HIV-1病毒样颗粒芽殖试验

Nathan H Vande Burgt; Luis J Cocka; Paul Bates

doi:10.21769/BioProtoc.830

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Peer-reviewed

HIV-1 Virus-like Particle Budding Assay

HIV-1病毒样颗粒芽殖试验

NB Nathan H Vande Burgt

L Luis J Cocka email

PB Paul Bates

发布: 2013年07月20日第3卷第14期 DOI: 10.21769/BioProtoc.830 浏览次数: 10452

评审: Fanglian He

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Cover of PLOS Pathogens, featuring study using the protocol.

Sep 2012

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Abstract

Viral replication culminates with the egress of the mature virion from the host cell. This step of the viral life cycle has recently garnered increased attention with the discovery of the cellular restriction factor, Tetherin, which tethers budded virions to the surface of infected cells and inhibits viral spread. The importance of this block in viral infections has been suggested by the discovery of viral antagonists, such as HIV-1 Vpu, which counteract Tetherin. This protocol describes a system to study HIV-1 budding under BSL-2 safety conditions. It takes advantage of the ability of many viral matrix/capsid proteins to generate non-infectious virus-like particles (VLPs) with the expression of a single viral protein (i.e. HIV-1 p24 Gag). This protocol was recently used to characterize the effect of Tetherin isoforms on VLP release in the presence of HIV-1 Vpu (Cocka and Bates, 2012). Simultaneous expression of Tetherin and other viral antagonists can be used to study Tetherin-mediated restriction on viral budding.

Keywords: HIV-1 (HIV-1)

Virus-like (病毒样)

Budding (萌芽)

Virus release (病毒的释放)

Materials and Reagents

HEK 293T cell line (ATCC, catalog number: CRL-11268 )
Antibody, HIV-1 p24 Gag (NIH AIDS Reagent Program, catalog number: 24-3 )
Antibody, HIV-1 Vpu (optional) (NIH AIDS Reagent Program, catalog number: 969 )
Antibody, human Tetherin (optional) (NIH AIDS Reagent Program, catalog number: 11721 )
Bromophenol Blue (Sigma-Aldrich, catalog number: B-5525 )
Complete Mini protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693132001 )
Criterion Precast Gels, 4-15% Tris-HCl (Bio-Rad Laboratories, catalog number: 345-0027 )
Dithiothreitol (DTT) (Thermo Fisher Scientific, catalog number: BP172-5 )
Dulbecco’s Modified Eagle Media (DMEM) (Life Technologies, Invitrogen^TM, catalog number: 11965-084 )
Dulbecco’s Phosphate-buffered saline (DPBS) (Life Technologies, Invitrogen^TM, catalog number: 14190-136 )
0.5 M EDTA solution (Life Technologies, Invitrogen^TM, catalog number: 15575-038 )
Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F2442-500ML )
Glycerol (Thermo Fisher Scientific, catalog number: BP229-1 )
Hydrochloric acid (HCl) (Thermo Fisher Scientific, catalog number: A144-212 )
Instant Nonfat Dry Milk (Nestle, catalog number: 050000033188 )
Lipofectamine 2000 (Life Technologies, Invitrogen^TM, catalog number: 11668-019 )
NaCl (Thermo Fisher Scientific, catalog number: BP 358-212 )
Opti-MEM (Life Technologies, Invitrogen^TM, catalog number: 31985-070 )
Plasmid, human Tetherin cDNA (optional) (OpenBiosystems, catalog number: 5217945 )
Plasmid, psPAX2 HIV Gag (Addgene, catalog number: 12260 )
Plasmid, Tetherin antagonist (HIV-1 Vpu) (optional)
Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: BP166-500 )
D-Sucrose (Thermo Fisher Scientific, catalog number: BP220-1 )
Tris Base (Thermo Fisher Scientific, catalog number: BP152-500 )
Triton X-100 (Thermo Fisher Scientific, catalog number: BP151-500 )
Tween 20 Thermo Fisher Scientific, catalog number: BP337-500 )
Cell culture media (see Recipes)
20% sucrose solution (see Recipes)
Triton X-100 lysis buffer (see Recipes)
Blocking solution (see Recipes)
6x protein gel loading buffer (see Recipes)

Equipment

24 well plate, cell culture treated (Corning, catalog number: 3524 )
1.7 ml Microcentrifuge tubes (GeneMate, catalog number: C-3260-1X )
8 x 34 mm Ultracentrifuge tubes (Beckman Coulter, catalog number: 343776 )
Tabletop Centrifuge, refrigerated (Eppendorf Centrifuge, model: 5417R )
TLA 120.1 rotor (Beckman Coulter, model: 357655 )
Ultracentrifuge, refrigerated (Beckman Coulter Optima, model: TLX-IM-3 )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Vande Burgt, N. H., Cocka, L. J. and Bates, P. (2013). HIV-1 Virus-like Particle Budding Assay. Bio-protocol 3(14): e830. DOI: 10.21769/BioProtoc.830.