Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA

Abstract

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell’s metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme’s function from transcription to replication.

D-mannose suppresses macrophage release of extracellular vesicles and ameliorates type 2 diabetes

“…Importantly, we revealed that CD36-OE reversed suppressive effects of D-mannose on PA-induced mEV release (Figure 6L and M), indicating that CD36 was the key to mediating D-mannose effects. To further investigate how D-mannose may regulate CD36 expression in macrophages, we applied chemical inhibitors of MPI and protein N-glycosylation to respectively block each of the two metabolic cascades of D-mannose 16,26,27 . We have also tested metabolites of D- mannose along the cascades, mannose-6-phosphate (M6P), mannose-1-phosphate (M1P) and fructose-6-phosphate (F6P), by treating PA-conditioned macrophages instead of D- mannose.…”

“…After internalization, D-mannose is phosphorylated by hexokinase to produce M6P, which undergo two major metabolic fates: a minor fraction (∼5%) is isomerized to M1P by PMM2 to be used in glycosylation pathways; the large majority (∼95%) is converted to F6P by MPI to be catabolized into glycolysis 16 . Therefore, to investigate the D-mannose metabolic effect, Tunicamycin (MedChemExpress, China) was used to block protein N-glycosylation 26 at 100 ng/mL for 48 h, and MLS0315771 (MedChemExpress, China) was applied to suppress MPI 27 at 5 mM for 48 h, the dose and duration were selected based on our preliminary dose-effect tests on macrophage viability (data not shown). Furthermore, to test the effects of D-mannose metabolites, M6P (Aladdin, China) was used at 25 mM (equal to the D-mannose concentration) for 48 h, M1P (Aladdin, China) was used at 1.25 mM (5% of the D-mannose dose) for 48 h, and F6P (Yingxinbio, China) was used at 23.75 mM (95% of the D-mannose dose) for 48 h. PA (Kunchuang Biotechnology, China) was added at 500 μM for 24 h based on our preliminary dose-effect tests on macrophage viability (data not shown) and previous research on regulating RAW 264.7 cells and EV release 28,29 .…”