scholarly journals Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Tina M. Cairns ◽  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Huan Lou ◽  
J. Charles Whitbeck ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Protein Interactions ◽  
Vaccine Development ◽  
Receptor Interaction ◽  
Receptor Binding Site ◽  
Interaction Site ◽  
Herpes Simplex Virus Glycoprotein ◽  
Simplex Virus

ABSTRACT 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.

scholarly journals Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus

2016 ◽  
Vol 90 (24) ◽  
pp. 11096-11105 ◽  
Author(s):  
Yu Okubo ◽  
Hiroaki Uchida ◽  
Aika Wakata ◽  
Takuma Suzuki ◽  
Tomoko Shibata ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Human Cancer ◽  
Specific Interaction ◽  
Lateral Spread ◽  
Receptor Interaction ◽  
Glycoprotein D ◽  
Single Chain ◽  
Simplex Virus

ABSTRACTMembrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors.IMPORTANCEHerpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

scholarly journals Immune Response to Herpes Simplex Virus Infection and Vaccine Development

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 302 ◽  
Author(s):  
Anthony C. Ike ◽  
Chisom J. Onu ◽  
Chukwuebuka M. Ononugbo ◽  
Eleazar E. Reward ◽  
Sophia O. Muo
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Infections ◽  
Recombinant Dna ◽  
Vaccine Development ◽  
The Body ◽  
Effective Vaccine ◽  
Subunit Vaccines ◽  
Simplex Virus ◽  
Hsv Infections

Herpes simplex virus (HSV) infections are among the most common viral infections and usually last for a lifetime. The virus can potentially be controlled with vaccines since humans are the only known host. However, despite the development and trial of many vaccines, this has not yet been possible. This is normally attributed to the high latency potential of the virus. Numerous immune cells, particularly the natural killer cells and interferon gamma and pathways that are used by the body to fight HSV infections have been identified. On the other hand, the virus has developed different mechanisms, including using different microRNAs to inhibit apoptosis and autophagy to avoid clearance and aid latency induction. Both traditional and new methods of vaccine development, including the use of live attenuated vaccines, replication incompetent vaccines, subunit vaccines and recombinant DNA vaccines are now being employed to develop an effective vaccine against the virus. We conclude that this review has contributed to a better understanding of the interplay between the immune system and the virus, which is necessary for the development of an effective vaccine against HSV.

scholarly journals Functional and Physical Interactions of the Herpes Simplex Virus Type 1 UL20 Membrane Protein with Glycoprotein K

2008 ◽  
Vol 82 (13) ◽  
pp. 6310-6323 ◽  
Author(s):  
Timothy P. Foster ◽  
Vladimir N. Chouljenko ◽  
K. G. Kousoulas
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Herpes Simplex Virus Type ◽  
Protein Protein Interactions ◽  
Glycoprotein K ◽  
Er Retention ◽  
Simplex Virus

ABSTRACT Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.

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