Glycoproteins and Entry into Host Cells – Structure and Function of the KSHV fusion machinery

• Applicant: Dr. Alexander Hahn
• Subject Area: Virology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 530009014

Kaposi’s sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma and two B cell malignancies. KSHV engages diverse cellular receptors to enter target cells via endocytosis. KSHV glycoproteins gB and gH/gL form the 'core fusion machinery' that induces merger of viral and endosomal membranes, allowing for the genome release into the cytosol. gB is the actual fusogen. The gH/gL complex regulates gB activity, in a still unknown manner. The fusion trigger for gB has not been identified either. In addition to its role in fusion, gB binds to integrins and DC-SIGN, and these interactions promote endocytosis. We discovered that the EphA receptors play an important role in KSHV entry by engaging gH/gL, and recently determined the structure of the gH/gL-EphA2 complex, but structural information on gB and complexes with its receptors is entirely missing. Since KSHV infection can occur independently of the known receptors, the list of the cellular receptors mentioned above is not exhaustive. This project will be carried out as a collaboration between Marija Backovic, a structural biologist specializing in viral glycoproteins, and Alexander Hahn, who is a molecular virologist and expert in genetic manipulation and biology of KSHV biology. Each partner is uniquely qualified and has a long-standing experience and technical know-how in different aspects of herpesvirus biology. Thus, we propose an integrated approach that relies on structural biology (X-ray crystallography and cryo-EM) and biophysics (Backovic), and functional studies to investigate virus entry, virus-receptor interactions, and host factor identification (Hahn). Our objectives are to 1) generate gB variants stabilized in the pre-fusion state, 2) search for new KSHV receptors by CRISPR/Cas9, 3) determine the interaction sites for gB bound to cellular receptors (known, and TIM-1 and NRP1, which were recently identified by Hahn group), 4) identify fusion trigger (pH and/or a receptor), 5) obtain a panel of neutralizing nanobodies (Nb) by immunizing llamas with stabilized gB and gH/gL, and 6) identify the best neutralizers and determine their structures in complexes with gB or gH/gL. The reason we plan to develop Nbs is the lack of monoclonal Abs (mAbs) against KSHV GPs. Thus, one of the most informative tools to study GP-mediated entry is largely missing in the field. Knowing the epitopes targeted by neutralizing Nbs will reveal vulnerabilities of the virus that can be exploited for the development of a prophylactic vaccine or therapeutic antibodies. Overall, these studies have a potential to significantly advance our understanding of the function of the fusion machinery and its interactions with cellular receptors, on a structural and on a functional level. The outcomes of this proposal will in addition inform us on the design of KSHV immunogens, opening roads to further studies focusing on the development of KSHV vaccine candidates.

• DFG Programme: Research Grants
• International Connection: France
• Cooperation Partner: Privatdozentin Dr. Marija Backovic, Ph.D.