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Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion.

ABSTRACT: Enveloped viruses typically encode their own fusion machinery to enter cells. Herpesviruses are unusual, as they fuse with a number of cellular compartments throughout their life cycles. As uncontrolled fusion of the host membranes should be avoided in these events, tight regulation of the viral fusion machinery is critical. While studying herpes simplex virus 1 (HSV-1) glycoprotein gM, we identified the cellular protein E-Syt1 (extended synaptotagmin 1) as an interaction partner. The interaction took place in both infected and transfected cells, suggesting other viral proteins were not required for the interaction. Most interestingly, E-Syt1 is a member of the synaptotagmin family of membrane fusion regulators. However, the protein is known to promote the tethering of the endoplasmic reticulum (ER) to the plasma membrane. We now show that E-Syt1, along with the related E-Syt3, negatively modulates viral release into the extracellular milieu, cell-to-cell viral spread, and viral entry, all processes that implicate membrane fusion events. Similarly, these E-Syt proteins impacted the formation of virus-induced syncytia. Altogether, these findings hint at the modulation of the viral fusion machinery by the E-Syt family of proteins.IMPORTANCE Viruses typically encode their own fusion apparatus to enable them to enter cells. For many viruses, this means a single fusogenic protein. However, herpesviruses are large entities that express several accessory viral proteins to regulate their fusogenic activity. The present study hints at the additional participation of cellular proteins in this process, suggesting the host can also modulate viral fusion to some extent. Hence E-Syt proteins 1 and 3 seem to negatively modulate the different viral fusion events that take place during the HSV-1 life cycle. This could represent yet another innate immunity response to the virus.

SUBMITTER: El Kasmi I

PROVIDER: S-EPMC5730791 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion.

El Kasmi Imane I Khadivjam Bita B Lackman Miki M Duron Johanne J Bonneil Eric E Thibault Pierre P Lippé Roger R

Journal of virology 20171214 1

Enveloped viruses typically encode their own fusion machinery to enter cells. Herpesviruses are unusual, as they fuse with a number of cellular compartments throughout their life cycles. As uncontrolled fusion of the host membranes should be avoided in these events, tight regulation of the viral fusion machinery is critical. While studying herpes simplex virus 1 (HSV-1) glycoprotein gM, we identified the cellular protein E-Syt1 (extended synaptotagmin 1) as an interaction partner. The interactio ...[more]

PMID: 29046455

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Project description:UnlabelledGlycoprotein B (gB), the fusogen of herpes simplex virus (HSV), is a class III fusion protein with a trimeric ectodomain of known structure for the postfusion state. Seen by negative-staining electron microscopy, it presents as a rod with three lobes (base, middle, and crown). gB has four functional regions (FR), defined by the physical location of epitopes recognized by anti-gB neutralizing monoclonal antibodies (MAbs). Located in the base, FR1 contains two internal fusion loops (FLs) and is the site of gB-lipid interaction (the fusion domain). Many of the MAbs to FR1 are neutralizing, block cell-cell fusion, and prevent the association of gB with lipid, suggesting that these MAbs affect FL function. Here we characterize FR1 epitopes by using electron microscopy to visualize purified Fab-gB ectodomain complexes, thus confirming the locations of several epitopes and localizing those of MAbs DL16 and SS63. We also generated MAb-resistant viruses in order to localize the SS55 epitope precisely. Because none of the epitopes of our anti-FR1 MAbs mapped to the FLs, we hyperimmunized rabbits with FL1 or FL2 peptides to generate polyclonal antibodies (PAbs). While the anti-FL1 PAb failed to bind gB, the anti-FL2 PAb had neutralizing activity, implying that the FLs become exposed during virus entry. Unexpectedly, the anti-FL2 PAb (and the anti-FR1 MAbs) bound to liposome-associated gB, suggesting that their epitopes are accessible even when the FLs engage lipid. These studies provide possible mechanisms of action for HSV neutralization and insight into how gB FR1 contributes to viral fusion.ImportanceFor herpesviruses, such as HSV, entry into a target cell involves transfer of the capsid-encased genome of the virus to the target cell after fusion of the lipid envelope of the virus with a lipid membrane of the host. Virus-encoded glycoproteins in the envelope are responsible for fusion. Antibodies to these glycoproteins are important biological tools, providing a way of examining how fusion works. Here we used electron microscopy and other techniques to study a panel of anti-gB antibodies. Some, with virus-neutralizing activity, impair gB-lipid association. We also generated a peptide antibody against one of the gB fusion loops; its properties provide insight into the way the fusion loops function as gB transits from its prefusion form to an active fusogen.

Project description:A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, "stabilizing" MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV.IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.

Dataset Information

Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion.

Publications

Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion.

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