Fig. 5: PMI affects virus entry via modulation of protein glycosylation.
From: PMI-controlled mannose metabolism and glycosylation determines tissue tolerance and virus fitness
a Time-dependent monitoring of PMI gene expression after virus infection. PMI mRNA was determined by RT-qPCR in indicated virus-infected cell models (all with 1MOI) (n = 4 biological repeats). b siRNA knockdown of PMI decreased viral replication in H1N1-infected A549 cells (0.1MOI, 24hpi), SARS-CoV-2-infected Caco2 cells (0.1MOI, 24hpi), and ZIKV-infected Huh7 cells (0.1MOI, 48hpi) (n = 4 biological repeats). c The PMI inhibitor MLS0315771 inhibits the replication of a panel of viruses including H1N1-infected MDCK cells (0.01 MOI, 24hpi), SARS-CoV-2-infected VeroE6 cells (0.01 MOI, 24hpi), and ZIKV-infected Huh7 cells (0.1 MOI, 48hpi) (n = 3 biological repeats). d Schematic illustration showing two strategies of SARS-CoV-2 spike-mediated entry measurement. e PMI inhibition either by siRNA knockdown or by inhibitor MLS0315771 reduced SARS-CoV-2 pseudovirus entry in HEK293T-ACE2 cells (strategy 1) (n = 4 biological repeats). f Overexpression (O/E) of PMI rescued SARS-CoV-2 entry (strategy 2). Both WT and Omicron BA.5 pseudovirus were examined (n = 4 biological repeats). g Addition of Tunicamycin (2 µg/ml) or MLS0315771 (5 µM) to HEK293T cells during packaging of pesudotyped SARS-CoV-2 decreased its entry efficacy VeroE6-TMPRSS2 cells (strategy 2) (n = 4 biological repeats). h PMI mediates high mannose-type N-glycosidic (HHL) glycosylation of host ACE2. After SARS-CoV-2 infection (0.1MOI, 24hpi) and pull-down experiment, full spectrum of host HHL glycosylation process particularly hACE2 were analyzed by Western blot and lectin blot analyses (n = 3 biological repeats). i Overexpression of PMI increased the hACE2 glycosylation. HEK293T cells were transfected with increasing concentrations of exogenous PMI plasmids followed by SARS-CoV-2 infection and analysis of hACE2 N- glycosylation as described in (h). j The PMI inhibitor MLS0315771 decreased the hACE2 glycosylation. HEK293T cells were transfected with hACE2 constructs and treated with MLS0315771 for 24 h before analysis of hACE2 N- glycosylation as described in (h) (n = 3 biological repeats). All the results are shown as mean ± SD. Unpaired two-sided student’s T test was used for figure (b, e) (left panel). One-way ANOVA with Dunnett’s post hoc test was used for (e) (right part), (f, g, h, i, j). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and n.s. indicates non-significant. Panel (d) was created with BioRender.com.
