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

POCOGNONI, CRISTIAN A.; MAYORGA, LUIS S.; DELGUI, LAURA R.

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

Zika virus (ZIKV) belongs to the Flaviviridae family and spreads mainly by the Aedes mosquitoes. ZIKV can also be transmitted sexually and by body fluids contact. ZIKV was originally detected in rhesus monkeys in 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 fuses with lysosomes and releases its genome into the cytoplasm. Afterwards, a viral polyprotein is synthetized by translation of the viral genome and, after proper processing throughout the secretory pathway, a mature virion is generated and released from the cells. A key step in this maturation process, involves the cleavage 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-infective particles lacking the viral genome but having key viral proteins anchored to their membranes. VLPs have been 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-expressed generate 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 up the use of ZIKV VLPs generated by co-transfecting HeLa cells with the ZO2 and ZO3 constructs. We used a time-lapse of 24, 48 and 72 h post-transfection (h p.t.) and by using an anti prM antibody, we detected its expression levels by Western Blot and intracellular distribution by Immunofluorescence. We found that prM localizes at ER, cis, medial and trans-Golgi compartments, as we assayed its co-localization with different components of the secretory pathway (calnexin, GBF1, BIG1, BIG2, and Furin) and they all showed good colocalization indexes (between 0.6-0.8 Pearson coefficient). Thus, suggesting that the nascent VLPs mimic the same ZIKV pathway. Finally, we tested ZO2 and ZO3 co-transfected HeLa cells (72 h p.t.) by electron microscopy. We identified “VLP-like structures” with an average diameter size of 60 nm and 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 novel antiviral drugs, contributing to stop the diseases that ZIKV infection causes.