Fig. 5: CREB1 is recruited to viral inclusion bodies upon trVLPs or EBOV infection.

a, b WT and AKIP1^−/− (KO) HepG2 cells were transfected with EBOV minigenome (p0) with or without 1 μM 666-15 or 10 μM H89 for 48 h and then immunostained with anti-VP35 (green) and anti-CREB1 (red) antibodies (a). The cytoplasmic/nuclear distribution of CREB1 in (a) was analyzed by ImageJ software (b). Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The ratio of CREB1 distribution in at least ten cells from two independent assays is presented as the mean ± s.e.m. (n = 10; ***P < 0.001). c, d HepG2 cells infected with live EBOV (MOI = 10) for 72 h were immunostained with anti-CREB1 (red) and anti-VP35 (green) antibodies. Arrows: CREB1 in VIBs. The ratio of cytoplasm/nuclei distributed CREB1 in (c) was analyzed by ImageJ software (d). The ratio of CREB1 distribution in at least ten cells from two independent assays is presented as the mean ± s.e.m. (n = 10; ***P < 0.001). Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. e Lysates of HepG2 cells transfected with the EBOV minigenome (p0) in the presence (upper panel) or absence (lower panel) of the T7 RNA polymerase expression plasmid pCAGGS-T7 were separated on a 25 to 60% (W/V) sucrose gradient. Fractions were collected and analyzed by immunoblotting. f The seventh and eighth fractions from (e, upper panel) were combined and subjected to immunoprecipitation and immunoblotting analysis. g WT and AKIP1^−/− (KO) HepG2 cells were transfected with the EBOV minigenome (p0). Cell lysates were subjected to anti-CREB1 (or IgG as a control) immunoprecipitation, and the coprecipitated viral RNA corresponding to 3Le or 5Tr was quantified by qRT-PCR. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The mean ± s.e.m. from three independent assays is presented (ns not significant; **P < 0.01; ***P < 0.001).