Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropods
Fig 4
AMt2 overexpression in Wolbachia-colonized cells rescues virus from endosymbiont-mediated inhibition.
C7/10 cells with Wolbachia were transfected with expression vectors FLAG-empty (w/ Wolb) or FLAG-AMt2 (w/ Wolb + AMt2) for 48 hours prior to infection with SINV-nLuc at MOI of 10. Wolbachia -free cells expressing FLAG-empty (w/o Wolb) were used as a positive control. (A) Schematic of experimental workflow. (B) Viral genome replication in C7/10 cells was quantified using qRT-PCR using extracted total RNA from infected cell lysates. One-way ANOVA with Tukey’s post-hoc test of multivariate comparison. (C) Specific Infectivity Ratios of progeny viruses produced from the aforementioned infection was calculated as described earlier [1]. Briefly, infectious progeny viruses collected from supernatants 48 hours post infection were quantified using plaque assays on BHK-21 cells, while total number of progeny virus particles was quantified via qRT-PCR of viral genome copies released into the supernatant. Error bars represent standard error of mean (SEM). One-way ANOVA with Tukey’s post-hoc test of multivariate comparison, w/ Wolb vs w/ Wolb + AMt2, p = 0.0003, w/o Wolb vs w/ Wolb, p < 0.0001. (D) C7/10 mosquito cells with Wolbachia were transfected with expression vectors FLAG-empty (w/ Wolb) or FLAG-AMt2 (w/ Wolb + AMt2) for 48 hours prior to quantification of endosymbiont titer via quantitative PCR using DNA from extracted cell lysates. Error bars represent standard error of mean (SEM). Unpaired, student’s t-test, p = 0.1316, t = 1.794, df = 5.097. Statistically non-significant values are indicated by ns. (E) Progeny viruses were used to synchronously infect naïve BHK-21 cells at equivalent MOIs of 5 particles/cell. Cell lysates were collected at indicated times post infection and luciferase activity (RLU), was used as a proxy for viral replication. Two-way ANOVA with Tukey’s post-hoc test of multivariate comparison, Time: p < 0.0001, Wolbachia/AMt2: p = 0.0003, Time x Wolbachia/AMt2: p < 0.0001. (F) Approximately 105 copies (determined using qRT-PCR) each of virion encapsidated RNA extracted from the aforementioned W+, W+ AMt2 and W- viruses were transfected into naïve BHK-21 cells and infectious titer was determined by the counting the number of plaques produced after 72 hours post transfection. Numbers above bars refer to the proportion of samples that formed quantifiable plaque-forming units on BHK-21 cells. One-way ANOVA with Tukey’s post-hoc test of multivariate comparison. (G) 105 copies each of virion encapsidated RNA extracted from the W+, W+ AMt2 and W- viruses were transfected into naïve BHK-21 cells and luciferase activity (RLU) was used as a proxy for viral replication at 9 hours post-transfection. Numbers above bars refer to the proportion of samples that produced luciferase signal above background levels, indicated by the dotted line. One-way ANOVA with Tukey’s post-hoc test of multivariate comparison, w/ Wolb vs w/ Wolb + AMt2: p < 0.00001, w/o Wolb vs w/ Wolb: p < 0.0001; w/o Wolb vs w/ Wolb + AMt2: p = 0.991. For all panels error bars represent standard error of mean (SEM). *P < 0.05; **P < 0.01; ****P < 0.0001. Graphical assets were made in BioRender (https://biorender.com).