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. 2022 Mar 16;18(3):e1010393. doi: 10.1371/journal.ppat.1010393

Fig 3. Pharmacological inhibition of mosquito DNMT2 reduces virus replication and per-particle infectivity.

Fig 3

Inhibition of mosquito DNMT2 in Wolbachia-free Aedes albopictus derived C7/10 cells was carried out using MTase inhibitors 5-Azacytidine (5-AZAC). Dimethyl-sulfoxide (DMSO) was used as the negative control. In each case, cells were pretreated with 5 μM inhibitors overnight prior to infections with SINV at MOI of 10. Cell lysates and supernatants were harvested at 24 hours post infection to quantify cellular viral RNA levels and infectious titer, respectively. (A) Levels of SINV RNA in mosquito cells treated with MTase inhibitor 5-AZAC were determined using quantitative RT-PCR. Unpaired two-tailed t-test with Welch’s correction, SINV: p = 0.0012, t = 6.742, df = 4.892. (B) Infectious SINV titers produced from mosquito cells treated with MTase inhibitor 5-AZAC were determined using plaque assays on BHK-21 cells. Unpaired two-tailed t-test with Welch’s correction, SINV: p = 0.0339, t = 4.541, df = 2.322. (C) Specific infectivity ratios of progeny SINV was calculated as the ratio of infectious plaque forming units (B) over total viral genome copies present in collected cell supernatants as quantified by qRT-PCR. Unpaired two-tailed t-test with Welch’s correction, SINV: p = 0.0002, t = 12.59, df = 3.946. Error bars represent standard error of mean (SEM) of three independent experimental replicates. (D) Schematic representation of live cell experiments. CHIKV expressing mKate fluorescent reporter protein was grown in C7/10 Aedes albopictus cells in the presence (W+ virus) or absence (W- virus) of Wolbachia (strain wStri). These progeny viruses were then used to infect naïve C7/10 cells without (E) and with (F) Wolbachia (strain wStri) pretreated with MTase inhibitor or DMSO synchronously at a MOI of 1 particle/cell. Virus growth in cells was measured in real time by imaging and quantifying the number of red cells (Virus Positive Cells/Image) expressing the virus encoded mKate protein over a period of forty-eight hours, using live cell imaging. Color of the data points distinguish treatment conditions; blue represent C7/10 Wolbachia-free cells treated with DMSO, red represent C7/10 Wolbachia-colonized cells treated with DMSO, black represent both C7/10 cell types treated with 5 μM DAC5. Shape of data points represent the progeny virus type used to initiate infection; boxes represent viruses derived from W- cells, circles represent viruses derived from W+ cells. The Y-axis label red object count/Image represent virus-positive cells in a single field of view, four of which were collected and averaged/sample at each two-hour time point over the course of infection. Three-way ANOVA with Tukey’s post hoc test for multivariate comparisons. Error bars represent standard error of mean (SEM) of independent experimental replicates (n = 3). Three-way ANOVA with Tukey’s multivariate analyses, DAC5: p = 0.1793, Virus Source: p = 0.5060, Time: p < 0.0001, DAC5 X Time: p > 0.99, Virus Source X Time: p > 0.99, Virus Source X DAC5: p = 0.1039, Virus Source X DAC5 X Time: p = 0.9804 (Fig 3E). *P < 0.05; **P < 0.01, ****P < 0.0001. Three-way ANOVA with Tukey’s multivariate analyses, DAC5: p < 0.0001, Virus Source: p < 0.0001, Time: p < 0.0001, DAC5 X Time: p < 0.0001, Source X Time: p < 0.0001, Virus Source X DAC5: p = 0.1148, Virus Source X DAC5 X Time: p > 0.999 (Fig 3F). Graphical assets were made in BioRender (https://biorender.com).