Imaging the biogenesis of individual HIV-1 virions in live cells

Abstract

Observations of individual virions in live cells have led to the characterization of their attachment, entry and intracellular transport1. However, the assembly of individual virions has never been observed in real time. Insights into this process have come primarily from biochemical analyses of populations of virions or from microscopic studies of fixed infected cells. Thus, some assembly properties, such as kinetics and location, are either unknown or controversial2,3,4,5. Here we describe quantitatively the genesis of individual virions in real time, from initiation of assembly to budding and release. We studied fluorescently tagged derivatives of Gag, the major structural component of HIV-1—which is sufficient to drive the assembly of virus-like particles6—with the use of fluorescence resonance energy transfer, fluorescence recovery after photobleaching and total-internal-reflection fluorescent microscopy in living cells. Virions appeared individually at the plasma membrane, their assembly rate accelerated as Gag protein accumulated in cells, and typically 5–6 min was required to complete the assembly of a single virion. These approaches allow a previously unobserved view of the genesis of individual virions and the determination of parameters of viral assembly that are inaccessible with conventional techniques.

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Figure 1: Two distinct behaviours of HIV-1 Gag puncta at the plasma membrane.
Figure 2: FRET analysis of individual Gag puncta.
Figure 3: FRAP analysis of individual Gag puncta.
Figure 4: Variation in HIV-1 assembly kinetics.

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Acknowledgements

We thank T. Zang for sharing the HeLa cell line stably expressing DsRed–clathrin light chain; A. Baraff for statistical analysis; members of the Bieniasz and Simon laboratories for discussions; and R. Y. Tsien, T. Kirchhausen and G. Miesenböck for plasmids. This work was supported by grants from the National Institutes of Health (to P.D.B. and S.M.S.) and the National Science Foundation (to S.M.S.). N.J. is supported by an amfAR Mathilde Krim Fellowship in Basic Biomedical Research.

Author information

Correspondence to Paul D. Bieniasz or Sanford M. Simon.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8 with Legends and Supplementary Methods. (PDF 1591 kb)

Supplementary Movie 1

This file contains Supplementary Movie 1. Cell expressing a 1:1 mixture of Gag and Gag-GFP, illustrating the transition from a diffuse to a punctate Gag distribution, observed over a 40 minute period. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 30 frames/sec. (MOV 7194 kb)

Supplementary Movie 2

This file contains Supplementary Movie 2. Example of 3 individual slowly appearing Gag puncta from a cell expressing a mixture of Gag and Gag-GFP observed for 40 min. The field of observation is 5.5x5.5μm. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 30 frames/sec. (MOV 15738 kb)

Supplementary Movie 3

This file contains Supplementary Movie 3. An example of a rapidly appearing/disappearing Gag punctum in a cell expressing a mixture of Gag and Gag-GFP observed for 10 minutes. The field of observation is 5.5x5.5μm. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 30 frames/sec. (MOV 7168 kb)

Supplementary Movie 4

This file contains Supplementary Movie 4. Appearance of Gag puncta in a cell stably expressing CD63-mCherry (red) and transiently expressing Gag and Gag-GFP (green), observed over a 40-minute period. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 30 frames/sec. (MOV 10308 kb)

Supplementary Movie 5

This file contains Supplementary Movie 5. Examples of rapidly appearing/disappearing Gag puncta in a cell stably expressing CD63-mCherry (red) and transiently expressing Gag and Gag-GFP (green) observed for 10 minutes. The field of observation is 5.5x5.5μm. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 15 frames/sec. (MOV 4284 kb)

Supplementary Movie 6

This file contains Supplementary Movie 6. Examples of a rapidly appearing/disappearing Gag puncta in a cell stably expressing DsRed-clathrin-light-chain (red) and transiently expressing Gag and Gag-GFP (green) observed over a 5 minute period. The field of observation is 5.5x5.5μm. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 15 frames/sec. (MOV 3995 kb)

Supplementary Movie 7

This file contains Supplementary Movie 7. Example of a slowly appearing Gag punctum in a cell stably expressing CD63-mCherry (red) and transiently expressing Gag and Gag-GFP (green) imaged over 30 minutes. The field of observation is 5.5x5.5μm. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 30 frames/sec. (MOV 12698 kb)

Supplementary Movie 8

This file contains Supplementary Movie 8. Example of a slowly appearing Gag punctum in a cell stably expressing DsRed-clathrin-light-chain (red) and transiently expressing Gag and Gag-GFP (green) observed over 15 minutes. The field of observation is 5.5x5.5μm. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 30 frames/sec. (MOV 3375 kb)

Supplementary Movie 9

This file contains Supplementary Movie 9. Cells expressing Gag and Gag-GFP imaged for a total of 28 minutes. The top cell is the control, non-bleached cell. The bottom cell was bleached for 2 minutes after a 5-minute imaging period and observed for 20 additional minutes after the bleach. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 30 frames/sec. (MOV 6666 kb)

Supplementary Movie 10

This file contains Supplementary Movie 10. View of a portion of a cell imaged for 5 minutes, bleached for 2 minutes and then observed for 20 additional minutes, illustrating both VLPs that recover after the bleach and VLPs that do not. The field of observation is 5.5x5.5μm. The movie was acquired at 1 frame every 5 sec, as described in ‘Materials and Methods’ and displayed at a rate of 30 frames/sec. (MOV 8363 kb)

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Jouvenet, N., Bieniasz, P. & Simon, S. Imaging the biogenesis of individual HIV-1 virions in live cells. Nature 454, 236–240 (2008) doi:10.1038/nature06998

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