During herpesvirus infection, egress of nascent viral capsids from the nucleus is mediated by the viral nuclear egress complex (NEC). NEC deforms the inner nuclear membrane (INM) around the capsid by forming a hexagonal array. However, how the NEC coat interacts with the capsid and how curved coats are generated to enable budding is yet unclear. Here, by structure-guided truncations, confocal microscopy, and cryoelectron tomography, we show that binding of the capsid protein UL25 promotes the formation of NEC pentagons rather than hexagons. We hypothesize that during nuclear budding, binding of UL25 situated at the pentagonal capsid vertices to the NEC at the INM promotes formation of NEC pentagons that would anchor the NEC coat to the capsid. Incorporation of NEC pentagons at the points of contact with the vertices would also promote assembly of the curved hexagonal NEC coat around the capsid, leading to productive egress of UL25-decorated capsids.

During viral replication, herpesviruses utilize a unique strategy, termed nuclear egress, to translocate capsids from the nucleus into the cytoplasm. This initial budding step transfers a newly formed capsid from within the nucleus, too large to fit through nuclear pores, through the inner nuclear membrane to the perinuclear space. The perinuclear enveloped virion must then fuse with the outer nuclear membrane to be released into the cytoplasm for further maturation, undergoing budding once again at the trans-Golgi network or early endosomes, and ultimately exit the cell non-lytically to spread infection. This first budding process is mediated by two conserved viral proteins, UL31 and UL34, that form a heterodimer called the nuclear egress complex (NEC). This review focuses on what we know about how the NEC mediates capsid transport to the perinuclear space, including steps prior to and after this budding event. Additionally, we discuss the involvement of other viral proteins in this process and how NEC-mediated budding may be regulated during infection. Introduction: overview of nuclear egressHerpesviruses replicate their double-stranded-DNA genomes and package them into capsids within the host cell nucleus. These progeny capsids must then exit the nucleus to form mature virions within the cytoplasm (Figure 1). The nucleus is caister.com/cimb 125 caister.com/cimb 126 Curr. Issues Mol. Biol. Vol. 41 Nuclear Egress DraganovaThis envelopment/de-envelopment model of nuclear egress is the most widely accepted model for capsid escape from the nucleus and is backed up by substantial data. First, enveloped virions in the PNS have been observed in cells infected with herpesvirus for several decades (Fuchs et al., 2002;Granzow et al., 1997;Reynolds et al., 2002;Stackpole, 1969). The phenotypes of viral gene deletions likewise provide compelling evidence in favor of the envelopment/deenvelopment model. Deletion of the US3 kinase gene from either HSV-1 or from the closely related pseudorabies virus (PRV) results in the accumulation of PEVs (Klupp et al., 2001;Reynolds et al., 2002;Ryckman and Roller, 2004;Sehl et al., 2020), which points to budding into the PNS being an intermediate stage in nuclear egress. Second, PEVs differ from mature virions in their morphology and protein composition. For example, UL31 and UL34, two viral proteins critical for nuclear egress, are absent from mature HSV-1 and PRV virions (Fuchs et al., 2002;Reynolds et al., 2002). Conversely, most tegument proteins found in mature virions are not found in the nucleus or in PEVs (Gershon et al., 1994;Klupp et al., 2000;Skepper et al., 2001). Furthermore, mature virions are studded with glycoprotein spikes and have a thick tegument while PEVs have a smooth envelope and thin tegument (Gershon et al., 1994;Granzow et al., 2001), consistent with the two types of particles being formed during different budding events. Third, deletions of particular tegument proteins, such as HSV-1 UL36 and UL37, or the glycoproteins gD and gE from either HSV-1 or PRV result ...

The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicate that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A reduces readily in the presence of dithionite. Raman frequencies of the FeCO distortions in WT, H136A, and Y235A HmuT–CO complexes provide further evidence for the axial ligand assignments. Additionally, the se frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A–CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen-bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen-bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

During replication, herpesviral capsids are translocated from the nucleus into the cytoplasm by an unusual mechanism, termed nuclear egress, that involves capsid budding at the inner nuclear membrane. This process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid. Although the NEC is essential for capsid nuclear egress across all three subfamilies of the Herpesviridae, most studies to date have focused on the NEC homologs from alpha- and beta- but not gammaherpesviruses. Here, we report the crystal structure of the NEC from Epstein-Barr virus (EBV), a prototypical gammaherpesvirus. The structure resembles known structures of NEC homologs yet is conformationally dynamic. We also show that purified, recombinant EBV NEC buds synthetic membranes in vitro and forms membrane-bound coats of unknown geometry. However, unlike other NEC homologs, EBV NEC forms dimers in the crystals instead of hexamers. The dimeric interfaces observed in the EBV NEC crystals are similar to the hexameric interfaces observed in other NEC homologs. Moreover, mutations engineered to disrupt the dimeric interface reduce budding. Putting together these data, we propose that EBV NEC-mediated budding is driven by oligomerization into membrane-bound coats.