Effect of the phosphatidylinositol 3-kinase/ Akt pathway on Junin virus replicaction

LINERO, FLORENCIA; SCOLARO, LUIS

Buenos Aires

Workshop; 1st Southamerican workshop on advanced fluorescence microscopy techniques; 2007

Facultad de Ciencias Exactas y Naturales, UBA

Most viruses have developed the ability to modulate a variety of host cell signalling pathways in order to improve the multiplicative cycle efficiency. The study of phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway has been addressed recently due to its central role in modulating several downstream signalling pathways associated with cell survival, proliferation, differentiation, morphology and apoptosis. Studies have demonstrated that the PI3K/Akt pathway is required for an efficient replication of human cytomegalovirus and coxsackievirus B3. In the present study, the modulation of the PI3K/Akt signalling pathway by Junin virus (JUNV) infection was investigated. To evaluate the participation of PI3K/Akt in JUNV replication, compounds Ly294002, specific inhibitor of the PI3K/Akt and caffeine, inhibitor of the phosphorylation of Akt, were employed. The effect of such compounds was evaluated by reduction of viral yield and the alteration in the expression of the viral nucleoprotein (N) detected by indirect inmunofluorescense (IFI) and western blot (WB). Vero and BHK-21 cells infected with JUNV at an moi of 0.01 – 0,5 PFU/cell and treated with different concentrations of caffeine, showed a significant inhibition in the titers of virus produced (>98%)  and a marked reduction of the viral nucleoprotein (N) detected by WB associated to a diminished number of N (+) cells detected by IFI. These data correlated with results previously obtained in our laboratory and suggested that phosphorylation of Akt would be necessary event for JUNV replication. Caffeine also showed a dramatic depolymerization of actin filaments observed by IFI. In order to circumvent the possibility that reduction of JUNV yield might be due to a secondary effect of caffeine on actin cytoskeleton, the compound Ly294002, specific inhibitor of PI3K, was assayed. JUNV infected Vero and BHK-21 cells, treated with different concentrations of this compound, ranging 5-30 µM, also showed a reduction in the amount of N, detected by WB, synthesized at 12, 24 and 48 h p.i.. This reduction was accompanied by a drop in the number of total cells due to the citotoxicity of the compound. The expression of N, determined by IFI, showed a low number of N (+) cells. To note, BHK-21 cells showed a higher viability percentage in comparison with Vero cells. Data of viability obtained by the method of MTT correlated with the number of total cells counted by IFI. In view of these results, short treatment periods, to maintain high viability percentages, will be considered for further experiments. These preliminary results would indicate that PI3K/Akt signalling pathway participates in the replication of JUNV, since its inhibition by caffeine and Ly294002, diminishes the multiplication of the virus in both cell lines.