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Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress-budding at the inner nuclear membrane-is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding., Competing Interests: Authors declare no competing interests., (Copyright: © 2024 Draganova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors have declared that no competing interests exist.

Human herpesviruses, classified into three subfamilies, are double-stranded DNA viruses that establish lifelong latent infections within most of the world's population and can cause severe disease, especially in immunocompromised people. There is no cure, and current preventative and therapeutic options are limited. Therefore, understanding the biology of these viruses is essential for finding new ways to stop them. Capsids play a central role in herpesvirus biology. They are sophisticated vehicles that shelter the pressurized double-stranded-DNA genomes while ensuring their delivery to defined cellular destinations on the way in and out of the host cell. Moreover, the importance of capsids for multiple key steps in the replication cycle makes their assembly an attractive therapeutic target. Recent cryo-electron microscopy reconstructions of capsids from all three subfamilies of human herpesviruses revealed not only conserved features but also remarkable structural differences. Furthermore, capsid assembly studies have suggested subfamily-specific roles of viral capsid protein homologs. In this review, we compare capsid structures, assembly mechanisms, and capsid protein functions across human herpesvirus subfamilies, highlighting the differences.

Herpesviruses infect a majority of the human population, establishing lifelong latent infections for which there is no cure. Periodic viral reactivation spreads infection to new hosts while causing various disease states particularly detrimental in the immunocompromised. Efficient viral replication, and ultimately the spread of infection, is dependent on the nuclear egress complex (NEC), a conserved viral heterodimer that helps translocate viral capsids from the nucleus to the cytoplasm where they mature into infectious virions. Here, we have identified peptides, derived from the capsid protein UL25, that are capable of inhibiting the membrane-budding activity of the NEC from herpes simplex virus type 1 in vitro. We show that the inhibitory ability of the peptides depends on their length and the propensity to form an α-helix but not on the exact amino acid sequence. Current therapeutics that target viral DNA replication machinery are rendered ineffective by drug resistance due to viral mutations. Our results establish a basis for the development of an alternative class of inhibitors against nuclear egress, an essential step in herpesvirus replication, potentially expanding the current repertoire of available therapeutics.

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.

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., Competing Interests: ED, JZ, ZZ, EH No competing interests declared, (© 2020, Draganova et al.)

The heme-binding protein HmuT is part of the Corynebacterium diphtheriae heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV-visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on π donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT-CO.

The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this report, we present the heme binding of the alanine mutants of the axial histidine (H229A) and methionine (M79A) ligands, as well as a lysine (K61A) and cysteine (C58A) located near the heme propionates (based on homology modeling) and a control mutant (C47A). pH titrations gave pKa values ranging from 9.0 to 9.5, close to the value of 9.7 for WT SiaA. Resonance Raman spectra of the mutants suggested that the ferric heme environment may be distinct from the wild-type; spectra of the ferrous states were similar. The midpoint reduction potential of the K61A mutant was determined by spectroelectrochemical titration to be 61±3mV vs. SHE, similar to the wild-type protein (68±3mV). The addition of guanidine hydrochloride showed two processes for protein denaturation, consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket (the half-life for the slower process ranged from less than half a day to two days). The ease of protein unfolding was related to the strength of interaction of the residues with the heme. We hypothesize that kinetically facile but only partial unfolding, followed by a very slow approach to the completely unfolded state, may be a fundamental attribute of heme trafficking proteins. Small motions to release/transfer the heme accompanied by resistance to extensive unfolding may preserve the three dimensional form of the protein for further uptake and release., (Copyright © 2015. Published by Elsevier Inc.)

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 (WT) 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 indicates 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 is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these 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.