Avaliação da inibição da atividade da fosfolipase citoplasmática A2 (cPLA2) sobre o ciclo de multiplicação do Vaccinia virus

Abstract

Vaccinia virus (VACV), the prototype virus of the Poxviridae family, is an enveloped virus that carries out its multiplicative cycle entirely in the cytoplasm of the host cell. Throughout its multiplication cycle, VACV produces two different types of virions that are classified according to the number of envelopes they present and their cellular location: intracellular mature viruses (IMVs), which have a single envelope, and the extracellular viruses (EVs), which have an additional lipid envelope. The existence of two enveloped VACV forms requires the host to undergo intense biogenesis and lipids rearrangement to meet the virus’ demand for membrane lipids. Cytosolic phospholipase A2 (cPLA2) is a lipolytic enzyme that is involved in phospholipid turnover, membrane remodeling and second messenger production that act on cell signaling pathways and host immune response, and, for these reasons, it may be related with virions morphogenesis during the VACV multiplication cycle. In order to confirm this hypothesis, we initially evaluated whether the virus was able to modify the activation pattern (phosphorylation) of cPLA2 in the host cell. We verified that the virus induces an increase in the activation of this protein by a mechanism dependent on the activation of cell signaling pathways (mainly through the MEK/ERK pathway). Subsequently, cPLA2 activity was inhibited, in HeLa cells, by pharmacological mechanisms (using RSC-3388 inhibitor) or genetically (transfection of cells with interference RNA against the gene encoding this enzyme) and the VACV multiplication cycle was evaluated in these conditions. It was observed that the treatment of the cells with the RSC-3388 inhibitor resulted in a significant dose-dependent reduction in the viral titers obtained, but gene silencing with interfering RNA did not have the same effect. Finally, we evaluated by fluorescence microscopy whether there was any alteration in the cellular sublocalization pattern of P-cPLA2 in the condition of infection by VACV, but no modification in this pattern was observed. These results suggest that cPLA2 is required for the VACV multiplication cycle to be optimally completed, but it was not possible to correlate the activity of this enzyme with the virus’ membrane acquisition process, as it was initially proposed.