The most comprehensive architectural model to date of the nuclear pore complex reveals previously unknown local interactions, and a role for nucleoporin 358 in Y-complex oligomerization. The transport of materials between the nucleus and cytoplasm in eukaryotic cells is controlled by the nuclear pore complex. Martin Beck and colleagues have used cryo-electron tomography, mass spectrometry and other analyses to generate the most comprehensive architectural model of the human nuclear pore complex to date. The model reveals previously unknown local interactions, and a role for the transport channel nucleoporin 358 (Nup358) in mediating oligomerization of the Y-complex within the nuclear pore complex. Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter1. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block—although compositionally identical—engage in different local sets of interactions and conformations.

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Significance: Successful host cell infection requires that viruses get various components—most importantly, their genomes—across the bounding membranes into the cytosol. For enveloped viruses, this crucial part of the entry process is achieved by merging the viral membrane with the host membrane, a process mediated by specialized virus-encoded fusion proteins residing on the virus envelope. Accordingly, these surface viral proteins constitute important targets for antiviral treatments as well as for prophylactic vaccine development. Herpesviruses are ubiquitous, opportunistic DNA viruses that have mastered immune system evasion to cause lifelong infections, with intermittent clinical and subclinical viral reactivation. The structural information on an effective glycoprotein B conformation reported here opens up overdue opportunities for targeted interventions in herpesvirus entry.

Elusive, pharmacologically relevant conformation of the herpesvirus fusion protein is revealed by a multi-methodological approach.

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be by-passed by membrane-tethering of pUL31 demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodelling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds which constrict to vesicles. Thus a single viral protein can mediate all events necessary for membrane budding and abscission.