Nature Methods
- 5, 75 - 85 (2008)
Published online: 2 December 2007; | doi:10.1038/nmeth1137
Resolution of de novo HIV production and trafficking in immature dendritic cellsStuart G Turville1, 3, Meropi Aravantinou1, Hella Stössel2, Nikolaus Romani2 & Melissa Robbiani11
Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10065, USA. 2
Department of Dermatology, Innsbruck Medical University, Schöpfstrasse 24, Innsbruck Austria A-6020. 3
Present address: Center for Virus Research, Westmead Millennium Institute, Westmead Hospital and University of Sydney, Sydney, NSW 2145, Australia.
Correspondence should be addressed to Stuart G Turville stuart_turville@wmi.usyd.edu.au The challenge in observing de novo virus production in human immunodeficiency virus (HIV)-infected dendritic cells (DCs) is the lack of resolution between cytosolic immature and endocytic mature HIV gag protein. To track HIV production, we developed an infectious HIV construct bearing a diothiol-resistant tetracysteine motif (dTCM) at the C terminus of HIV p17 matrix within the HIV gag protein. Using this construct in combination with biarsenical dyes, we observed restricted staining of the dTCM to de novo–synthesized uncleaved gag in the DC cytosol. Co-staining with HIV gag antibodies, reactive to either p17 matrix or p24 capsid, preferentially stained mature virions and thus allowed us to track the virus at distinct stages of its life cycle within DCs and upon transfer to neighboring DCs or T cells. Thus, in staining HIV gag with biarsenical dye system in situ, we characterized a replication-competent virus capable of being tracked preferentially within infected leukocytes and observed in detail the dynamic nature of the HIV production and transfer in primary DCs.
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