Role of lipid droples in viral replication

VÁZQUEZ CA; PEÑA CÁRCAMO JR; MORELL ML; CORDO SM; GARCÍA C

Sao Jose do Rio Preto

Workshop; Sao Paulo School of Advanced Science in Arbovirology; 2017

FAMERP

Lipid droplets (LD) are organelles that consist of a neutral lipids core, surrounded by a phospholipid monolayer, in which proteins are embedded. It has been well documented that LD play important roles in viral replication of viruses, such as hepatitis C, dengue and rotavirus. Our lab is focused on the study of RNA virus relevant to human health mainly those greatly affecting the region. The goal of this work was to study the importance of LD in Junin virus (JUNV), dengue virus (DENV), zika virus (ZIKV) and yellow fever virus (YFV) infection. In this work we explored diverse techniques to study these organelles in the context of viral infection.Human HepG2 (hepatocellular carcinoma) and A549 ( lung carcinoma) cell lines were used. Cells monolayers were treated with oleic acid (OA) and C75 to stimulate and inhibit LD morphogenesis, respectively. The effect of this modulation was evaluated microscopically, by LD staining with Bodipy 493/503 and Oil Red-O. Flow cytometry was also used to determine changes in cellular complexity. All of the techniques allowed us to determine the conditions of stimulation and inhibition of LD morphogenesis in the used model. We established the conditions necessary to isolate LD by cellular fractionation in density gradients. To confirm the isolation, fractions were analyzed by mass spectrometry. LD markers, perilipin 2 and 3, were found in the top fraction, but not in the bottom fractions. Afterwards, immunofluorescence suggested that in JUNV infected and OA stimulated cultures, the viral nucleoprotein N localizes in LD. We are currently verifying this observation by isolating LD in a subcellular fractionation of infected cells.Having implemented techniques which allow us to study LD, we will expand our study and compare the importance of this organelle in the replication of JUNV, DENV, ZIKV and YFV.