Dynamics of ESCRT protein recruitment during retroviral assembly

Abstract

The ESCRT (endosomal sorting complex required for transport) complexes and associated proteins mediate membrane scission reactions, such as multivesicular body formation, the terminal stages of cytokinesis and retroviral particle release. These proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the ATPase Vps4A. However, these events have never been observed in living cells, and their dynamics are unknown. By quantifying the recruitment of several ESCRT and associated proteins during the assembly of two retroviruses, we show that Alix progressively accumulated at viral assembly sites, coincident with the accumulation of the main viral structural protein, Gag, and was not recycled after assembly. In contrast, ESCRT-III and Vps4A were transiently recruited only when the accumulation of Gag was complete. These data indicate that the rapid and transient recruitment of proteins that act late in the ESCRT pathway and carry out membrane fission is triggered by prior and progressive accumulation of proteins that bridge viral proteins and the late-acting ESCRT proteins.

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Figure 1: Characterization of the GFP–Chmp4b-expressing cell clone.
Figure 2: Effect of stably expressed GFP-fused ESCRT proteins on cell proliferation, cytokinesis and virion assembly and release.
Figure 3: Catalytically inactive Vps4A increases localization of stably expressed GFP-tagged ESCRT-III proteins at sites of HIV-1 assembly.
Figure 4: Imaging Chmp1b, Chmp4b, Chmp4c and Vps4A recruitment during HIV-1 Gag assembly.
Figure 5: Imaging Chmp1b, Chmp4b, Chmp4c and Vps4A recruitment during EIAV Gag assembly.
Figure 6: Imaging Alix recruitment during EIAV Gag assembly.
Figure 7: Dynamics and pattern of ESCRT protein recruitment during retroviral assembly.

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Acknowledgements

We thank W. Sundquist for reagents, J. Martin-Serrano for discussions, V. Sahi for fluorescence-activated cell sorting analysis and A. Mattheyses for TIR-FM settings. This work was supported by NIH grant K99AI87368 (to N.J.), NIH grant R01AI50111 and R01AI52774 (to P.D.B.) and NSF grant BES-0620813 and NIH GM87977 and R01 AI089844 (to S.M.S.). P.D.B. is a Howard Hughes Medical Institute investigator.

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N.J., S.M.S. and P.D.B. conceived and designed the experiments. N.J. carried out the experiments with help from M.Z. (Figs 2d and 5). N.J., S.M.S. and P.D.B. analysed the data and wrote the manuscript.

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

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The authors declare no competing financial interests.

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Jouvenet, N., Zhadina, M., Bieniasz, P. et al. Dynamics of ESCRT protein recruitment during retroviral assembly. Nat Cell Biol 13, 394–401 (2011). https://doi.org/10.1038/ncb2207

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