Grantor: Croatian Science Foundation
Project duration: 1 January 2020 – 31 December 2023
Total funding: 1.355.937,50 HRK (180.000 EUR)
Principal investigator: Prof. Pero Lučin, MD, PhD
Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Research team
Prof. Hana Mahmutefendić Lučin, MD, PhD, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Prof. Gordana Blagojević Zagorac, MD, PhD, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Prof. Kristina Grabušić, PhD, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Prof. Sandra Kraljević Pavelić, PhD, Faculty of Health Studies, University of Rijeka, Croatia
Prof. Martin Messerle, PhD, Hannover Medical School, Germany
Prof. Zsolt Ruzsics, MD, PhD, University Medical Center Freiburg, Institute of Virology, Germany
Asst. Prof. Tamara Gulić, PhD, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia, project associate since June 2022
Asst. Prof. Ljerka Karleuša, PhD, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Natalia Jug Vučko, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia, project associate until June 2023
Valentino Pavišić, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia, project associate until May 2022
Silvija Lukanović Jurić, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Marina Marcelić, PhD, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Ingrid Šutić, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia, project associate until May 2022
Igor Štimac, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Ivona Viduka, Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka, Croatia
Summary
Beta-herpesviruses extensively reorganize the membranous system of the host cell which is exploited for the final stages of virus manufacturing, the secondary envelopment and virion egress. The site and mechanisms of these processes remain unresolved due to the unrevealed complexity of the membranous system, insufficiently developed methods for quantification of released virions, and the inability of single virus- and/or organelle-tracking inside the cell. In this project we will use recombinants of the murine cytomegalovirus (MCMV), a member of the beta-herpesvirus family, to improve methods of extracellular virus quantification, distinguishing virus replication from production, and virus tracking. Recombinant MCMVs with fluorescently tagged small capsid protein (SCP), the only available capsid-tagged viruses in the beta-herpesvirus family, will enable colocalization analysis and time-lapse tracking during processes of the secondary envelopment. Given that the CMV egress is a rare event we will use digital holotomographic microscopy (DHTM) to track MCMV egress and to develop an assay for egress monitoring. Using systems approaches, such as analysis of local interactome using computational methods and proximity-dependent biotin identification assay, analysis of transcriptome and linear motif mimicry, we will focus on significant host-cell factors that control the processes of envelopment and egress. The significant host-cell factors will be screened using the newly developed two-step siRNA screening assays and those that are essential for the final stages will be identified. To gain mechanistic insights, a small GTPase from Rab and/or Arf family that controls each of these processes will be analyzed in-depth using the improved methodology and knock-down in infected cells. The data obtained will serve for the construction of the roadmap of cellular changes required for the final stages of beta-herpesvirus maturation and for the identification of potential antiviral targets.