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. 2020 Oct 30;10(26):12223–12240. doi: 10.7150/thno.50992

Figure 6.

Figure 6

P38α phosphorylates the HCV core protein and promotes core oligomerization. (A) In vitro phosphorylation of the HCV core protein by p38α. The purified Flag-p38α protein from HEK293T cells was incubated with the purified GST-HCV core protein in kinase reaction buffer. After the reaction, the samples were subjected to western blotting with anti-HCV-core and anti-p38 antibodies. (B) Representative mass spectrometry identification of HCV core protein phosphorylation sites catalyzed by p38α. (C) Summary and statistics of the sites in the HCV core protein phosphorylated by p38α and their flanking sequences identified by mass spectrometry. Mass spectrometry of the GST-HCV core protein was used as a control. n.d., not detected. (D) Illustration of the mutant HCV core protein with p38α-catalyzed phosphorylation sites. (E) Impairment by phosphorylation site mutations of HCV core protein oligomerization. HEK293T cells were co-transfected with WT and mutant Flag- and HA-tagged HCV core protein expression plasmids as indicated. Cell lysates were prepared for co-IP (left panel). The fold change in (IP-GST/Input-GST)/(IP-His/Input-His) reflecting the binding efficiency of HCV core protein dimerization was measured by ImageJ software, and the rate for the WT was normalized to 1 (right panel). (F) Dimerization defect of the 8A mutant HCV core protein. Experiment was performed as described in (E), and immunoblotting was carried out (top panel). The fold-change analysis was similar to the description of (E). (G) Reduced aggregation of 8A mutant HCV core protein in HEK293T cells. EGFP-HCV core-WT or EGFP- HCV core-8A plasmids were transfected into HEK293T cells. EGFP-core-WT and EGFP-core-8A fluorescence aggregation (green) and nuclear fluorescence (blue) were quantified by ImageJ software. (H, I) Inhibitory effect of the 8A mutant core protein on HCV replication. Full-length JFH1 RNA containing the GND, WT or 8A mutant HCV core protein sequence was electroporated into Huh7.5.1 cells. The intracellular (H) and extracellular (I) HCV RNA levels on day 1 and day 3 were analyzed by qRT-PCR.