THE HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN K IS A CELL FACTOR REQUIRED FOR DENGUE VIRUS TYPE 2 MULTIPLICATION

CASTILLA, VIVIANA; BRUNETTI, JESÚS EMANUEL; SCOLARO, LUIS

Belem

Simposio; IV Pan American dengue research network meeting; 2014

Pan American dengue research network

Heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and K are cellular RNA-binding proteins that participate in mRNA splicing, trafficking and translation and have been implicated in the multiplication of several cytoplasmic RNA viruses. To identify cellular factors involved in dengue virus (DENV) replication, here we characterized hnRNP A2 and K expression in Vero and A549 cells infected with DENV type 2 and we evaluated the effect of hnRNP K silencing on DENV-2 multiplication. We analyzed hnRNP intracellular localization by indirect immunofluorescence assays, using anti-hnRNP and anti-viral glycoprotein E antibodies, at 2 or 5 days postinfection (dpi). Whereas hnRNP A2 showed a clear nuclear localization, similar to that observed in uninfected cells, the cytoplasmic translocation of hnRNP K was evident at 5 dpi in both cell types. We also performed A549 cell transfection with a plasmid that allows the expression of T7-tagged hnRNP K and the re-localization of transiently overexpressed hnRNP K to the cytoplasm was observed at 2dpi. The analysis by western blot showed no significant differences in the total levels of hnRNP K expression between uninfected and DENV-2 infected Vero or A549 cells. Finally, A549 cells were transfected with small interfering RNAs (siRNAs) that target hnRNP K or small non-interfering RNA used as control, and at 24 h post-transfection cells were infected. The effectiveness of siRNA silencing was corroborated by western blot and at 1 and 5 dpi, virus yield and the amount of glycoprotein E expressing cells were assessed by plaque assay and immunofluorescence technique, respectively. A significant reduction of 66.6% and 96.0% of virus yield was achieved at 1 and 5 dpi, respectively, and the amount of fluorescent cells was also inhibited. The results obtained indicate that DENV-2 infection alters hnRNP K intracellular distribution and this hnRNP would play an important role in DENV-2 multiplication in cell cultures.