Study of cell lipid droplets during viral infection

VAZQUEZ CA 1,2, PEÑA CÁRCAMO JR 1, MORELL ML 1,2, CORDO, SM 2, GARCÍA, C1

Bernal, Buenos Aires

Jornada; II Argentine Meeting on Biology of Non-Coding RNAs; 2018

(P37) Study of cell lipid droplets during viral infectionVazquez CA 1,2, Peña Cárcamo JR 1, Morell ML 1,2, Cordo, SM 2, García, C11 Laboratory of Antiviral Strategies, Biochemistry Department, School of Sciences, University of Buenos Aires- IQUIBICEN, CONICET. 2 Laboratory of Biochemistry and Biology of Junín virus, Biochemistry Department, School of Sciences, University of Buenos Aires- IQUIBICEN, CONICET.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. 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 immunofluorescence microscopy 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. 66ABSTRACT BOOKII Argentine Meeting on Biology of Non-Coding RNAs July 30th-31st, 2018. UNQ, Bernal, Argentina (P38) Comparative analysis of sRNAs profiles associated with the response to contrasting environments in potatoZavallo D1*, Leone M1*, Ibañez V3, Crescente JM2, Marfil C3, Masuelli R3, Asurmendi S1,4.1 Instituto de Biotecnología CICVyA-INTA, Hurlingham, Argentina. 2 Grupo de Biotecnologia y Recursos Genéticos, EEA INTA Marcos Juarez, Argentina. 3 Instituto de Biología Agrícola de Mendoza, FCA, UNCuyo, Mendoza, Argentina. 4CONICET Argentina.Potato is the main horticultural crop worldwide and third as human food after rice and wheat. Argentina ranks the 22th in terms of production, is therefore of great interest to understand the response and adaptation to the environment in order to have the capacity to expand the arable areas. Wild potato species, which are adapted to diverse environments along the Andes, represents an invaluable source of heritable variability for crop improvement. These species present high phenotypic plasticity and little is known about the responses to stresses or environmental fluctuation. During evolution, plants have acquired a series of adaptive strategies that allow them to survive under adverse environmental conditions. One of the central principles of biology is that evolutionary processes act on genetic variability in natural populations; however epigenetic mechanisms also play an important role in reprogramming gene expression in response to environmental cues. Thus, epigenetic marks can serve as a priming mechanism to prepare future generations to better withstand biotic and abiotic stresses. Epigenetic modification often results in silencing gene expression in a process called RNA-directed DNA Methylation (RdDM) and one of the key components in that process involved small RNAs (sRNAs). The extent about the role of sRNAs in the origin of phenotypic variation and their adaptive consequences has been little explored. In this work, the Argentine wild potato species best adapted to arid regions growing in their natural habitat was used as experimental model: biological replicates (clones) of Solanum kurtzianum were in situ cultivated in the Villavicencio Natural Reserve (Mendoza) in two experimental gardens (EG) at contrasting elevation (i.e. 1200 m vs. 2200 m a.s.l). This altitude gradient generates differences, among others, in ultraviolet-B radiation levels (10%) and mean temperature (35%). Morphological, productivity and biochemical characters measured in leaves and tubers showed significant differences including increased leaf area, tuber number and mean tuber weight in 2200 m respect to 1200 m clones. In order to assess the role of epigenetic mechanisms in the origin the documented phenotypic plasticity, a sRNA-seq experiment was perform on leaves from clones cultivated in the two EG. Bioinformatic analysis was implemented to profile 21 and 24nt sRNAs, the latter ones are known to be the responsible of the RdDM and two independent programs were used to account for the differentially accumulated sRNAs. Distributions of different features across the potato genome were performed and highlighted those with differentially accumulated sRNAs. Several features presented differentially accumulated sRNAs between the two environments which they could have an impact on the epigenome and in consequence in the transcriptome.