izvor podataka: poirot

Reorganizacija sučelja između ranih endosoma i endosomalnog reciklirajućeg odjeljka

Cytomegalovirus reorganization of the interface between early endosomes and endosomal recycling compartment


Biogeneza cVAC (engl. cytoplasmic virion assembly compartment) predstavlja važan, i još nerazjašnjen problem za razumijevanje biologije herpesvirusa. U ovom projektnom prijedlogu ćemo proučavati najranije faze reorganizacije mambranskih organela koje nazivamo preAC (engl. pre-assembly compartment). Ovaj istraživački projekt predstavlja nastavak i produbljenje našeg prethodnoga rada i fokusira se na stanične mehanizme koje koristi mišji citomegalovirus (MCMV) na sučelju između ranih endosoma i endosomalnog reciklirajućeg odjeljaka (EE-ERC), odnosno mjestu za koje pretpostavljamo da predstavlja inicijacijski korak cVAC biogeneze. U tu svrhu, MCMV može ciljati (1) domaćinske efektore koji reguliraju procese sortiranja i recikliranja na EE-ERC sučelju, (2) Rab10 i njegove partnere, regulatorne proteine koji kontroliraju EE-ERC prometovanje, i (3) procese ubikvitinilacije koji kontroliraju endosomalno recikliranje, a djeluju prema principu reostata. Ovi potencijalni učinci MCMV djelovanja predstavljaju tri cilja ovog projektnog prijedloga. Kako bismo ih ispunili testirat ćemo skoro 50 staničnih efektora i regulatora na EE-ERC sučelju i njihovu ulogu u nastanku preAC. Stanice će biti inficirane divljim tipom i različitim MCMV rekombinantama, a domaćinski proteini će biti analizirani knock-down (siRNA, inhibitori) i knock-out eksperimentima (CRISPR-Cas9). Nastanak preAC će se pratiti konfokalnom mikroskopijom i rekonstrukcijom mape interakcija na EE-ERC sučelju pomoću BioID testa, imunoprecipitacije, Western blot analize i subcelularnog frakcioniranja. MCMV učinak na kompleksnu mrežu regulatornih proteina i efektora na EE-ERC sučelju će predstavljati značajan znanstveni doprinos ovog projektnog prijedloga. On će razjasniti ne samo najranije stadije patogeneze beta-herpesvirusne infekcije, već i prostorno-vremenske odnose u neinficiranim stanicama, dva nedovoljno razjašnjena fundamentalna biološka procesa.

The biogenesis of cVAC (cytoplasmic virion assembly compartment) is an outstanding and unresolved issue in the biology of herpesviruses. In this research proposal we will study the earliest stages of host-cell membranous-organelle reorganization which we considers as preAC (pre-assembly compartment). This project represents an extension of our previous work and focuses on cellular mechanisms exploited by murine cytomegalovirus (MCMV) at the interface between early endosomes and endosomal recycling compartment (EE-ERC), since the targeting the EE-ERC interface may be initiation step in the cVAC biogenesis. To achieve this, MCMV may target (1) host-cell effectors that regulates processes of sorting and retrieving at the EE-ERC interface, (2) Rab10 and its interacting partners, regulatory proteins that controls EE-ERC trafficking, and (3) ubiquitination processes that act as a regulatory rheostat mechanism in the endocytic recycling. These potential MCMV targets are aligned as three objectives of the research proposal. To address them, we will test almost 50 host-cell effectors and regulators of the EE-ERC interface and their role in the preAC development. Cells will be infected with wild type and various MCMV recombinants, and host-cell factors analyzed by knock-down (siRNA, inhibitors) and knock-out experiments (CRISPR-Cas9). The preAC development will be monitored by confocal microscopy and reconstruction of the roadmap at the EE-ERC interface by BioID assay, immunoprecipitation, Western blot analysis, and subcellular fractionation. The effect of MCMV on a complex network of host-cell regulatory proteins and effectors at the EE-ERC interface will be a significant research outcome of the project proposal. This outcome may resolve both, the earliest stages in the pathogenesis of beta-herpesvirus infection, and the spatial-temporal physiology of the EE-ERC interface in non-infected cells, the two insufficiently understood fundamental biological processes.


citomegalovirus, odjeljak za sklapanje virusnih čestica, rani endosomi, endosomalni reciklirajući odjeljak, ubikvitinilacija

cytomegalovirus, viral assembly compartment, early endosomes, endosomal recycling compartment, ubiquitination

Znanstveno-istraživački projekti

CREERC

IP-2020-02-2916

01.01.2021

31.12.2024

nije evidentirano

HRK 912.000,00