Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1

Hübner W, Chen P, Del Portillo A, Liu Y, Gordon RE, Chen BK (2007)
Journal of virology 81(22): 12596-12607.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
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Hübner, WolfgangUniBi ; Chen, Ping; Del Portillo, Armando; Liu, Yuxin; Gordon, Ronald E; Chen, Benjamin K
Abstract / Bemerkung
The assembly of infectious human immunodeficiency virus (HIV) requires that Gag transport and oligomerization be coordinated with its association with other viral proteins, viral RNAs, and cellular membranes. We have developed a replication-competent HIV type 1 molecular clone that carries a Gag-internal or interdomain green fluorescent protein (iGFP) fusion to reveal a physiologically accurate temporal sequence of Gag localization and oligomerization during the formation of infectious HIV. This recombinant HIV is as infectious as native HIV in single-round infectivity assays, validating its use for trafficking studies. It replicates robustly in permissive MT4 cells and is infectious, yet it spreads poorly in other T-cell lines. Immunofluorescence of Gag-iGFP showed a pattern very similar to that of native Gag. However, the intense plasma membrane Gag-iGFP fluorescence contrasts markedly with its immunofluorescence at this site, indicating that many Gag epitopes can be masked by oligomerization. Consistent with this, fluorescence resonance energy transfer studies visualized intense Gag oligomerization at the plasma membrane and weaker oligomerization at cytoplasmic sites. Four-dimensional, time-lapse confocal imaging reveals a temporal progression of Gag distribution over hours in which Gag is initially diffusely localized within the cytoplasm. Plasma membrane signals then accumulate as Gag levels increase and vesicular association appears late, only after plasma membrane site signals have reached high intensity. Lastly, the cell rounds up and HIV protease activation induces diffuse fluorescence throughout the cell. These distinct phases reveal a natural progression of Gag trafficking during the viral gene expression program. HIV Gag-iGFP is a useful tool for dissecting mechanisms of viral assembly and transmission.
Recombinant Fusion Proteins/metabolism; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/analysis; Molecular Sequence Data; Confocal; Microscopy; Humans; HIV-1/physiology; Green Fluorescent Proteins/metabolism; Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/analysis; Amino Acid Sequence; Fluorescence Resonance Energy Transfer; Virus Replication; gag Gene Products; Human Immunodeficiency Virus/analysis; gag Gene Products; Human Immunodeficiency Virus/genetics; gag Gene Products; Human Immunodeficiency Virus/metabolism
Journal of virology
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Hübner W, Chen P, Del Portillo A, Liu Y, Gordon RE, Chen BK. Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1. Journal of virology. 2007;81(22):12596-12607.
Hübner, W., Chen, P., Del Portillo, A., Liu, Y., Gordon, R. E., & Chen, B. K. (2007). Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1. Journal of virology, 81(22), 12596-12607. doi:10.1128/JVI.01088-07
Hübner, Wolfgang, Chen, Ping, Del Portillo, Armando, Liu, Yuxin, Gordon, Ronald E, and Chen, Benjamin K. 2007. “Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1”. Journal of virology 81 (22): 12596-12607.
Hübner, W., Chen, P., Del Portillo, A., Liu, Y., Gordon, R. E., and Chen, B. K. (2007). Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1. Journal of virology 81, 12596-12607.
Hübner, W., et al., 2007. Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1. Journal of virology, 81(22), p 12596-12607.
W. Hübner, et al., “Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1”, Journal of virology, vol. 81, 2007, pp. 12596-12607.
Hübner, W., Chen, P., Del Portillo, A., Liu, Y., Gordon, R.E., Chen, B.K.: Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1. Journal of virology. 81, 12596-12607 (2007).
Hübner, Wolfgang, Chen, Ping, Del Portillo, Armando, Liu, Yuxin, Gordon, Ronald E, and Chen, Benjamin K. “Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1”. Journal of virology 81.22 (2007): 12596-12607.

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Dong X, Li H, Derdowski A, Ding L, Burnett A, Chen X, Peters TR, Dermody TS, Woodruff E, Wang JJ, Spearman P., Cell 120(5), 2005
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Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling.
Rudner L, Nydegger S, Coren LV, Nagashima K, Thali M, Ott DE., J. Virol. 79(7), 2005
PMID: 15767407
Involvement of HIV-1 protease in virus-induced cell killing.
Ventoso I, Navarro J, Munoz MA, Carrasco L., Antiviral Res. 66(1), 2005
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HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent.
Jin YJ, Cai CY, Zhang X, Zhang HT, Hirst JA, Burakoff SJ., J. Immunol. 175(5), 2005
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Plasma membrane is the site of productive HIV-1 particle assembly.
Jouvenet N, Neil SJ, Bess C, Johnson MC, Virgen CA, Simon SM, Bieniasz PD., PLoS Biol. 4(12), 2006
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In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53.
Deneka M, Pelchen-Matthews A, Byland R, Ruiz-Mateos E, Marsh M., J. Cell Biol. 177(2), 2007
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HIV-1 buds predominantly at the plasma membrane of primary human macrophages.
Welsch S, Keppler OT, Habermann A, Allespach I, Krijnse-Locker J, Krausslich HG., PLoS Pathog. 3(3), 2007
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Visualization of retrovirus budding with correlated light and electron microscopy.
Larson DR, Johnson MC, Webb WW, Vogt VM., Proc. Natl. Acad. Sci. U.S.A. 102(43), 2005
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The pericentriolar recycling endosome plays a key role in Vpu-mediated enhancement of HIV-1 particle release.
Varthakavi V, Smith RM, Martin KL, Derdowski A, Lapierre LA, Goldenring JR, Spearman P., Traffic 7(3), 2006
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Vpu and Tsg101 regulate intracellular targeting of the human immunodeficiency virus type 1 core protein precursor Pr55gag.
Harila K, Prior I, Sjoberg M, Salminen A, Hinkula J, Suomalainen M., J. Virol. 80(8), 2006
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Fanciful FRET.
Vogel SS, Thaler C, Koushik SV., Sci. STKE 2006(331), 2006
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HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane.
Neil SJ, Eastman SW, Jouvenet N, Bieniasz PD., PLoS Pathog. 2(5), 2006
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