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- Loving, R, et al.
(författare)
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Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering
- 2012
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 109:20, s. 7735-7740
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Tidskriftsartikel (refereegranskat)abstract
- The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU–TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.
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- Wu, S.R., et al.
(författare)
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Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure
- 2010
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:44, s. 18844-18849
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Tidskriftsartikel (refereegranskat)abstract
- The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus-cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N- and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.
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