Figure 3.

Effect of geneticin on viral RNA synthesis and viral protein accumulation. A) Quantitative RT-PCR analysis of intracellular viral RNA in BHK cells infected with DENV-2 in the presence of 25 μg/ml of geneticin, at 6, 12, 24, and 48 hpi. Viral RNA expression is defined as a percent of viral RNA collected from geneticin-treated infected cells over RNA detected from untreated infected cells. Data are presented as means ± SE for each specified condition (n=4). B) Western blot representative of DENV-2 E protein expression inhibited by geneticin. BHK cells were infected with DENV-2 (MOI of 1) in the absence or presence of 25μg/ml of geneticin, and at 6,12, 24, and 48 hpi (see Experimental Procedures). Cell lysates were subjected to SDS-PAGE and Western blotting analysis using primary antibody (D1-4G2) for E protein. C) Quantification of Viral E protein expression 48 hpi on Western blots was performed with the Kodak molecular imaging systems software (Carestream Health, Inc. Rochester, NY). The DENV-2-infected control, which was not treated by geneticin, was set as 100% of viral E protein expression. The percentage of inhibition of E protein accumulation was expressed as E protein from geneticin-treated infected cells over viral E protein from untreated infected cells. Data are presented as means ± SE for each specified condition (n=3).