0
actin binding LIM protein 1	96 kDa	0	0	0	43%	0
guanine nucleotide binding protein-like 2 (nucleolar)	84 kDa	40%	0	0	0	0
high density lipoprotein binding protein	141 kDa	0	0	34%	0	0
adenosine deaminase, RNA-specific, B2	81 kDa	0	0	28%	0	0
cystatin E/M	17 kDa	0	27%	0	0	0
zinc finger protein 786	80 kDa	0	0	23%	0	0
pleckstrin homology-like domain, family B, member 1	145 kDa	0	0	0	95%	0
protein phosphatase methylesterase 1	44 kDa	0	0	94%	0	0
janus kinase and microtubule interacting protein 2	95 kDa	0	0	0	94%	0
signal recognition particle 68kDa	67 kDa	0	0	0	93%	0
TBC1 domain family, member 24	63 kDa	0	0	0	89%	0
mitochondrial ribosomal protein L27	16 kDa	0	0	0	89%	0
mitochondrial ribosomal protein L2	24 kDa	0	0	0	88%	0
mitochondrial ribosomal protein S2	33 kDa	0	0	0	87%	0
Pentatricopeptide repeat domain 3	79 kDa	0	0	0	84%	0
ribosomal protein, large, P2	12 kDa	0	0	0	76%	0
IMP (inosine 5'-monophosphate) dehydrogenase 2	56 kDa	0	0	76%	0	0
tubulin, beta 4B class IVb	50 kDa	0	0	76%	0	0
mitochondrial ribosomal protein L23	19 kDa	0	0	0	74%	0
mitochondrial ribosomal protein S31	45 kDa	0	0	0	74%	0
galactose-3-O-sulfotransferase 1	49 kDa	74%	0	0	0	0
suppressor of variegation 4-20 homolog 1 (Drosophila)	99 kDa	0	72%	0	0	0
mitochondrial ribosomal protein S25	20 kDa	0	0	0	70%	0
ribosomal L1 domain containing 1	55 kDa	0	0	0	70%	0
family with sequence similarity 110, member D	29 kDa	69%	0	0	0	0
ribosomal protein L36a-like	12 kDa	0	0	0	66%	0
cerebellin 4 precursor	22 kDa	0	0	0	64%	0
N-acetylglucosamine-1-phosphate transferase, alpha and beta subunits	144 kDa	64%	0	0	0	0
RAD51 homolog B (S. cerevisiae)	38 kDa	0	0	0	64%	0
transcription elongation regulator 1	124 kDa	63%	0	0	0	0
homeobox A1	15 kDa	0	0	0	62%	0
phospholipid transfer protein	49 kDa	0	0	0	62%	0
Rho GTPase activating protein 33	137 kDa	0	0	54%	0	0
mitochondrial ribosomal protein S18B	29 kDa	0	0	0	52%	0
endoplasmic reticulum aminopeptidase 2	106 kDa	51%	0	0	0	0
tripartite motif containing 28	89 kDa	0	50%	0	0	0
immature colon carcinoma transcript 1	24 kDa	0	0	0	50%	0
AT rich interactive domain 1A (SWI-like)	242 kDa	0	0	49%	0	0
mitochondrial ribosomal protein S17	15 kDa	0	0	0	48%	0
pinin, desmosome associated protein	82 kDa	0	0	0	48%	0
protein phosphatase, Mg2+/Mn2+ dependent, 1G	59 kDa	0	0	0	45%	0
G patch domain and ankyrin repeats 1	39 kDa	45%	0	0	0	0
mitochondrial ribosomal protein L3	39 kDa	0	0	0	44%	0
budding uninhibited by benzimidazoles 3 homolog (yeast)	37 kDa	0	44%	0	0	0
WD and tetratricopeptide repeats 1	76 kDa	0	0	0	44%	0
protein disulfide isomerase family A, member 2	58 kDa	0	0	0	42%	0
kazrin, periplakin interacting protein	86 kDa	0	41%	0	0	0
coiled-coil-helix-coiled-coil-helix domain containing 2	16 kDa	0	0	40%	0	0
heat shock protein 90kDa alpha (cytosolic), class A member 1	85 kDa	0	0	40%	0	0
retinitis pigmentosa GTPase regulator	83 kDa	0	0	40%	0	0
CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) phosphatase, subunit 1	104 kDa	0	39%	0	0	0
mitochondrial ribosomal protein L37	48 kDa	0	0	0	39%	0
C2CD2-like	76 kDa	0	38%	0	0	0
DnaJ (Hsp40) homolog, subfamily C, member 6	106 kDa	0	0	38%	0	0
mitochondrial ribosomal protein L51	15 kDa	0	0	0	38%	0
bystin-like	50 kDa	0	0	0	38%	0
huntingtin-associated protein 1	76 kDa	0	0	0	37%	0
zinc finger protein 263	77 kDa	0	36%	0	0	0
cell division cycle and apoptosis regulator 1	133 kDa	0	0	34%	0	0
protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated phosphatase)	256 kDa	0	0	34%	0	0
KRAB-A domain containing 2	56 kDa	0	0	0	31%	0
activating signal cointegrator 1 complex subunit 2	28 kDa	0	0	0	31%	0
centrosomal protein 76kDa	74 kDa	0	30%	0	0	0
polymerase (RNA) III (DNA directed) polypeptide C (62kD)	61 kDa	0	0	0	30%	0
5-hydroxytryptamine (serotonin) receptor 6	47 kDa	0	0	0	29%	0
T cell receptor alpha joining 56	2 kDa	0	26%	0	0	0
biorientation of chromosomes in cell division 1-like	330 kDa	0	0	0	25%	0
Ras association and DIL domains	114 kDa	0	0	25%	0	0
WD repeat domain 66	130 kDa	0	0	0	24%	0
chromosome 6 open reading frame 25	13 kDa	0	0	22%	0	0
transmembrane protein 177	34 kDa	0	0	0	21%	0
neural precursor cell expressed, developmentally down-regulated 4-like	101 kDa	0	20%	0	0	0

Table 2: Identification of cellular host factors complexed with nucleolar-localizing Rev mutations 4, 5, and 6 during HIV-1 production. The Coomassie-stained SDS-PAGE gel in Figure 6 was processed for tandem mass spectrometry. Protein interaction results of WT Rev versus nucleolar mutations M4, M5, and M6 are summarized by protein identification probability (in percentages).

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 or multiple arginine substitutions, was assessed through immunoprecipitation of Rev-3'flag during viral production (Figure 2; WT and mutations M2, M6, and M9). B23 affinity to single-point Rev-NoLS mutations M4, M5, and M6 were previously examined in the presence of HIV-1 replication. In the previous study, IP eluates of M4, M5, and M6 were subjected to western immunoblotting with an antibody specific to α-flag for Rev and B23 affinity¹. In the background of Rev single-point mutations, B23 binding affinity was significantly reduced. B23 maintained affinity with WT Rev during HIV replication. Single-point mutations induced within Rev-NoLS were expected to decrease binding affinity to other cellular host factors facilitating HIV mRNA binding and transport. Rev-NoLS single- and multiple-point mutations in this model abolished nucleophosmin B23 affinity to Rev, indicating a disruption in nucleocytoplasmic shuttling and HIV mRNA transport. It is likely that nucleolar factors (nucleolin C23 and nucleosome assembly protein 1), transport factors (ARHGEF1 and TCB1D24), and splicing factors (hnRNPC and PNN) are involved in the HIV-1 infectious cycle through interaction with Rev protein complexes. Table 1 and Table 2 reveal various other cellular factors, both nucleolar and nonnucleolar in pattern, that are potentially involved in the HIV-1 replication cycle through Rev. The nucleolar factor – snoRNA C/D box 58, implicated in snoRNA processing, snoRNA transport to the nucleolus, and 2'-O-methylation of ribosomal RNA – was identified yet the function of this protein in the HIV-1 replication cycle remains unknown. The specific roles of these cellular factors in the HIV-1 infectious cycle are currently being investigated.

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. Viral/host nucleolar factors that participate in other disease models could be identified using this approach. It is further likely that one nucleolar factor could involve multifunctional roles in various disease models. For example, B23 has been implicated in the transport of nucleolar viral proteins, viral assembly, encapsidation, replication, and latency in other viral infectious models. B23 is characterized in interactions with NoLS of cellular factors for nucleocytoplasmic transport – p120 growth factor (amino acids 40-57)²³ and C23 pre-rRNA processor (amino acids 540–628)²⁴. B23 is also documented to interact with the human T-cell lymphotropic virus (HTLV-1) protein Rex (amino acids 1-22)²⁵, HIV-1 Tat (amino acids 49–57)², and HIV-1 Rev (amino acids 37-47)²⁶. The Japanese encephalitis virus (JEV) genome encodes a nucleolar-localizing core protein, through which amino acids Gly42 and Pro43 interact with the N-terminal region of B23 during JEV infection, resulting in the transportation of viral core protein/B23 into the nucleus²⁷. The single-stranded RNA genome of the Hepatitis B virus (HBV) is composed partially of double-stranded DNA, which encodes a nucleolar core protein. The HBV core protein associates with nucleolin and B23 in the nucleolus²⁸; B23 was demonstrated in the HBV assembly through interaction with the core protein N-terminal domain. Specifically, B23 amino acids 259-294 bind to the N-terminal domain of the HBV core protein to allow viral encapsidation²⁹. The negative-sense, single-stranded RNA hepatitis D virus (HDV) expresses HDVAg antigen in two isoforms; the small isoform aids in RNA replication, and the large isoform facilitates viral assembly. RNA replication takes place within the nucleolus and requires B23 interaction with HDVAg³⁰^,³¹. HDV infection causes an upregulation of B23, which interacts mostly with the small HDVAg isoform and less with the large HDVAg isoform. Interactions take place through the small HDVAg NLS domain, through which B23 binds and achieves nuclear accumulation. Upon deletion of the HDV binding site to B23, RNA replication was impaired. HDVAg was shown to colocalize with B23 and nucleolin in the nucleolus. Nucleolin was discovered to possess transcriptional properties as a repressor³², revealing the nucleolus as a compartment for regulation of HDV replication. B23 is also involved in the latency of the Kaposi’s sarcoma-associated herpesvirus (KSHV) genome. KSHV latent protein – v-cyclin – with host CDK6 kinase, phosphorylates B23 at Thr199, facilitating B23 interaction with latency-associated nuclear antigen³³. The latency-associated nuclear antigen acts to prevent viral lytic replication. Depletion of B23 leads to KSHV reactivation, revealing B23 as a regulator of KSHV latency. B23 function in the HIV-1 replication cycle is characterized in the nucleocytoplasmic transport activity of Tat and Rev, and it is unknown if B23 can induce latency during HIV infection. B23’s involvement in the replication, encapsidation, and assembly of HIV-1 is currently unknown.

Adaptation of this method to other disease models would require much effort and time for the generation of protein-deficient infectious backbones expressed in the appropriate cell lines. The advantage of this Rev-deficient HIV-1_HXB2 backbone is the ability to examine Rev-NoLS mutations in the presence of the full viral backbone, viral infectious factors, and host factors involved in the HIV-1 replication cycle. Other studies have examined Rev nucleolar function in the absence of the full HIV-1 infectious system. Thorough characterization of infectious and disease pathways must include representative environments that support the natural course of disease progression. Two different types of analyses were conducted and compared in the ability to identify nucleolar factors. Table 1 lists factors that remained bound to WT Rev as a result of direct mass spectrometry analysis of protein eluate. This analysis yielded several known factors of HIV-1 Rev. This direct method was compared to another process involving the extraction of protein separated within SDS-PAGE gels and mass spectrometry analysis of such proteins. This second method yielded a variety of known and potential factors that are bound to the Rev protein complex but lacked the following proteins previously identified in Table 1: signal recognition particle 14 kDa, eukaryotic translation initiation factor 4B, ribosomal protein L22, small nucleolar RNA C/D box 58B, and zinc finger CCHC domain containing 11. Ultimately, the superior method chosen for mass spectrometry analyses in this protocol involved the extraction of peptides from SDS-PAGE gels. The first direct method included 2x sample buffer in the eluate without bromophenol blue; the remaining components of the 2x sample buffer could interfere with complete trypsin treatment and could yield incompletely processed peptides for mass spectrometry analysis. The second indirect method was able to purify trypsin-treated peptides from potential contaminants of SDS-PAGE.

The mass spectrometry preparation methods described here could be utilized for the identification of therapeutic interventions to eradicate HIV-1 infection through targeting Rev. All deletion and single-point Rev-NoLS mutations could be examined for dominant-negative activity and utilized in the arrest of Rev function. Dominant negative characteristics of interest for Rev functional arrest are the following: Rev/RRE binding affinity; relocalization of nucleolar WT Rev through multimerization with Rev mutants; loss in affinity to key cellular factors involved in HIV-1 mRNA transport and splicing. Rev multimerization involving the coexpression of Rev-NoLS mutations with WT Rev could be examined. Dominant negative mutations in this model are expected to multimerize with WT Rev and shift nucleolar patterns toward the nucleus and cytoplasm, leading to a Rev functional arrest. Mass spectrometry could be used to identify the loss of key cellular factors involved in HIV-1 mRNA splicing and transport. The identification of missing interactions with WT Rev as a result of coexpression with dominant-negative Rev-NoLS mutations would reveal the involvement of nucleolar-specific pathways in HIV-1 pathogenesis. Alternatively, viral HIV-1_NL4-3 particles generated in the background of Rev-NoLS mutations could be investigated for all packaged cellular factors. Cellular and viral factors packaged within viral particles may be further identified through mass spectrometry. This would reveal the presence of nucleolar factors within viral particles and the role of identified nucleolar factors in viral infection. The methods described are applicable to other viral and disease models for the identification and characterization of understudied pathways. This would allow the development of therapeutic interventions against diseases by which limited treatments are available.

開示

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Arizala, J. A. C., Chomchan, P., Li, H., Moore, R., Ge, H., Ouellet, D. L., Rossi, J. J. Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry. J. Vis. Exp. (148), e59329, doi:10.3791/59329 (2019).

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