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. 2020 Mar 18;12(3):328. doi: 10.3390/v12030328

Figure 1.

Figure 1

The tick-borne flavivirus Langat virus (LGTV) infection activates host unfolded protein response (UPR) and autophagy pathways. (A) LGTV infection (MOI = 10) in HEK293T cells generated an increase in binding immunoglobulin protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) expression, but not activating transcription factor 6 (ATF6) cleavage, suggesting active protein kinase R-like endoplasmic reticulum kinase PERK signaling. Tunicamycin (10 µg/mL) control included for BiP, ATF6, and CHOP. LGTV E expression increased throughout infection time course. (B) Basal autophagic flux of HEK293T cells measured by microtubule-associated protein light chain 3 (LC3)-I and LC3-II expression. Earle’s balanced salt solution (EBSS) starvation increases flux, depleting LC3-I and LC3-II. Blocking autolysosomal degradation with bafilomycin A1 (BafA1) increased the abundance of LC3-II, more so under EBSS starvation. Total signal normalized to mock control and reported below bands. (C) LGTV infection (MOI = 10) alters autophagic flux in HEK293T cells 24 hpi (hours post infection). Infected samples had higher overall LC3 expression, but the majority was unlipidated LC3-I, suggesting inhibition of LC3 lipidation. (D) Representative images of HEK293T cells treated with EBSS, BafA1, or LGTV infection. DAPI represented in blue, LC3 in green, and LGTV NS3 in red. Images acquired at 63x magnification. (E) Average autophagosomes per cell identified by LC3 fluorescent signal normalized to number of nuclei. Very few autophagosomes recorded in non-BafA1-treated samples.