Study of cell lipid droplets during viral infection

VÁZQUEZ CA; PEÑA CARCAMO J R; MORELL M L; CORDO S M; GARCÍA C C

Bernal

Encuentro; II Argentine Meeting on Biology of Non-coding RNAs; 2018

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 affectingthe region. Junín virus (JUNV) is an enveloped RNA virus, which causes Argentine hemorrhagic fever. The goal of this work was to study the importance of LD in JUNV infection. In this work we explored diverse techniques to study LD in the context of viral infection. HepG2 (human hepatocellular carcinoma) and A549 (human lung carcinoma) cell lines were used. First, cells monolayers were treated with oleic acid (OA) and C75 to stimulate and inhibit LD morphogenesis, respectively. The effect of this modulation was evaluated by immunofluorescencemicroscopy and Oil Red-O staining. Flow cytometry was also used to determine changes in cellular complexity. A quantification of the number of LD per cell, done by means of indirect immunofluorescence and confocal microscopy, showed a reduction in the number of LD in infected cells, when compared to non-infected cells. 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. 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. Having implemented techniques which allow us to study LD, we will expand our study to understand the role of this organelle during JUNV replication. This understanding will help us find potential targets for antiviral therapy.