Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating
Fig 2
Rotavirus entry and infection depend on Rab5 and Rab7.
(A) MA104 cells were transfected with either scrambled siRNA or siRNAs against Rab5 or Rab7. Afterwards, cells were exposed to Alexa 594-labeled DS-1 (approximately 595 particles/cell) or NCDV (approximately 790 particles/cell) for 30 min at 4°C. Unbound virus was washed off and the cells were shifted to 37°C for 90 min. Cells were then fixed and processed for confocal microscopy. Cells treated in parallel were analyzed by Western blot analysis to ensure effective knockdown of protein levels. (B-E) MA104 cells transfected with siRNAs against Rab5 or Rab7 were infected with the DS-1 and NCDV strains (MOI = 10 FFU/cell). The total viral RNA (B), antigen-positive cells (using anti-RVA VP6 Mab) (C), and VP6 protein (D) were determined by real-time RT-PCR, immunofluorescence, and Western blot analyses, respectively. GAPDH was used as a loading control. The intensity of pPI3K, pAkt, and pERK relative to GAPDH was determined by densitometric analysis and is indicated above each lane. (E) The virus titer was determined by cell culture immunofluorescence assay using cell lysates produced by 3 cycles of freezing and thawing, and is expressed as FFU. All experiments were performed in triplicate and panels A and C show a representative set of results. Data are presented as means ± standard error of the mean from three independent experiments. Differences were evaluated using the One-Way ANOVA. *p<0.05; **p<0.001; ***p<0.0001. The scale bars in panels A and C correspond to 20 μm.