Biochemical, Biophysical and Structural Characterization of the Conserved Herpesviral Nuclear Egress Complex

To exit infected cells, progeny virions must pass through host cell membrane barriers. Herpesviruses are masters of membrane manipulation as they deform membranes at multiple stages while exiting a cell. The first instance occurs when the virally encoded nuclear egress complex (NEC) bends the nuclear membrane around nucleocapsids, assembled entirely in the nucleus and too large to fit through nuclear pores, resulting in their envelopment and trafficking through the nuclear membrane. Despite being essential for herpesvirus replication, how the NEC bends the nuclear membrane around capsids is not well understood. In this work, we used biochemical, biophysical, and structural approaches to further our mechanistic understanding of NEC-mediated membrane deformation. Here, we report that electrostatics govern NEC-membrane interactions leading to membrane budding. We propose that the virus uses phosphorylation to control NEC-mediated membrane budding during infection as phosphomimicking mutations block budding in vivo and in vitro. Furthermore, we establish that the membrane-proximal regions (MPRs) of the NEC order lipid headgroups and acyl chains to generate local areas of negative membrane curvature, important for formation of the body of the bud, and negative Gaussian curvature (NGC), important for scission of the budding membrane. We propose that NEC oligomerization, known to occur and required for budding, creates a rigid, closely packed scaffold on the membrane increasing the area of local negative membrane curvature thereby forming the body of the bud. At the edges of the scaffold, non-oligomerized NEC generates NGC to form the neck of the bud. We also report the crystal structure of the NEC from Epstein-Barr virus (EBV), a gammaherpesvirus. Comparison to NECs from alpha- and betaherpesviruses show that the overall structure is well conserved across all three herpesvirus subfamilies. We show, experimentally, that the EBV NEC is conformationally dynamic which we hypothesize is important for function. Furthermore, we establish that the EBV NEC is a self-contained membrane budding machine which also requires oligomerization for function. We propose that MPR-mediated membrane ordering and NEC oligomerization function in concert to mediate nuclear egress for all herpesviruses.