Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Herpes Simplex Virus Glycoprotein B 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 sizes. 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 diseases 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 forms 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 locations 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.

Entry of Alphaherpesviruses.

Alphaherpesviruses are enveloped viruses that enter cells by fusing the viral membrane with a host cell membrane, either within an endocytic vesicle or at the plasma membrane. This entry event is mediated by a set of essential entry glycoproteins, including glycoprotein D (gD), gHgL, and gB. gHgL and gB are conserved among herpesviruses, but gD is unique to the alphaherpesviruses and is not encoded by all alphaherpesviruses. gD is a receptor-binding protein, the heterodimer gHgL serves as a fusion regulator, and gB is a class III viral fusion protein. Sequential interactions among these glycoproteins are thought to trigger the virus to fuse at the right place and time. Structural studies of these glycoproteins from multiple alphaherpesviruses has enabled the design and interpretation of functional studies. The structures of gD in a receptor- bound and in an unliganded form reveal a conformational change in the C terminus of the gD ectodomain upon receptor binding that may serve as a signal for fusion. By mapping neutralizing antibodies to the gHgL structures and constructing interspecies chimeric forms of gHgL, interaction sites for both gD and gB on gHgL have been proposed. A comparison of the post fusion structure of gB and an alternative conformation of gB visualized using cryo- electron tomography suggests that gB undergoes substantial refolding to execute membrane fusion. Although these structures have provided excellent insights into the entry mechanism, many questions remain about how these viruses coordinate the interactions and conformational changes required for entry.

A Functional Interaction between Herpes Simplex Virus 1 Glycoprotein gH/gL Domains I and II and gD Is Defined by Using Alphaherpesvirus gH and gL Chimeras.

UNLABELLED: Whereas most viruses require only a single protein to bind to and fuse with cells, herpesviruses use multiple glycoproteins to mediate virus entry, and thus communication among these proteins is required. For most alphaherpesviruses, the minimal set of viral proteins required for fusion with the host cell includes glycoproteins gD, gB, and a gH/gL heterodimer. In the current model of entry, gD binds to a cellular receptor and transmits a signal to gH/gL. This signal then triggers gB, the conserved fusion protein, to insert into the target membrane and refold to merge the viral and cellular membranes. We previously demonstrated that gB homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1), were interchangeable. In contrast, neither gD nor gH/gL functioned with heterotypic entry glycoproteins, indicating that gD and gH/gL exhibit an essential type-specific functional interaction. To map this homotypic interaction site on gH/gL, we generated HSV-1/SaHV-1 gH and gL chimeras. The functional interaction with HSV-1 gD mapped to the N-terminal domains I and II of the HSV-1 gH ectodomain. The core of HSV-1 gL that interacts with gH also was required for functional homotypic interaction. The N-terminal gH/gL domains I and II are the least conserved and may have evolved to support species-specific glycoprotein interactions. IMPORTANCE: The first step of the herpesvirus life cycle is entry into a host cell. A coordinated interaction among multiple viral glycoproteins is required to mediate fusion of the viral envelope with the cell membrane. The details of how these glycoproteins interact to trigger fusion are unclear. By swapping the entry glycoproteins of two alphaherpesviruses (HSV-1 and SaHV-1), we previously demonstrated a functional homotypic interaction between gD and gH/gL. To define the gH and gL requirements for homotypic interaction, we evaluated the function of a panel of HSV-1/SaHV-1 gH and gL chimeras. We demonstrate that domains I and II of HSV-1 gH are sufficient to promote a functional, albeit reduced, interaction with HSV-1 gD. These findings contribute to our model of how the entry glycoproteins cooperate to mediate herpesvirus entry into the cell.

Substitution of herpes simplex virus 1 entry glycoproteins with those of saimiriine herpesvirus 1 reveals a gD-gH/gL functional interaction and a region within the gD profusion domain that is critical for fusion.

UNLABELLED: To gain insight into the mechanism of herpesvirus entry into cells, the four glycoproteins that are necessary for herpes simplex virus (HSV) fusion were cloned from the saimiriine herpesvirus 1 (SaHV-1) genome, a primate member of the alphaherpesvirus family. Cell-cell fusion assays indicate that SaHV-1 entry glycoproteins function with the previously identified alphaherpesvirus entry receptors nectin-1 and CD155 but not with herpesvirus entry mediator (HVEM) or paired immunoglobulin-like type 2 receptor alpha (PILRα). Replacement of HSV-1 gD with the SaHV-1 gD homolog resulted in a complete loss of fusion function when coexpressed with HSV-1 gB and gH/gL. HSV-1 gD was also unable to substitute for SaHV-1 gD when coexpressed with SaHV-1 gB and gH/gL. Similarly, the gH/gL heterodimers from HSV-1 and SaHV-1 were not interchangeable. In contrast, both the HSV-1 and SaHV-1 gB homologs retained function in a heterotypic context. These results suggest that an essential interaction between homotypic gD and gH/gL occurs during both HSV-1 and SaHV-1 entry. To map the site of this homotypic interaction, we created a series of gD chimeras, focusing on the "profusion domain" (PFD) that consists of HSV-1 gD residues 261 to 305 or SaHV-1 gD residues 264 to 307. We identified a seven-amino-acid stretch (264 RTLPPPK 270) at the N terminus of the SaHV-1 gD PFD that contributes to homotypic fusion. Finally, we found that the gD receptor-binding region and PFD cannot function independently but that both can inhibit the function of wild-type gD. IMPORTANCE: The herpesvirus entry machinery requires the concerted action of at least four glycoproteins; however, details of the interactions among these glycoproteins are not well understood. Like HSV-1, SaHV-1 belongs to the alphaherpesvirus subfamily. Using cell-cell fusion experiments, we found that SaHV-1 uses the entry receptors nectin-1 and CD155 but not HVEM or PILRα. By swapping the entry glycoproteins between HSV-1 and SaHV-1, we revealed a functional interaction between gD and gH/gL. To examine the homotypic interaction site on gD, we evaluated the function of a panel of HSV-1/SaHV-1 gD chimeras and identified a small region in the SaHV-1 gD profusion domain that is critical for SaHV-1 fusion. This study contributes to our understanding of the molecular mechanisms of herpesvirus entry and membrane fusion.

Residues within the C-terminal arm of the herpes simplex virus 1 glycoprotein B ectodomain contribute to its refolding during the fusion step of virus entry.

Herpesvirus entry into cells requires coordinated interactions among several viral glycoproteins. The final membrane fusion step of entry is executed by glycoprotein B (gB), a class III viral fusion protein that is conserved across all herpesviruses. Fusion proteins are metastable proteins that mediate fusion by inserting into a target membrane and refolding from a prefusion to postfusion conformation to bring the viral and cell membranes together. Although the structure of gB has been solved in a conformation that likely represents its postfusion form, its prefusion structure and the details of how it refolds to execute fusion are unknown. The postfusion gB structure contains a trimeric coiled-coil at its core and a long C-terminal arm within the ectodomain packs against this coil in an antiparallel manner. This coil-arm complex is reminiscent of the six-helix bundle that provides the energy for fusion in class I fusogens. To determine the role of the coil-arm complex, we individually mutated residues in the herpes simplex virus 1 gB coil-arm complex to alanine and assessed the contribution of each residue to cell-cell and virus-cell fusion. Several coil mutations resulted in a loss of cell surface expression, indicating that the coil residues are important for proper processing of gB. Three mutations in the arm region (I671A, H681A, and F683A) reduced fusion without affecting expression. Combining these three arm mutations drastically reduced the ability of gB to execute fusion; however, fusion function could be restored by adding known hyperfusogenic mutations to the arm mutant. We propose that the formation of the coil-arm complex drives the gB transition to a postfusion conformation and the coil-arm complex performs a function similar to that of the six-helix bundle in class I fusion. Furthermore, we suggest that these specific mutations in the arm may energetically favor the prefusion state of gB.

Reversible inhibition of fusion activity of a paramyxovirus fusion protein by an engineered disulfide bond in the membrane-proximal external region.

Cysteines were introduced into the membrane-proximal external region (MPER) of the paramyxovirus F protein. A disulfide bond formed, and the mutant protein was expressed at the cell surface but was fusion inactive. Reduction of the disulfide bond restored fusion activity. The data indicate that in addition to dissociation of the three-helix bundle stalk domain of prefusion F, the MPER region also needs to separate for F to be able to refold and cause fusion.

Multiple Sites on Glycoprotein H (gH) Functionally Interact with the gB Fusion Protein to Promote Fusion during Herpes Simplex Virus (HSV) Entry.

Enveloped virus entry requires fusion of the viral envelope with a host cell membrane. Herpes simplex virus 1 (HSV-1) entry is mediated by a set of glycoproteins that interact to trigger the viral fusion protein glycoprotein B (gB). In the current model, receptor-binding by gD signals a gH/gL heterodimer to trigger a refolding event in gB that fuses the membranes. To explore functional interactions between gB and gH/gL, we used a bacterial artificial chromosome (BAC) to generate two HSV-1 mutants that show a small plaque phenotype due to changes in gB. We passaged the viruses to select for restoration of plaque size and analyzed second-site mutations that arose in gH. HSV-1 gB was replaced either by gB from saimiriine herpesvirus 1 (SaHV-1) or by a mutant form of HSV-1 gB with three alanine substitutions in domain V (gB3A). To shift the selective pressure away from gB, the gB3A virus was passaged in cells expressing gB3A. Sequencing of passaged viruses identified two interesting mutations in gH, including gH-H789Y in domain IV and gH-S830N in the cytoplasmic tail (CT). Characterization of these gH mutations indicated they are responsible for the enhanced plaque size. Rather than being globally hyperfusogenic, both gH mutations partially rescued function of the specific gB version present during their selection. These sites may represent functional interaction sites on gH/gL for gB. gH-H789 may alter the positioning of a membrane-proximal flap in the gH ectodomain, whereas gH-S830 may contribute to an interaction between the gB and gH CTs. IMPORTANCE Enveloped viruses enter cells by fusing their envelope with the host cell membrane. Herpes simplex virus 1 (HSV-1) entry requires the coordinated interaction of several viral glycoproteins, including gH/gL and gB. gH/gL and gB are essential for virus replication and both proteins are targets of neutralizing antibodies. gB fuses the membranes after being activated by gH/gL, but the details of how gH/gL activates gB are not known. This study examined the gH/gL-gB interaction using HSV-1 mutants that displayed reduced virus entry due to changes in gB. The mutant viruses were grown over time to select for additional mutations that could partially restore entry. Two mutations in gH (H789Y and S830N) were identified. The positions of the mutations in gH/gL may represent sites that contribute to gB activation during virus entry.

Trained immunity is induced in humans after immunization with an adenoviral vector COVID-19 vaccine.

BackgroundHeterologous effects of vaccines are mediated by "trained immunity," whereby myeloid cells are metabolically and epigenetically reprogrammed, resulting in heightened responses to subsequent insults. Adenovirus vaccine vector has been reported to induce trained immunity in mice. Therefore, we sought to determine whether the ChAdOx1 nCoV-19 vaccine (AZD1222), which uses an adenoviral vector, could induce trained immunity in vivo in humans.MethodsTen healthy volunteers donated blood on the day before receiving the ChAdOx1 nCoV-19 vaccine and on days 14, 56, and 83 after vaccination. Monocytes were purified from PBMCs, cell phenotype was determined by flow cytometry, expression of metabolic enzymes was quantified by RT-qPCR, and production of cytokines and chemokines in response to stimulation ex vivo was analyzed by multiplex ELISA.ResultsMonocyte frequency and count were increased in peripheral blood up to 3 months after vaccination compared with their own prevaccine controls. Expression of HLA-DR, CD40, and CD80 was enhanced on monocytes for up to 3 months following vaccination. Moreover, monocytes had increased expression of glycolysis-associated enzymes 2 months after vaccination. Upon stimulation ex vivo with unrelated antigens, monocytes produced increased IL-1ß, IL-6, IL-10, CXCL1, and MIP-1α and decreased TNF, compared with prevaccine controls. Resting monocytes produced more IFN-γ, IL-18, and MCP-1 up to 3 months after vaccination compared with prevaccine controls.ConclusionThese data provide evidence for the induction of trained immunity following a single dose of the ChAdOx1 nCoV-19 vaccine.FundingThis work was funded by the Health Research Board (EIA-2019-010) and Science Foundation Ireland Strategic Partnership Programme (proposal ID 20/SPP/3685).

The structural basis of herpesvirus entry.

Herpesviruses are ubiquitous, double-stranded DNA, enveloped viruses that establish lifelong infections and cause a range of diseases. Entry into host cells requires binding of the virus to specific receptors, followed by the coordinated action of multiple viral entry glycoproteins to trigger membrane fusion. Although the core fusion machinery is conserved for all herpesviruses, each species uses distinct receptors and receptor-binding glycoproteins. Structural studies of the prototypical herpesviruses herpes simplex virus 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) entry glycoproteins have defined the interaction sites for glycoprotein complexes and receptors, and have revealed conformational changes that occur on receptor binding. Recent crystallography and electron microscopy studies have refined our model of herpesvirus entry into cells, clarifying both the conserved features and the unique features. In this Review, we discuss recent insights into herpesvirus entry by analysing the structures of entry glycoproteins, including the diverse receptor-binding glycoproteins (HSV-1 glycoprotein D (gD), EBV glycoprotein 42 (gp42) and HCMV gH-gL-gO trimer and gH-gL-UL128-UL130-UL131A pentamer), as well gH-gL and the fusion protein gB, which are conserved in all herpesviruses.

Bimolecular complementation of paramyxovirus fusion and hemagglutinin-neuraminidase proteins enhances fusion: implications for the mechanism of fusion triggering.

For paramyxoviruses, entry requires a receptor-binding protein (hemagglutinin-neuraminidase [HN], H, or G) and a fusion protein (F). Like other class I viral fusion proteins, F is expressed as a prefusion metastable protein that undergoes a refolding event to induce fusion. HN binding to its receptor triggers F refolding by an unknown mechanism. HN may serve as a clamp that stabilizes F in its prefusion state until HN binds the target cell (the "clamp model"). Alternatively, HN itself may undergo a conformational change after receptor binding that destabilizes F and causes F to trigger (the "provocateur model"). To examine F-HN interactions by bimolecular fluorescence complementation (BiFC), the cytoplasmic tails of parainfluenza virus 5 (PIV5) F and HN were fused to complementary fragments of yellow fluorescent protein (YFP). Coexpression of the BiFC constructs resulted in fluorescence; however, coexpression with unrelated BiFC constructs also produced fluorescence. The affinity of the two halves of YFP presumably superseded the F-HN interaction. Unexpectedly, coexpression of the BiFC F and HN constructs greatly enhanced fusion in multiple cell types. We hypothesize that the increase in fusion occurs because the BiFC tags bring F and HN together more frequently than occurs in a wild-type (wt) scenario. This implies that normally much of wt F is not associated with wt HN, in conflict with the clamp model for activation. Correspondingly, we show that wt PIV5 fusion occurs in an HN concentration-dependent manner. Also inconsistent with the clamp model are the findings that BiFC F does not adopt a postfusion conformation when expressed in the absence of HN and that HN coexpression does not provide resistance to the heat-induced triggering of F. In support of a provocateur model of F activation, we demonstrate by analysis of the morphology of soluble F trimers that the hyperfusogenic mutation S443P has a destabilizing effect on F.

Natural Selection of Glycoprotein B Mutations That Rescue the Small-Plaque Phenotype of a Fusion-Impaired Herpes Simplex Virus Mutant.

Glycoprotein B (gB) is a conserved viral fusion protein that is required for herpesvirus entry. To mediate fusion with the cellular membrane, gB refolds from a prefusion to a postfusion conformation. We hypothesize that an interaction between the C-terminal arm and the central coiled coil of the herpes simplex virus 1 (HSV-1) gB ectodomain is critical for fusion. We previously reported that three mutations in the C-terminal arm (I671A/H681A/F683A, called gB3A) greatly reduced cell-cell fusion and that virus carrying these mutations had a small-plaque phenotype and delayed entry into cells. By serially passaging gB3A virus, we selected three revertant viruses with larger plaques. These revertant viruses acquired mutations in gB that restore the fusion function of gB3A, including gB-A683V, gB-S383F/G645R/V705I/A855V, and gB-T509M/N709H. V705I and N709H are novel mutations that map to the portion of domain V that enters domain I in the postfusion structure. S383F, G645R, and T509M are novel mutations that map to an intersection of three domains in a prefusion model of gB. We introduced these second-site mutations individually and in combination into wild-type gB and gB3A to examine the impact of the mutations on fusion and expression. V705I and A855V (a known hyperfusogenic mutation) restored the fusion function of gB3A, whereas S383F and G645R dampened fusion and T509M and N709H worked in concert to restore gB3A fusion. The results identify two regions in the gB ectodomain that modulate the fusion activity of gB, potentially by impacting intramolecular interactions and stability of the prefusion and/or postfusion gB trimer.IMPORTANCE Glycoprotein B (gB) is an essential viral protein that is conserved in all herpesviruses and is required for virus entry. gB is thought to undergo a conformational change that provides the energy to fuse the viral and cellular membranes; however, the details of this conformational change and the structure of the prefusion and intermediate conformations of gB are not known. Previously, we demonstrated that mutations in the gB "arm" region inhibit fusion and impart a small-plaque phenotype. Using serial passage of a virus carrying these mutations, we identified revertants with restored plaque size. The revertant viruses acquired novel mutations in gB that restored fusion function and mapped to two sites in the gB ectodomain. This work supports our hypothesis that an interaction between the gB arm and the core of gB is critical for gB refolding and provides details about the function of gB in herpesvirus-mediated fusion and subsequent virus entry.

Mapping sites of herpes simplex virus type 1 glycoprotein D that permit insertions and impact gD and gB receptors usage.

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) is one of four glycoproteins essential for HSV entry and cell fusion. The purpose of this study was to determine the plasticity of gD to tolerate insertion or deletion mutations and to construct an oncolytic HSV-1 that utilizes the disialoganglioside GD2 as a HSV-1 entry receptor. We found that the N-terminus of gD tolerates long insertions, whereas residues adjacent to the gD Ig-like V-type core tolerated shorter insertions (up to 15 amino acids), but not greater than 60 amino acids. Recombinant HSV-1 containing the ch14.18 single chain variable fragment (scFv) at the N-terminus of gD failed to mediate entry, even though the ch14.18 scFv-gD chimera Fc bound to neuroblastoma cells expressing GD2. Finally, we found that hyperfusogenic gB mutants enhanced fusion to a greater degree with the gB receptor the paired immunoglobulin-like type 2 receptor alpha (PILRα) than with gD receptors HVEM and nectin-1. Hyperfusogenic gB could restore the fusion function with PILRα when a gD constructed contained only the "profusion domain" (PFD), suggesting the hyperfusogenic form of gB may regulate fusion of PILRα via a novel mechanism through gH/gL and the gD PFD.

Structure-Based Mutations in the Herpes Simplex Virus 1 Glycoprotein B Ectodomain Arm Impart a Slow-Entry Phenotype.

Glycoprotein B (gB) is the conserved herpesvirus fusion protein, and it is required for the entry of herpesviruses. The structure of the postfusion conformation of gB has been solved for several herpesviruses; however, the gB prefusion crystal structure and the details of how the protein refolds from a prefusion to a postfusion form to mediate fusion have not been determined. Using structure-based mutagenesis, we previously reported that three mutations (I671A, H681A, and F683A) in the C-terminal arm of the gB ectodomain greatly reduced cell-cell fusion. This fusion deficit could be rescued by the addition of a hyperfusogenic mutation, suggesting that the gB triple mutant was not misfolded. Using a bacterial artificial chromosome (BAC), we constructed two independent herpes simplex virus 1 mutant strains (gB 3A) carrying the three arm mutations. The gB 3A viruses have 200-fold smaller plaques than the wild-type virus and demonstrate remarkably delayed entry into cells. Single-step and multistep growth curves show that gB 3A viruses have delayed replication kinetics. Interestingly, incubation at 40°C promoted the entry of the gB 3A viruses. We propose that the gB 3A viruses' entry deficit is due to a loss of interactions between residues in the gB C-terminal arm and the coiled-coil core of gB. The results suggest that the triple alanine mutation may destabilize the postfusion gB conformation and/or stabilize the prefusion gB conformation and that exposure to elevated temperatures can overcome the defect in gB 3A viruses.IMPORTANCE Because of its complexity, the mechanism of herpesvirus entry into cells is not well understood. Our study investigated one of the most important unanswered questions about herpesvirus entry; i.e., how does the herpesvirus fusion protein gB mediate membrane fusion? gB is an essential protein that is conserved in all herpesviruses and is thought to undergo a conformational change to provide the energy to fuse the viral and cellular membranes. Using our understanding of the structure of gB, we designed mutations in the gB "arm" region that we predicted would impede gB function. We introduced these mutations into herpes simplex virus 1 by using a bacterial artificial chromosome, and the mutant virus exhibited a drastically delayed rate of entry. This entry defect was rescued by incubation at elevated temperatures, supporting a hypothesis that the engineered mutations altered the energetics of gB refolding. This study supports our hypothesis that an interaction between the gB arm and the core of gB is critical for gB refolding and the execution of membrane fusion, thus providing key details about the function of gB in herpesvirus-mediated fusion and subsequent virus entry.

Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins.

Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

The fusion loops and membrane proximal region of Epstein-Barr virus glycoprotein B (gB) can function in the context of herpes simplex virus 1 gB when substituted individually but not in combination.

Among the herpesvirus glycoprotein B (gB) fusion proteins, the hydrophobic content of fusion loops and membrane proximal regions (MPRs) are inversely correlated with each other. We examined the functional importance of the hydrophobicity of these regions by replacing them in herpes simplex virus type 1 gB with corresponding regions from Epstein-Barr virus gB. We show that fusion activity is dependent on the structural context in which the specific loops and MPR sequences exist, rather than a simple hydrophobic relationship.

A soluble form of Epstein-Barr virus gH/gL inhibits EBV-induced membrane fusion and does not function in fusion.

We investigated whether soluble EBV gH/gL (sgH/gL) functions in fusion and made a series of truncations of gH/gL domains based on the gH/gL crystal structure. We found sgH/gL failed to mediate cell-cell fusion both when co-expressed with the other entry glycoproteins and when added exogenously to fusion assays. Interestingly, sgH/gL inhibited cell-cell fusion in a dose dependent manner when co-expressed. sgH/gL from HSV was unable to inhibit EBV fusion, suggesting the inhibition was specific to EBV gH/gL. sgH/gL stably binds gp42, but not gB nor gH/gL. The domain mutants, DI/gL, DI-II/gL and DI-II-III/gL were unable to bind gp42. Instead, DI-II/gL, DI-II-III/gL and sgH/gL but not DI/gL decreased the expression of gp42, resulting in decreased overall fusion. Overall, our results suggest that domain IV may be required for proper folding and the transmembrane domain and cytoplasmic tail of EBV gH/gL are required for the most efficient fusion.

Fusing structure and function: a structural view of the herpesvirus entry machinery.

Herpesviruses are double-stranded DNA, enveloped viruses that infect host cells through fusion with either the host cell plasma membrane or endocytic vesicle membranes. Efficient infection of host cells by herpesviruses is remarkably more complex than infection by other viruses, as it requires the concerted effort of multiple glycoproteins and involves multiple host receptors. The structures of the major viral glycoproteins and a number of host receptors involved in the entry of the prototypical herpesviruses, the herpes simplex viruses (HSVs) and Epstein-Barr virus (EBV), are now known. These structural studies have accelerated our understanding of HSV and EBV binding and fusion by revealing the conformational changes that occur on virus-receptor binding, depicting potential sites of functional protein and lipid interactions, and identifying the probable viral fusogen.

Paramyxovirus fusion: real-time measurement of parainfluenza virus 5 virus-cell fusion.

Although cell-cell fusion assays are useful surrogate methods for studying virus fusion, differences between cell-cell and virus-cell fusion exist. To examine paramyxovirus fusion in real time, we labeled viruses with fluorescent lipid probes and monitored virus-cell fusion by fluorimetry. Two parainfluenza virus 5 (PIV5) isolates (W3A and SER) and PIV5 containing mutations within the fusion protein (F) were studied. Fusion was specific and temperature-dependent. Compared to many low pH-dependent viruses, the kinetics of PIV5 fusion was slow, approaching completion within several minutes. As predicted from cell-cell fusion assays, virus containing an F protein with an extended cytoplasmic tail (rSV5 F551) had reduced fusion compared to wild-type virus (W3A). In contrast, virus-cell fusion for SER occurred at near wild-type levels, despite the fact that this isolate exhibits a severely reduced cell-cell fusion phenotype. These results support the notion that virus-cell and cell-cell fusion have significant differences.

Refolding of a paramyxovirus F protein from prefusion to postfusion conformations observed by liposome binding and electron microscopy.

For paramyxoviruses, two viral glycoproteins are key to the entry process: an attachment protein (HN, H, or G) and the fusion protein (F). The F protein folds to a metastable state that can be triggered to undergo large conformational rearrangements to a fusogenic intermediate and a more stable postfusion state. The triggering mechanism that controls paramyxovirus fusion has not been elucidated. To correlate the molecular structure of a soluble form of the prefusion F (PIV5 F-GCNt) with the biological function of F, soluble F protein was triggered to refold. In the absence of HN, heat was found to function as a surrogate F trigger, and F associated with liposomes and aggregated on sucrose density gradients. Electron microscopy data showed that triggered F formed rosettes. Taken together these data suggest that release and membrane insertion of the hydrophobic fusion peptide require both cleavage of F and heat. Heating of cleaved F causes conversion to a postfusion form as judged by its "golf tee" morphology in the electron microscope. Heating of uncleaved F also causes conversion of F to a morphologically similar form. The reactivity of the F protein with conformation-specific mAbs and peptide binding suggest that soluble F-GCNt and membrane-bound F proteins refold through a comparable pathway.