Sequence of human immunodeficiency virus type 1 (HIV-1) gag localization and oligomerization monitored with live Confocal Imaging of a replication-competent, fluorescently tagged EIV-1

被引:181
作者
Huebner, Wolfgang [1 ]
Chen, Ping [1 ]
Del Portillo, Armando [1 ]
Liu, Yuxin [1 ]
Gordon, Ronald E. [2 ]
Chen, Benjamin K. [1 ]
机构
[1] Mt Sinai Sch Med, Dept Med, Div Infect Dis, Inst Immunol, New York, NY 10029 USA
[2] Mt Sinai Sch Med, Dept Pathol, New York, NY 10029 USA
关键词
D O I
10.1128/JVI.01088-07
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
引用
收藏
页码:12596 / 12607
页数:12
相关论文
共 57 条
[1]   PRODUCTION OF ACQUIRED IMMUNODEFICIENCY SYNDROME-ASSOCIATED RETROVIRUS IN HUMAN AND NONHUMAN CELLS TRANSFECTED WITH AN INFECTIOUS MOLECULAR CLONE [J].
ADACHI, A ;
GENDELMAN, HE ;
KOENIG, S ;
FOLKS, T ;
WILLEY, R ;
RABSON, A ;
MARTIN, MA .
JOURNAL OF VIROLOGY, 1986, 59 (02) :284-291
[2]   Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles [J].
Basyuk, E ;
Galli, T ;
Mougel, M ;
Blanchard, JM ;
Sitbon, M ;
Bertrand, E .
DEVELOPMENTAL CELL, 2003, 5 (01) :161-174
[3]   FRET or no FRET: A quantitative comparison [J].
Berney, C ;
Danuser, G .
BIOPHYSICAL JOURNAL, 2003, 84 (06) :3992-4010
[4]   Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses [J].
Chen, Ping ;
Huebner, Wolfgang ;
Spinelli, Matthew A. ;
Chen, Benjamin K. .
JOURNAL OF VIROLOGY, 2007, 81 (22) :12582-12595
[5]   Leucine-specific, functional interactions between human immunodeficiency virus type 1 Nef and adaptor protein complexes [J].
Coleman, SH ;
Van Damme, N ;
Day, JR ;
Noviello, CM ;
Hitchin, D ;
Madrid, R ;
Benichou, S ;
Guatelli, JC .
JOURNAL OF VIROLOGY, 2005, 79 (04) :2066-2078
[6]   In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53 [J].
Denek, Magdalena ;
Pelchen-Matthews, Annegret ;
Byland, Rahel ;
Ruiz-Mateos, Ezequiel ;
Marsh, Mark .
JOURNAL OF CELL BIOLOGY, 2007, 177 (02) :329-341
[7]   A novel fluorescence resonance energy transfer assay demonstrates that the human immunodeficiency virus type 1 Pr55GagI domain mediates Gag-Gag interactions [J].
Derdowski, A ;
Ding, LM ;
Spearman, P .
JOURNAL OF VIROLOGY, 2004, 78 (03) :1230-1242
[8]   AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly [J].
Dong, XH ;
Li, H ;
Derdowski, A ;
Ding, LM ;
Burnett, A ;
Chen, XM ;
Peters, TR ;
Dermody, TS ;
Woodruff, E ;
Wang, JJ ;
Spearman, P .
CELL, 2005, 120 (05) :663-674
[9]   Mutation of the SP1 sequence impairs both multimerization and membrane-binding activities of human immunodeficiency virus type 1 Gag [J].
Guo, XF ;
Roldan, A ;
Hu, J ;
Wainberg, MA ;
Liang, C .
JOURNAL OF VIROLOGY, 2005, 79 (03) :1803-1812
[10]   Rapid localization of Gag/GagPol complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus type 1 [J].
Halwani, R ;
Khorchid, A ;
Cen, S ;
Kleiman, L .
JOURNAL OF VIROLOGY, 2003, 77 (07) :3973-3984