Abstract
SummaryVesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.
Highlights
Intracytoplasmic transport between compartments is primarily mediated by vesicles (Schekman and Orci, 1996)
In Situ Structural Characterization of nuclear egress complex (NEC)-Mediated Capsid Envelopment at the inner nuclear membrane (INM) by cryo-electron microscopy of vitreous sections (CEMOVIS) To analyze the NEC coat formed in situ during viral infection in its most native environment, we used electron cryo-microscopy and tomography (Hoenger, 2014)
In electron cryo-microscopy of vitreous sections (CEMOVIS), a method for imaging hydrated and unstained cellular ultrastructural detail (Dubochet, 2012), NECs were observed as electron-dense coats at the nucleoplasmic side of the INM and in the perinuclear space of herpes simplex virus 1 (HSV-1)-infected Vero cells (Figures 1B–1F and Movies S1 and S2)
Summary
A multi-modal live-cell and cryo-imaging approach reveals how vesicles assemble at the inner nuclear membrane for transport to the cytoplasm during herpesvirus maturation. This suggests a functional model for counterparts in uninfected cells that mediate nuclear egress of large cargo like ribonucleoprotein particles.
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