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. 2020 Jan 14;11:260. doi: 10.1038/s41467-019-14115-z

Fig. 5. AaVA-1 competes with Beclin-1 from the LRPPRC-mediated inhibition.

Fig. 5

a The localization of AaVA-1 in human moMØ cells. Purified AaVA-1 (5 μg/ml) was incubated with human moMØ cells at 4 °C or 37 °C. Both AaVA-1 and specific markers of the endocytosis pathways were stained, and nuclei were stained with DAPI. Scale bars, 10 μm. b, c Suppression of RhoA by the antagonist Rhosin (b) and siRNA-mediated silencing (c) restored AaVA-1-mediated ZIKV enhancement in THP-1 cells. Gene quantities were normalized against human actin (NM_001101.4). d Colocalization of AaVA-1 and LRPPRC on mitochondria in SGE-treated human moMØ cells. Purified AaVA-1 (biotin-labeled, 5 μg/ml) was incubated with human moMØ for 6 h. The cells were stained with anti-LRPPRC and anti-Tom20, as well as streptavidin Alexa-633. The nuclei were stained with DAPI. Images were examined using a Zeiss LSM 780 meta confocal microscope in multi-track mode. Scale bars, 10 μm. e Purification of a recombinant LRPPRC protein in 293F cells. The protein was detected by staining with Coomassie blue in an SDS–PAGE gel. f AaVA-1 directly binds LRPPRC. This interaction was detected by ELISA. g AaVA-1 impaired the binding between LRPPRC and Beclin-1. SGE or ΔAaVA-1-SGE was incubated with the lysates from Beclin-1 (Myc tag)-expressing cells to investigate protein interactions. The protein complex was pulled down with anti-Myc antibodies. h Treatment of SGE dissociated Beclin-1 from LRPPRC on mitochondria. The human moMØ cells were infected ZIKV (0.1 MOI) in combination with either ΔAaVA-1-SGE or WT-SGE. After 6 h of incubation, the cells were stained with anti-Beclin-1, anti-LRPPRC, and anti-Tom20. The nuclei were stained with DAPI. Images were examined using a Zeiss LSM 780 meta confocal microscope. Scale bars, 10 μm. The colocalization of Beclin-1 and LRPPRC on mitochondria was quantified in the white rectangle (right panel). b, c, f The data are presented as the mean ± SEM. A nonparametric Mann–Whitney test was used for the statistical analysis. *p < 0.05. n = 4 independent samples. The experiments were repeated two times (a, e–g) or three times (b–d, h) with the similar results. Source data are provided as a Source Data file.