Figure 3. Induction of autophagy and endoplasmic reticulum stress in chikungunya virus infected U-87 MG cells.

(a) Punctae formation indicating the autophagosomes. U-87 MG cells stably transfected with a GFP-tagged light chain 3 (LC3) expression-construct, and cells transiently transfected with GFP expressing plasmid alone were infected with CHIKV, and observed 24h post-infection. Distinct punctae formation by LC3 recruitment to autophagosomes is visible in the infected cells expressing GFP-LC3. (b) Quantitation of the autophagy response. The average number of puncta/field in infected and uninfected cells is given. Values are Mean ±SD from four different microscopic fields and two independent experiments. ‘**’ indicates significance level p<0.005 (c) XBP-1 mRNA splicing indicating ER stress in infected cells. Total RNA was isolated from infected and uninfected cells at different time points and subjected to reverse-transcription PCR and agarose gel electrophoresis as described in the methods. The splice variant of XBP-1 (263bp) along with the original mRNA (289bp) is detectable in CHIKV infected cells from as early as 12h post-infection, but is more pronounced at 72h post infection in cells infected at an MOI of 10.Amplification of β-actin mRNA serves as the loading control. (d) eIF2α phosphorylation in CHIKV infected U-87 MG cells. Total cell lysate from the infected and uninfected cells was isolated 24h, 48 and 72h p.i. 50µg of total protein was subjected to SDS-PAGE and western blot as described in the methods using anti- eIF2α and anti-phopho-eIF2α antibodies. The band intensities of phopho-eIF2α and eIF2α expression, determined from densitometric analysis, were initially normalised independently to that of corresponding β-Actin, and the ratio of phopho- eIF2α to eIF2α expression was calculated. The fold change in expression was calculated relative to the protein expression in uninfected controls at the respective time points. Values are Mean ± SD from three independent experiments.