ZIKA VIRUS LIKE PARTICLES CHARACTERIZATION AS A USEFUL STRATEGY TO STUDY THE VIRAL MATURATION PROCESS.

POCOGNONI, CRISTIAN A.; MAYORGA, LUIS; DELGUI, LAURA RUTH

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

SAIB

Zika virus (ZIKV) belongs to the Flaviviridae family and spreads mainly by the Aedes mosquitoes. ZIKV canalso be transmitted sexually and by body fluids contact. ZIKV was originally detected in rhesus monkeysin Uganda in 1947, but was really put under the scope after its last outbreak in North and South America(2015-2017). ZIKV can cause an autoimmune disorder in adults that affects the nervous system (Guillain-Barré syndrome); and a severe brain damage in newborns from infected women (Microcephaly).Unfortunately, no vaccines or effective treatments to prevent ZIKV-related diseases are available,highlighting the urgent necessity of compelling research in this field.ZIKV enters the host cell by specific receptor mediated endocytosis: once inside the cell, the virion fuseswith lysosomes and releases its genome into the cytoplasm. Afterwards, a viral polyprotein is synthetizedby translation of the viral genome and, after proper processing throughout the secretory pathway, amature virion is generated and released from the cells. A key step in this maturation process, involves thecleavage of the viral protein prM by the host cell endopeptidase Furin to produce the small M protein(involved in ZIKV-host cell recognition) and the fragment pr. Viral Like Particles (VLPs) are non-infectiveparticles lacking the viral genome but having key viral proteins anchored to their membranes. VLPs havebeen widely used as a model to study different aspects of Flavivirus biology.Dr. Jose Galarza (CEO, TechnoVax, Inc, USA) gently transferred us two constructs, that when co-expressedgenerate ZIKV VLPs: i) ZO2, to express the ZIKA capsid, prM, and the E envelope protein (CprME), and ii)ZO3, to express the viral protease NS3 and its cofactor NS2B (NS2B/NS3P). In the present work, we set upthe use of ZIKV VLPs generated by co-transfecting HeLa cells with the ZO2 and ZO3 constructs. We used atime-lapse of 24, 48 and 72 h post-transfection (h p.t.) and by using an anti prM antibody, we detected itsexpression levels by Western Blot and intracellular distribution by Immunofluorescence. We found thatprM localizes at ER, cis, medial and trans-Golgi compartments, as we assayed its co-localization withdifferent components of the secretory pathway (calnexin, GBF1, BIG1, BIG2, and Furin) and they allshowed good colocalization indexes (between 0.6-0.8 Pearson coefficient). Thus, suggesting that thenascent VLPs mimic the same ZIKV pathway. Finally, we tested ZO2 and ZO3 co-transfected HeLa cells (72h p.t.) by electron microscopy. We identified “VLP-like structures” with an average diameter size of 60 nmand a morphology that correlates with the data published before.Summarizing, here we show a useful strategy that can be developed in a regular class I biosafety cabinet,to eventually study the ZIKV maturation process. Our findings set the scene for the further testing of novelantiviral drugs, contributing to stop the diseases that ZIKV infection causes.