This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rudner, L.
Right arrow Articles by Ott, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rudner, L.
Right arrow Articles by Ott, D. E.

 Previous Article  |  Next Article 

Journal of Virology, April 2005, p. 4055-4065, Vol. 79, No. 7
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.7.4055-4065.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Dynamic Fluorescent Imaging of Human Immunodeficiency Virus Type 1 Gag in Live Cells by Biarsenical Labeling

Lynnie Rudner,1 Sascha Nydegger,1 Lori V. Coren,2 Kunio Nagashima,3 Markus Thali,1 and David E. Ott2*

Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont,1 Basic Research,2 Research Technology Programs, SAIC—Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland3

Received 9 September 2004/ Accepted 12 November 2004

Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both Pr55Gag expression and full-length proviral constructs. Membrane-permeable biarsenical compounds FlAsH and ReAsH covalently bond to this tetracysteine sequence and specifically fluoresce, effectively labeling Gag in the cell. Biarsenical labeling readily and specifically detected a tetracysteine-tagged HIV-1 Gag protein (Gag-TC) in HeLa, Mel JuSo, and Jurkat T cells by deconvolution fluorescence microscopy. Gag-TC was localized primarily at or near the plasma membrane in all cell types examined. Fluorescent two-color analysis of Gag-TC in HeLa cells revealed that nascent Gag was present mostly at the plasma membrane in distinct regions. Intracellular imaging of a Gag-TC myristylation mutant observed a diffuse signal throughout the cell, consistent with the role of myristylation in Gag localization to the plasma membrane. In contrast, mutation of the L-domain core sequence did not appreciably alter the localization of Gag, suggesting that the PTAP L domain functions at the site of budding rather than as a targeting signal. Taken together, our results show that Gag concentrates in specific plasma membrane areas rapidly after translation and demonstrate the utility of biarsenical labeling for visualizing the dynamic localization of Gag.

* Corresponding author. Mailing address: Basic Research Program, SAIC—Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702-1201. Phone: (301) 846-5723. Fax: (301) 846-5588. E-mail: ott{at}ncifcrf.gov.

Journal of Virology, April 2005, p. 4055-4065, Vol. 79, No. 7
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.7.4055-4065.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Beaumont, E., Vendrame, D., Verrier, B., Roch, E., Biron, F., Barin, F., Mammano, F., Brand, D. (2009). Matrix and Envelope Coevolution Revealed in a Patient Monitored since Primary Infection with Human Immunodeficiency Virus Type 1. J. Virol. 83: 9875-9889 [Abstract] [Full Text]  
  • Ott, D. E., Coren, L. V., Shatzer, T. (2009). The Nucleocapsid Region of Human Immunodeficiency Virus Type 1 Gag Assists in the Coordination of Assembly and Gag Processing: Role for RNA-Gag Binding in the Early Stages of Assembly. J. Virol. 83: 7718-7727 [Abstract] [Full Text]  
  • Molle, D., Segura-Morales, C., Camus, G., Berlioz-Torrent, C., Kjems, J., Basyuk, E., Bertrand, E. (2009). Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress. J. Biol. Chem. 284: 19727-19743 [Abstract] [Full Text]  
  • Joshi, A., Ablan, S. D., Soheilian, F., Nagashima, K., Freed, E. O. (2009). Evidence that Productive Human Immunodeficiency Virus Type 1 Assembly Can Occur in an Intracellular Compartment. J. Virol. 83: 5375-5387 [Abstract] [Full Text]  
  • Das, S. C., Panda, D., Nayak, D., Pattnaik, A. K. (2009). Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells. J. Virol. 83: 2611-2622 [Abstract] [Full Text]  
  • Chan, R., Uchil, P. D., Jin, J., Shui, G., Ott, D. E., Mothes, W., Wenk, M. R. (2008). Retroviruses Human Immunodeficiency Virus and Murine Leukemia Virus Are Enriched in Phosphoinositides. J. Virol. 82: 11228-11238 [Abstract] [Full Text]  
  • Thomas, J. A., Bosche, W. J., Shatzer, T. L., Johnson, D. G., Gorelick, R. J. (2008). Mutations in Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Zinc Fingers Cause Premature Reverse Transcription. J. Virol. 82: 9318-9328 [Abstract] [Full Text]  
  • Lin, M. Z., Wang, L. (2008). Selective Labeling of Proteins with Chemical Probes in Living Cells. Physiology 23: 131-141 [Abstract] [Full Text]  
  • Hubner, W., Chen, P., Portillo, A. D., 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. J. Virol. 81: 12596-12607 [Abstract] [Full Text]  
  • Jolly, C., Sattentau, Q. J. (2007). Human Immunodeficiency Virus Type 1 Assembly, Budding, and Cell-Cell Spread in T Cells Take Place in Tetraspanin-Enriched Plasma Membrane Domains. J. Virol. 81: 7873-7884 [Abstract] [Full Text]  
  • Finzi, A., Orthwein, A., Mercier, J., Cohen, E. A. (2007). Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane. J. Virol. 81: 7476-7490 [Abstract] [Full Text]  
  • Chatel-Chaix, L., Abrahamyan, L., Frechina, C., Mouland, A. J., DesGroseillers, L. (2007). The Host Protein Staufen1 Participates in Human Immunodeficiency Virus Type 1 Assembly in Live Cells by Influencing pr55Gag Multimerization. J. Virol. 81: 6216-6230 [Abstract] [Full Text]  
  • Nydegger, S., Khurana, S., Krementsov, D. N., Foti, M., Thali, M. (2006). Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1. JCB 173: 795-807 [Abstract] [Full Text]  
  • Harila, K., Prior, I., Sjoberg, M., Salminen, A., Hinkula, J., Suomalainen, M. (2006). Vpu and tsg101 regulate intracellular targeting of the human immunodeficiency virus type 1 core protein precursor pr55gag.. J. Virol. 80: 3765-3772 [Abstract] [Full Text]  
  • Ott, D. E., Coren, L. V., Gagliardi, T. D. (2005). Redundant Roles for Nucleocapsid and Matrix RNA-Binding Sequences in Human Immunodeficiency Virus Type 1 Assembly. J. Virol. 79: 13839-13847 [Abstract] [Full Text]  
  • Ott, D. E., Coren, L. V., Gagliardi, T. D., Nagashima, K. (2005). Heterologous Late-Domain Sequences Have Various Abilities To Promote Budding of Human Immunodeficiency Virus Type 1. J. Virol. 79: 9038-9045 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»