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Resolution of de novo HIV production and trafficking in immature dendritic cells

Nature Methods volume 5, pages 75–85 (2008)Cite this article

Abstract

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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Figure 1: Introduction of dTCM in the matrix-capsid protein spacer region has limited effects on HIV replication.
Figure 2: Maximizing signal-to-noise ratio and staining restriction with FlAsH in immature DCs.
Figure 3: Dichotomy of FlAsH and mAb staining is a function of dTCM redox state and gag cleavage.
Figure 4: FlAsH-Matrix staining is absent in DC endosomes.
Figure 5: HIV production and release at the surface of DCs.
Figure 6: Aggregation and transfer of HIV in DC-DC conjugates.
Figure 7: HIV mobilization in DCs after CD4+ T cell contact FlAsH-matrix mobilization from HIV-infected immature DC toward CD4+ T cell contact points in the presence or absence of HIV envelope.
Figure 8: Schematic summary of FlAsH and mAb bias in the viral life cycle.

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Acknowledgements

We acknowledge assistance from the members of the Rockefeller University's Bioimaging Facility and core director A. North, F. Brilot for help with immunofluorescence staining, bioimaging analysis and editing the manuscript, S. Trapp for assistance with protein chemistry and Luminex Analyses, and I. Frank for critical evaluation of the manuscript. We thank M. Federico (Istituto Superiore di Sanità) for the deltaenvNL43 plasmid, E. Freed (US National Institute of Allergy and Infectious Diseases) for the pEnvAD8, pEnvNL43p6L1Term and pEnvNL43p6PTAP- NL43 plasmids and T. Hope (Feinberg School of Medicine, Northwestern University) for the mrfp-VPR plasmid. The TZM-bl cell line and The pHEF-VSV-G envelope plasmid was obtained from the AIDS Reagent Program, National Institute of Allergy and Infectious Diseases). This work was outlined and supported by a CJ Martin Fellowship of the National Health and Medical Research Council of Australia (S.G.T.). Also supported by National Institute of Health grants AI040877, HD041752, AI052048, AI065413, DE015512 and AI065412. H.S. and N.R. are supported by the Tilak—The Health Company (Innsbruck, Austria).

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  1. Stuart G Turville

    Present address: Present address: Center for Virus Research, Westmead Millennium Institute, Westmead Hospital and University of Sydney, Sydney, NSW 2145, Australia.,

Authors and Affiliations

  1. Center for Biomedical Research, Population Council, 1230 York Avenue, New York, 10065, New York, USA

    Stuart G Turville, Meropi Aravantinou & Melissa Robbiani

  2. Department of Dermatology, Innsbruck Medical University, Schöpfstrasse 24, A-6020, Innsbruck, Austria

    Hella Stössel & Nikolaus Romani

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Contributions

S.G.T. conceived and designed the study, acquired, analyzed and interpreted data, and wrote the manuscript; M.A. acquired and analyzed data; H.S. acquired and analyzed electron microscopy data; N.R. analyzed electron microscopy data and revised the manuscript; M.R. supervised the project and wrote the manuscript.

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Correspondence to Stuart G Turville.

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Turville, S., Aravantinou, M., Stössel, H. et al. Resolution of de novo HIV production and trafficking in immature dendritic cells. Nat Methods 5, 75–85 (2008). https://doi.org/10.1038/nmeth1137

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