Herpesvirus Nuclear Egress across the Outer Nuclear Membrane

Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.

Herpes simplex virus glycoprotein B (gB) mutations define structural sites in domain I, the membrane proximal region, and the cytodomain that regulate entry.

The viral fusion protein glycoprotein B (gB) is conserved in all herpesviruses and is essential for virus entry. During entry, gB fuses viral and host cell membranes by refolding from a prefusion to a postfusion form. We previously introduced three structure-based mutations (gB-I671A/H681A/F683A) into the domain V arm of the gB ectodomain that resulted in reduced cell-cell fusion. A virus carrying these three mutations (called gB3A) displayed a small plaque phenotype and remarkably delayed entry into cells. To identify mutations that could counteract this phenotype, we serially passaged the gB3A virus and selected for revertant viruses with increased plaque size. Genomic sequencing revealed that the revertant viruses had second-site mutations in gB, including E187A, M742T, and S383F/G645R/V705I/V880G. Using expression constructs encoding these mutations, only gB-V880G was shown to enhance cell-cell fusion. In contrast, all of the revertant viruses showed enhanced entry kinetics, underscoring the fact that cell-cell fusion and virus-cell fusion are different. The results indicate that mutations in three different regions of gB (domain I, the membrane proximal region, and the cytoplasmic tail domain) can counteract the slow entry phenotype of gB3A virus. Mapping these compensatory mutations to prefusion and postfusion structural models suggests sites of intramolecular functional interactions with the gB domain V arm that may contribute to the gB fusion function. Importance The nine human herpesviruses are ubiquitous and cause a range of disease in humans. Glycoprotein B (gB) is an essential viral fusion protein that is conserved in all herpesviruses. During host cell entry, gB mediates virus-cell membrane fusion by undergoing a conformational change. Structural models for the prefusion and postfusion form of gB exist, but the details of how the protein converts from one to the other are unclear. We previously introduced structure-based mutations into gB that inhibited virus entry and fusion. By passaging this entry-deficient virus over time, we selected second-site mutations that partially restore virus entry. The location of these mutations suggest regulatory sites that contribute to fusion and gB refolding during entry. gB is a target of neutralizing antibodies and defining how gB refolds during entry could provide a basis for the development of fusion inhibitors for future research or clinical use.

Cell Mechanics and Signalization: SARS-CoV-2 Hijacks Membrane Liquid Crystals and Cytoskeletal Fractal Topology

We highlight changes to cell signaling under virus invasion (with the example of SARS-CoV-2), involving disturbance of membranes (plasma, mitochondrial, endothelial-alveolar) and of nanodomains, modulated by the cytoskeleton. Virus alters the mechanical properties of the membranes, impairing mesophase structures mediated by the fractal architecture initiated by actomyosin. It changes the topology of the membrane and its lipid composition distribution. Mechano-transduction, self-organization and topology far from equilibrium are omnipresent. We propose that the actomyosin contractility generates the cytoskeletons fractal organization. We focus on three membranar processus: The transition from lamellar configuration in cell and viral membranes to a bi-continuous organization in the presence of ethanolamine. (The energy for this transition is provided by change of the folding of the viral fusion protein from metastable to stable state). The action of mitochondrial antiviral signaling protein on the external mitochondrial envelope in contact with mitochondrial-associated membranes, modified by viral endoribonuclease, distorting innate immune response. The increased permeability of the epithelial-alveolar-pulmonary barrier involves the cytoskeleton membranes. The pulmonary surfactant is also perturbed in its liquid crystal state. Viral subversion disorganizes membrane structure and functions and thus the metabolism of the cell. We advocate systematic multidisciplinary exploration of membrane mesophases and their links with fractal dynamics, to enable novel therapies for SARS-CoV-2 infection.

A trimeric hydrophobic zipper mediates the intramembrane assembly of SARS-CoV-2 spike

The S protein of the SARS-CoV-2 is a Type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the nuclear magnetic resonance (NMR) structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane α-helix (TMH) trimer that assembles spontaneously in membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeat. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchor of viral fusion protein can form highly specific oligomers, but the exact function of these oligomers remain unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.

Application of error-prone PCR to functionally probe the morbillivirus Haemagglutinin protein

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4. Receptor binding is initiated by the viral attachment protein Haemagglutinin (H), with the viral Fusion protein (F) driving membrane fusion. Crystal structures of the prototypic morbillivirus measles virus H with either SLAMF1 or Nectin-4 are available and have served as the basis for improved understanding of this interaction. However, whether these interactions remain conserved throughout the morbillivirus genus requires further characterisation. Using a random mutagenesis approach, based on error-prone PCR, we targeted the putative receptor binding site for SLAMF1 interaction on peste des petits ruminants virus (PPRV) H, identifying mutations that inhibited virus-induced cell-cell fusion. These data, combined with structural modelling of the PPRV H and ovine SLAMF1 interaction, indicate this region is functionally conserved across all morbilliviruses. Error-prone PCR provides a powerful tool for functionally characterising functional domains within viral proteins.

Critical Residues and Contacts within Domain IV of Autographa californica Multiple Nucleopolyhedrovirus GP64 Contribute to Its Refolding during Membrane Fusion

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 is a class III viral fusion protein that mediates low-pH-triggered membrane fusion during virus entry. Although the structure of GP64 in a postfusion conformation has been solved, its prefusion structure and the mechanism of how the protein refolds to execute fusion are unknown. In its postfusion structure, GP64 is composed of five domains (domains I to V). Domain IV (amino acids [aa] 374 to 407) contains two loops (loop 1 and loop 2) that form a hydrophobic pocket at the membrane-distal end of the molecule. To determine the roles of domain IV, we used alanine-scanning mutagenesis to replace each of the individual residues and the contact-forming residues within domain IV and evaluate their contributions to GP64-mediated membrane fusion and virus infection. In many cases, replacement of a single amino acid had no significant impact on GP64. However, replacement of R392 or disruption of the N381-N385, N384-Y388, N385-W393, or K389-W393 contact resulted in poor cell surface expression and fusion loss of the modified GP64, whereas replacement of E390 or G391 or disruption of the N381-K389, N381-Q401, or N381-I403 contact reduced the cell surface expression level of the constructs and the ability of GP64 to mediate fusion pore expansion. In contrast, replacement of N407 or disruption of contact D404-S406 appeared to restrict fusion pore expansion without affecting expression. Combined with the finding that these constructs remain in the prefusion conformation or have a dramatically less efficient transition from the prefusion to the postfusion state under acidic conditions, we proposed that domain IV is necessary for refolding of GP64 during membrane fusion. IMPORTANCE Baculovirus GP64 is grouped with rhabdovirus G, herpesvirus gB, and thogotovirus glycoproteins as a class III viral fusion protein. In their postfusion structures, these proteins contain five domains (domains I to V). Distinct from domain IV of rhabdovirus G and herpesvirus gB proteins, which is composed of β-sheets, domain IV of GP64 is a loop region; the same domain in thogotovirus glycoproteins has not been solved. In addition, domain IV is proximal to domain I (fusion domain) in prefusion structures of vesicular stomatitis virus (VSV) G and human cytomegalovirus (HCMV) gB but resides at the domain I-distal end of the molecule in a postfusion conformation. In this study, we identified that highly conserved residues and contacts within domain IV of AcMNPV GP64 are necessary for low-pH-triggered conformational change and fusion pore expansion. Our results highlight the roles of domain IV of class III viral fusion proteins in refolding during membrane fusion.

Cell Fusion Induced by a Fusion-Active Form of Human Cytomegalovirus Glycoprotein B (gB) Is Inhibited by Antibodies Directed at Antigenic Domain 5 in the Ectodomain of gB

ABSTRACT Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero. Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB’s fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection. IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB’s fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.

Effects of Single α-to-β Residue Replacements on Recognition of an Extended Segment in a Viral Fusion Protein

Partial replacement of α-amino acid residues with β-amino acid residues has been established as a strategy for preserving target-engagement by helix-forming polypeptides while suppressing susceptibility to proteolysis. The impact of β-residue incorporation within polypeptides that adopt less regular conformations, however, has received less attention. The HRC domains of fusion glycoproteins from pathogenic paramyxoviruses contain a segment that must adopt an extended conformation in order to engage the HRN domain in the post-fusion state and drive merger of the viral envelope with a target cell membrane. Here we examine the impact of single α-to-β substi-tutions within this extended N-terminal segment of the engineered HRC peptide VIQKI. Stabilities of helix-bundles formed with a native viral HRN have been evaluated, the structures of five helix-bundles have been determined, and antiviral efficacies have been measured. Many sites within the extended segment show functional tolerance of α-to-β substitution. These results offer a basis for future develop-ment of protease-resistant inhibitors of paramyxovirus infection.

Effects of Single α-to-β Residue Replacements on Recognition of an Extended Segment in a Viral Fusion Protein

Partial replacement of α-amino acid residues with β-amino acid residues has been established as a strategy for preserving target-engagement by helix-forming polypeptides while suppressing susceptibility to proteolysis. The impact of β-residue incorporation within polypeptides that adopt less regular conformations, however, has received less attention. The HRC domains of fusion glycoproteins from pathogenic paramyxoviruses contain a segment that must adopt an extended conformation in order to engage the HRN domain in the post-fusion state and drive merger of the viral envelope with a target cell membrane. Here we examine the impact of single α-to-β substi-tutions within this extended N-terminal segment of the engineered HRC peptide VIQKI. Stabilities of helix-bundles formed with a native viral HRN have been evaluated, the structures of five helix-bundles have been determined, and antiviral efficacies have been measured. Many sites within the extended segment show functional tolerance of α-to-β substitution. These results offer a basis for future develop-ment of protease-resistant inhibitors of paramyxovirus infection.