Name: in vitro uncoating of hiv-1 cores
Uploaded: 2011-11-08T13:00:00
Description: Watch this Scientific Journal Video about In vitro Uncoating of HIV-1 Cores at JoVE.com

HIV-1 uncoating studies in the Aiken laboratory were supported by NIH grants AI40364, AI50423 and AI076121. Several key materials, including reagents used in the p24 ELISA, were provided by the NIH AIDS Research and Reference Program, Division of AIDS, NIAID, NIH.

1. Production of HIV-1 particles
Before proceeding, you must obtain permission and follow the guidelines from the Biosafety office of your institution to work in a biosafety facility approved for infectious HIV. You must ensure that proper care and safety precautions are followed during working in the biosafety laboratory.
Production of HIV-1 particles is typically performed by transient transfection of 293T cells with HIV-1 proviral DNA using a calcium phosphate tran...

<ol>
	<li>Ganser, B. K., Li, S., Klishko, V. Y., Finch, J. T., Sundquist, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+analysis+of+conical+models+for+the+HIV-1+core.">Assembly and analysis of conical models for the HIV-1 core.</a> Science. 283, 80-83 (1999).</li><li>Li, S., Hill, C. P., Sundquist, W. I., Finch, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+reconstructions+of+helical+assemblies+of+the+HIV-1+CA+protein.">Image reconstructions of helical assemblies of the HIV-1 CA protein.</a> Nature. 407, 409-413 (2000).</li><li>Aiken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+and+cellular+factors+that+regulate+HIV-1+uncoating.">Viral and cellular factors that regulate HIV-1 uncoating.</a> Curr. Opin. HIV. AIDS. 1, 194-199 (2006).</li><li>Forshey, B. M., Schwedler, U. v. o. n., Sundquist, W. I., Aiken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+a+human+immunodeficiency+virus+type+1+core+of+optimal+stability+is+crucial+for+viral+replication.">Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication.</a> J. Virol. 76, 5667-5677 (2002).</li><li>Forshey, B. M., Aiken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disassembly+of+human+immunodeficiency+virus+type+1+cores+in+vitro+reveals+association+of+Nef+with+the+subviral+ribonucleoprotein+complex.">Disassembly of human immunodeficiency virus type 1 cores in vitro reveals association of Nef with the subviral ribonucleoprotein complex.</a> J. Virol. 77, 4409-4414 (2003).</li><li>Wacharapornin, P., Lauhakirti, D., Auewarakul, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+capsid+mutations+on+HIV-1+uncoating.">The effect of capsid mutations on HIV-1 uncoating.</a> Virology. 358, 48-54 (2007).</li><li>Chen, C., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transformation+of+mammalian+cells+by+plasmid+DNA.">High-efficiency transformation of mammalian cells by plasmid DNA.</a> Mol Cell Biol. 7, 2745-2752 (1987).</li><li>Aiken, C., Prasad, V. R., Ganjam, K. 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N., Emerman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vpx+association+with+mature+core+structures+of+HIV-2.">Vpx association with mature core structures of HIV-2.</a> Virology. 218, 159-168 (1996).</li><li>Wehrly, K., Chesebro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p24+antigen+capture+assay+for+quantification+of+human+immunodeficiency+virus+using+readily+available+inexpensive+reagents.">p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents.</a> Methods. 12, 288-293 (1997).</li><li>Welker, R., Hohenberg, H., Tessmer, U., Huckhagel, C., Kr&#228;usslich, H. 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The method described here to purify HIV-1 cores to study uncoating in vitro is useful for studying this phase of the HIV-1 life cycle, especially due to unavailability of a validated method to analyze HIV-1 uncoating in target cells. Although this method uses equilibrium ultracentrifugation to purify cores, others have used direct pelleting after brief lysis with 1% Triton-X-10012, 13 or pelleting through a sucrose cushion overlaid with 0.1% Triton-X-10014, 15.
T...

<table border="1">
	<tbody>
		<tr>
			<td width="190">Name of the reagent</td>
			<td width="197">Company</td>
			<td width="114">Catalog number</td>
			<td width="161">Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cellgro</td>
			<td>21-031-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Mallinckrodt Baker Inc</td>
			<td>9002-93-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Acros</td>
			<td>9005-64-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Cellgro</td>
			<td>25-053-CI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2XBBS</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Composition: 50mM BES [pH 6.95], 280 mM NaCl and 1.5 mM Na2HPO4. Adjust pH to 6.95 at room temperature. Filter sterilize &amp; store in aliquots at -20&deg;C.</td>
		</tr>
		<tr>
			<td>Coating antibody: Monoclonal antibody to p24 (183-H12-5C)</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>3537</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Primary antibody: HIV-Ig (hyperimmune human patient serum)</td>
			<td>
			NIH AIDS Researchand Reference Reagent Program</td>
			<td>3957</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>
			Secondary antibody:Goat anti-human IgG, peroxidase conjugated</td>
			<td>Pierce</td>
			<td>31130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HRP substrate</td>
			<td>KPL Inc</td>
			<td>50-76-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Immulon 2HB 96 well plates</td>
			<td>Thermo Scientific</td>
			<td>3455</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SW32Ti and SW32.1 Ti rotors and compatible ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TLA-55 rotor and tabletop ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>High speed microfuge tubes</td>
			<td>Beckman Coulter</td>
			<td>326823, 358123, 357448</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Auto-Densi Flow density gradient fractionator</td>
			<td>Labconco Corp.</td>
			<td>4517000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gradient Former</td>
			<td>CBS Scientific Co. Inc.</td>
			<td>GM-20</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro uncoating of hiv-1 cores

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

Watch this Scientific Journal Video about In vitro Uncoating of HIV-1 Cores at JoVE.com

In vitro Uncoating of HIV-1 Cores

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

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 ...

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