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

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

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

<ol>
	<li>Arizala, J. A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleolar+Localization+of+HIV-1+Rev+Is+Required,+Yet+Insufficient+for+Production+of+Infectious+Viral+Particles.">Nucleolar Localization of HIV-1 Rev Is Required, Yet Insufficient for Production of Infectious Viral Particles.</a> AIDS Research and Human Retroviruses. , (2018).</li><li>Li, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+B23+is+an+important+human+factor+for+the+nucleolar+localization+of+the+human+immunodeficiency+virus+protein+Tat.">Protein B23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein Tat.</a> Journal of Virology. 71, 4098-4102 (1997).</li><li>Szebeni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleolar+protein+B23+stimulates+nuclear+import+of+the+HIV-1+Rev+protein+and+NLS-conjugated+albumin.">Nucleolar protein B23 stimulates nuclear import of the HIV-1 Rev protein and NLS-conjugated albumin.</a> Biochemistry. 36, 3941-3949 (1997).</li><li>Truant, R., Cullen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+arginine-rich+domains+present+in+human+immunodeficiency+virus+type+1+Tat+and+Rev+function+as+direct+importin+beta-dependent+nuclear+localization+signals.">The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals.</a> Molecular and Cellular Biology. 19, 1210-1217 (1999).</li><li>Eirin-Lopez, J. M., Frehlick, L. J., Ausio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+evolution+and+functional+diversification+in+the+members+of+the+nucleophosmin/nucleoplasmin+family+of+nuclear+chaperones.">Long-term evolution and functional diversification in the members of the nucleophosmin/nucleoplasmin family of nuclear chaperones.</a> Genetics. 173, 1835-1850 (2006).</li><li>Frehlick, L. J., Eirin-Lopez, J. M., Ausio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+nucleophosmin/nucleoplasmin+family+of+nuclear+chaperones.">New insights into the nucleophosmin/nucleoplasmin family of nuclear chaperones.</a> BioEssays. 29, 49-59 (2007).</li><li>Okuwaki, M., Matsumoto, K., Tsujimoto, M., Nagata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+nucleophosmin/B23,+a+nucleolar+acidic+protein,+as+a+histone+chaperone.">Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone.</a> FEBS Letters. 506, 272-276 (2001).</li><li>Szebeni, A., Olson, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleolar+protein+B23+has+molecular+chaperone+activities.">Nucleolar protein B23 has molecular chaperone activities.</a> Protein Science. 8, 905-912 (1999).</li><li>Hingorani, K., Szebeni, A., Olson, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+functional+domains+of+nucleolar+protein+B23.">Mapping the functional domains of nucleolar protein B23.</a> Journal of Biological Chemistry. 275, 24451-24457 (2000).</li><li>Borer, R. A., Lehner, C. F., Eppenberger, H. M., Nigg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+nucleolar+proteins+shuttle+between+nucleus+and+cytoplasm.">Major nucleolar proteins shuttle between nucleus and cytoplasm.</a> Cell. 56, 379-390 (1989).</li><li>Yun, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophosmin/B23+is+a+proliferate+shuttle+protein+associated+with+nuclear+matrix.">Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix.</a> Journal of Cellular Biochemistry. 90, 1140-1148 (2003).</li><li>Savkur, R. S., Olson, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+cleavage+in+pre-ribosomal+RNA+byprotein+B23+endoribonuclease.">Preferential cleavage in pre-ribosomal RNA byprotein B23 endoribonuclease.</a> Nucleic Acids Research. 26, 4508-4515 (1998).</li><li>Herrera, J. E., Savkur, R., Olson, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribonuclease+activity+of+nucleolar+protein+B23.">The ribonuclease activity of nucleolar protein B23.</a> Nucleic Acids Research. 23, 3974-3979 (1995).</li><li>Grisendi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nucleophosmin+in+embryonic+development+and+tumorigenesis.">Role of nucleophosmin in embryonic development and tumorigenesis.</a> Nature. 437, 147-153 (2005).</li><li>Itahana, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+ARF+degrades+B23,+a+nucleolar+protein+involved+in+ribosome+biogenesis+and+cell+proliferation.">Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation.</a> Molecular Cell. 12, 1151-1164 (2003).</li><li>Ye, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophosmin/B23,+a+multifunctional+protein+that+can+regulate+apoptosis.">Nucleophosmin/B23, a multifunctional protein that can regulate apoptosis.</a> Cancer Biology &amp; Therapy. 4, 918-923 (2005).</li><li>Kuo, M. L., den Besten, W., Bertwistle, D., Roussel, M. F., Sherr, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-terminal+polyubiquitination+and+degradation+of+the+Arf+tumor+suppressor.">N-terminal polyubiquitination and degradation of the Arf tumor suppressor.</a> Genes &amp; Development. 18, 1862-1874 (2004).</li><li>Kuo, M. L., den Besten, W., Thomas, M. C., Sherr, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arf-induced+turnover+of+the+nucleolar+nucleophosmin-associated+SUMO-2/3+protease+Senp3.">Arf-induced turnover of the nucleolar nucleophosmin-associated SUMO-2/3 protease Senp3.</a> Cell Cycle. 7, 3378-3387 (2008).</li><li>Horn, H. F., Vousden, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer:+guarding+the+guardian.">Cancer: guarding the guardian.</a> Nature. 427, 110-111 (2004).</li><li>Grisendi, S., Mecucci, C., Falini, B., Pandolfi, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophosmin+and+cancer.">Nucleophosmin and cancer.</a> Nature Reviews Cancer. 6, 493-505 (2006).</li><li>Dingwall, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoplasmin+cDNA+sequence+reveals+polyglutamic+acid+tracts+and+a+cluster+of+sequences+homologous+to+putative+nuclear+localization+signals.">Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals.</a> The EMBO Journal. 6, 69-74 (1987).</li><li>Fankhauser, C., Izaurralde, E., Adachi, Y., Wingfield, P., Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+complex+of+human+immunodeficiency+virus+type+1+rev+and+nucleolar+B23+proteins:+dissociation+by+the+Rev+response+element.">Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element.</a> Molecular and Cellular Biology. 11, 2567-2575 (1991).</li><li>Valdez, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+nuclear+and+nucleolar+localization+signals+of+the+protein+p120.+Interaction+with+translocation+protein+B23.">Identification of the nuclear and nucleolar localization signals of the protein p120. Interaction with translocation protein B23.</a> Journal of Biological Chemistry. 269, 23776-23783 (1994).</li><li>Li, Y. P., Busch, R. K., Valdez, B. C., Busch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C23+interacts+with+B23,+a+putative+nucleolar-localization-signal-binding+protein.">C23 interacts with B23, a putative nucleolar-localization-signal-binding protein.</a> European Journal of Biochemistry/FEBS. 237, 153-158 (1996).</li><li>Adachi, Y., Copeland, T. D., Hatanaka, M., Oroszlan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleolar+targeting+signal+of+Rex+protein+of+human+T-cell+leukemia+virus+type+I+specifically+binds+to+nucleolar+shuttle+protein+B-23.">Nucleolar targeting signal of Rex protein of human T-cell leukemia virus type I specifically binds to nucleolar shuttle protein B-23.</a> Journal of Biological Chemistry. 268, 13930-13934 (1993).</li><li>Szebeni, A., Herrera, J. E., Olson, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+nucleolar+protein+B23+with+peptides+related+to+nuclear+localization+signals.">Interaction of nucleolar protein B23 with peptides related to nuclear localization signals.</a> Biochemistry. 34, 8037-8042 (1995).</li><li>Tsuda, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleolar+protein+B23+interacts+with+Japanese+encephalitis+virus+core+protein+and+participates+in+viral+replication.">Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication.</a> Microbiology and Immunology. 50, 225-234 (2006).</li><li>Ning, B., Shih, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleolar+localization+of+human+hepatitis+B+virus+capsid+protein.">Nucleolar localization of human hepatitis B virus capsid protein.</a> Journal of Virology. 78, 13653-13668 (2004).</li><li>Lee, S. J., Shim, H. Y., Hsieh, A., Min, J. Y., Jung, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+core+interacts+with+the+host+cell+nucleolar+protein,+nucleophosmin+1.">Hepatitis B virus core interacts with the host cell nucleolar protein, nucleophosmin 1.</a> Journal of Microbiology. 47, 746-752 (2009).</li><li>Huang, W. H., Yung, B. Y., Syu, W. J., Lee, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleolar+phosphoprotein+B23+interacts+with+hepatitis+delta+antigens+and+modulates+the+hepatitis+delta+virus+RNA+replication.">The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication.</a> Journal of Biological Chemistry. 276, 25166-25175 (2001).</li><li>Li, Y. J., Macnaughton, T., Gao, L., Lai, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-templated+replication+of+hepatitis+delta+virus:+genomic+and+antigenomic+RNAs+associate+with+different+nuclear+bodies.">RNA-templated replication of hepatitis delta virus: genomic and antigenomic RNAs associate with different nuclear bodies.</a> Journal of Virology. 80, 6478-6486 (2006).</li><li>Yang, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+nucleolin+and+its+identification+as+a+transcription+repressor.">Purification and characterization of nucleolin and its identification as a transcription repressor.</a> Molecular and Cellular Biology. 14, 6068-6074 (1994).</li><li>Sarek, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophosmin+phosphorylation+by+v-cyclin-CDK6+controls+KSHV+latency.">Nucleophosmin phosphorylation by v-cyclin-CDK6 controls KSHV latency.</a> PLoS Pathogens. 6, 1000818 (2010).</li></ol>

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

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

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

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

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

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

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

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

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

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

Research

JoVE Journal

Immunology and Infection

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

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

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

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

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

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

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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

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

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

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

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

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

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

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

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

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

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

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

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...