Journal: Science / Year: 2008 Title: Structure of the immature dengue virus at low pH primes proteolytic maturation. Authors: I-Mei Yu / Wei Zhang / Heather A Holdaway / Long Li / Victor A Kostyuchenko / Paul R Chipman / Richard J Kuhn / Michael G Rossmann / Jue Chen / Abstract: Intracellular cleavage of immature flaviviruses is a critical step in assembly that generates the membrane fusion potential of the E glycoprotein. With cryo-electron microscopy we show that the ...Intracellular cleavage of immature flaviviruses is a critical step in assembly that generates the membrane fusion potential of the E glycoprotein. With cryo-electron microscopy we show that the immature dengue particles undergo a reversible conformational change at low pH that renders them accessible to furin cleavage. At a pH of 6.0, the E proteins are arranged in a herringbone pattern with the pr peptides docked onto the fusion loops, a configuration similar to that of the mature virion. After cleavage, the dissociation of pr is pH-dependent, suggesting that in the acidic environment of the trans-Golgi network pr is retained on the virion to prevent membrane fusion. These results suggest a mechanism by which flaviviruses are processed and stabilized in the host cell secretory pathway.
History
Deposition
Feb 26, 2008
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Header (metadata) release
Mar 4, 2008
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Map release
Apr 22, 2009
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Update
Jul 8, 2011
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Current status
Jul 8, 2011
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 6 Details: The virus was mixed in NTE buffer (10 mM Tris, 120 mM NaCl, and 1 mM EDTA at pH 8) with 50 mM MES, 120 mM NaCl at pH 5.6 to yield a final pH of 6
Grid
Details: 400 mesh copper grid
Vitrification
Cryogen name: ETHANE / Instrument: OTHER Method: A small vial of ethane is placed inside a larger liquid nitrogen reservoir. The grid holding a few microliters of the sample is held in place at the bottom of a plunger by the means of fine ...Method: A small vial of ethane is placed inside a larger liquid nitrogen reservoir. The grid holding a few microliters of the sample is held in place at the bottom of a plunger by the means of fine tweezers. Once the ethane in the vial is completely frozen, it needs to be slightly melted. When the liquid ethane is ready, a piece of filter paper is then pressed against the sample to blot of excess buffer, sufficient to leave a thin layer on the grid. After a predetermined time, the filter paper is removed, and the plunger is allowed to drop into the liquid ethane. Once the grid enters the liquid ethane, the sample is rapidly frozen, and the grid is transferred under liquid nitrogen to a storage box immersed liquid nitrogen for later use in the microscope.
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Electron microscopy
Microscope
FEI/PHILIPS CM200FEG
Temperature
Average: 98 K
Image recording
Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 14 / Average electron dose: 17 e/Å2 / Od range: 1 / Bits/pixel: 12
Tilt angle min
0
Tilt angle max
0
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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