Journal: Proc Natl Acad Sci U S A / Year: 2014 Title: Furin cleavage of the Moloney murine leukemia virus Env precursor reorganizes the spike structure. Authors: Mathilda Sjöberg / Shang-Rung Wu / Robin Löving / Kimmo Rantalainen / Birgitta Lindqvist / Henrik Garoff / Abstract: The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell ...The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion.
History
Deposition
Mar 28, 2014
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Header (metadata) release
Apr 9, 2014
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Map release
Apr 9, 2014
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Update
May 14, 2014
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Current status
May 14, 2014
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Legacy - Astigmatism: Objective lens astigmatism was corrected using online FFT.
Date
Jan 17, 2014
Image recording
Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 3.5 µm / Number real images: 30 / Average electron dose: 9 e/Å2 / Bits/pixel: 14
Electron beam
Acceleration voltage: 200 kV / Electron source: TUNGSTEN HAIRPIN
The particles were selected using a semi-automatic selection program in EMAN.
CTF correction
Details: Each particle
Final reconstruction
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: EMAN1, EMAN2 Details: Final maps were calculated from a single dataset. The particles were selected using an automatic selection program. Damaged particles were removed by visual inspection. Number images used: 1329
Final angle assignment
Details: EMAN
Final two d classification
Number classes: 20
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