JUNIN VIRUS INFECTION TRIGGERS THE AUTOPHAGY PATHWAY IN A HUMAN CELL LINE

J. S. ROLDÁN; L.R. DELGUI; N. CANDURRA

Philadelphia

Congreso; The American Society for Cell Biology Annual Meeting; 2014

The American Society for Cell Biology

JUNIN VIRUS INFECTION TRIGGERS THE AUTOPHAGY PATHWAY IN A HUMAN CELL LINE J. S. Roldán1, L. R. Delgui2, N. A. Candurra1 1Dpto de Qca. Biológica, IQUIBICEN, FCEyN-UBA, Buenos Aires 2IHEM, UNCuyo-CONICET, Mendoza, Argentina. Autophagy is a conserved degradative pathway that plays a key role in maintaining cellular homeostasis through eliminating unwanted proteins and damaged organelles by cellular self-digestion in the lysosome to fulfil the demand for aminoacids required for cell survival. The first step of this process is the formation of double membrane vesicles, denominated autophagosomes, around portions of cytoplasm which then fuse with lysosomes where enzymatic degradation occurs. This process has a crucial role in protecting cells from viral invasion. However, viruses have been able to subvert this pathway in their own benefit. Junin virus (JUNV) belongs to the phylogenetic clade B of the Arenaviridae family. It is the aetiological agent of Argentine haemorrhagic fever, an endemo-epidemic disease affecting the population of the most fertile farming land of Argentina. We analysed the role of autophagy in the course of JUNV infection employing human cell line A549. We used LC3 as a protein marker of autophagy pathway modulation after viral infection. Mammalian LC3 is a cytoplasmic protein (LC3-I) that becomes lipidated and membrane associated (LC3-II) upon induction of autophagy. When detected by confocal laser scanning microscopy, vesicles associated LC3-II appears as discrete green dots. We have observed that cells overexpressing EGFP-LC3 and infected with JUNV showed an increased number of LC3 dots similar to what we have shown after starvation- or BafilomycinA1- treatment, which leads to autophagosome formation induction or accumulation, respectively. We have monitored the conversion of LC3-I (cytosolic) to LC3-II (associated to autophagosomes) by Western blot technique observing that levels of LC3-II in JUNV-infected cells were similar to that observed in starved cells. Moreover, cells pre-treated with rapamycin, a pharmacological autophagy inductor, enhanced virus yield with respect to the control situation. In addition, we have assayed the replication capacity of JUNV in Atg5 knock-out cells (a key molecular component of the autophagic pathway), but no differences were found when compared to the wild type Atg5 cells. At last, we have kinetically studied the number of LC3 dots after JUNV infection over a period of 24 h post infection (p.i.) and found that the pathway was activated by the presence of the virus since 2 h p.i. and remained activated until the end of the mentioned lapse of timer monitored. All together, these results allowed us to conclude that JUNV infection leads to an autophagic response in the infected cells. However, a functional autophagy pathway does not seem to be required for efficient virus replication.