Sequential activation of the three protomers in the Moloney murine leukemia virus Env

Sjöberg, Mathilda; Löving, Robin; Lindqvist, Birgitta; Garoff, Henrik

doi:10.1073/pnas.1617264114

Cited by 3 publications

(3 citation statements)

References 35 publications

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“…Asymmetric structural intermediates during the activation of viral fusion machines may be more common than previously thought. Single receptor engagement and sequential shedding has been observed for the Murine Leukemia Virus (MLV) Env ( Riedel et al, 2017 ; Sjöberg et al, 2017 ). And a recent study on the activation of pre-fusion HIV-1 gp41 to downstream intermediate states suggested that gp41 also opens through asymmetric intermediates ( Khasnis et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations

Gorman

et al. 2018

eLife

139

201

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HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions shows Env trimers to be dynamic, spontaneously transitioning between three distinct well-populated conformational states: a pre-triggered Env (State 1), a default intermediate (State 2) and a three-CD4-bound conformation (State 3), which can be stabilized by binding of CD4 and coreceptor-surrogate antibody 17b. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we show the default intermediate configuration to be asymmetric, with individual protomers adopting distinct conformations. During entry, this asymmetric intermediate forms when a single CD4 molecule engages the trimer. The trimer can then transition to State 3 by binding additional CD4 molecules and coreceptor.

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Section: Discussionmentioning

confidence: 99%

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations

Gorman

et al. 2018

eLife

139

201

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“…In support of this model, we note that the three-RBD-out conformation is only observed in 0.3% of our cryo-EM data. Further experiments will be required to test this proposed model of receptor-induced triggering, but sequential activation of protomers in a class-I fusion protein has recently been demonstrated for Moloney murine leukemia virus (39).…”

Section: Engineering Of Coronavirus S Proteins That Retain the Prefusionmentioning

confidence: 99%

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

Pallesen

Wang

Corbett

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

1,049

1,259

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Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined highresolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptoraccessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.coronavirus | neutralizing antibody | cryo-EM | X-ray crystallography | peplomer

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“…During membrane fusion in influenza virus and herpes simplex virus, a V-or Y-shaped density was observed in the intermediate state by cryoEM (14,15), and in the retroviral Env trimer, an asymmetric intermediate appeared to emanate from a single protomer in cryo-electron tomography experiments (16). For the Moloney murine leukemia virus Env protein, the sequential release of the surface subunit from the transmembrane subunit induces the formation of an asymmetric trimer (17). Adoption of an asymmetric conformation presumably overcomes steric hindrance within the three fusion protein subunits.…”

mentioning

confidence: 99%

Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

Kawase

Kataoka

Shirato

et al. 2019

J Virol

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A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre-and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses. IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre-to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.

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Sequential activation of the three protomers in the Moloney murine leukemia virus Env

Cited by 3 publications

References 35 publications

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

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