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Volume 4 issue 11 November 2003
Visualization of retroviral replication in living cells reveals budding into multivesicular bodies
Nathan M. Sherer*, Maik J. Lehmann*, Luisa F. Jimenez-Soto*, Alyssa Ingmundson*, Stacy M. Horner*, Gregor Cicchetti#, Philip G. Allen#, Marc Pypaert&, James M. Cunningham$, and Walther Mothes*
*Section of Microbial Pathogenesis and &Department of Cell Biology, Yale University School of Medicine, 295 Congress Ave, New Haven, CT 06536, USA
#Division of Hematology, Brigham and Women's Hospital, Boston, MA, 02115
$Howard Hughes Medical Institute and Division of Hematology, Brigham and Women's Hospital, Boston, MA, 02115
Key words: retroviral budding and assembly, murine leukemia virus (MLV), human immunodeficiency virus (HIV), multivesicular body (MVB), imaging of viral replication.
Running title: retroviral budding
Abbreviations: HIV, human immunodeficiency virus; MLV, murine leukemia virus; MVB, multivesicular body; vps, vacuolar protein sorting; TSG101, tumor susceptibility gene 101.
To whom correspondence should be addressed:
email: walther.mothes@yale.edu
Supplemental Movies
Supplemental Movie for Figure 2D. Gag localizes to vesicles. MLV Gag-RFP was expressed under conditions of infectious particle production in 293 cells expressing cytoplasmic YFP. A time lapse is presented of living cells, images taken every 5 s. RFP and YFP channels were consecutively recorded in multitrack mode using an LSM 510 confocal microscope and subsequently pseudo-colored in red and green, respectively.
View Movie
Supplemental Movie for Figure 2E. HIV Gag-GFP was expressed in 293 cells. Cells were fixed 30 hours post-transfection for analysis by confocal z-sectioning at a step size of 0.4 µm.
View Movie
Supplemental Movies a and b for Figure 3. (a) A time-lapse movie (2 s / frame) is presented showing MLV Gag-CFP (green) and Lamp1-YFP (red) co-expressed in 293 cells. White dashes indicate highly motile Lamp1+ and Gag+ vesicles and tubules. (b) A time-lapse movie as in (a) showing HIV Gag-CFP (green) co-expressed with Lamp1-YFP (red).
Supplemental Movies a and b for Figure 4D. HIV Gag-CFP localizes to the cytoplasmic face of Lamp1 vesicles. (a) A complete z-stack for the images presented in Figure 4D. (b) A short time-lapse (2 s / frame) of a similar experiment as in (a).
Supplemental Movies a and b for Appendix Figure 2. (a) Short time lapse for the image shown in panel B (2 s / frame). (b) The accumulation of Gag in large intracellular vesicles is particularly pronounced for mutant Gag impaired in budding. Short time lapse of two cells expressing HIV Gag-GFP where the budding motif PTAP has been mutated.
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