Influenza A Virus (H1N1) Infection Induces Glycolysis to Facilitate Viral Replication

Abstract

Viruses depend on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. In this study, we observed that influenza A virus (H1N1), a single-stranded, negative-sense RNA virus with an eight-segmented genome, enhanced glycolysis both in mouse lung tissues and in human lung epithelial (A549) cells. In detail, the expression of hexokinase 2 (HK2), the first enzyme in glycolysis, was upregulated in H1N1-infected A549 cells, and the expression of pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase kinase 3 (PDK3) was upregulated in H1N1-infected mouse lung tissues. Pharmacologically inhibiting the glycolytic pathway or targeting hypoxia-inducible factor 1 (HIF-1), the central transcriptional factor critical for glycolysis, significantly reduced H1N1 replication, revealing a requirement for glycolysis during H1N1 infection. In addition, pharmacologically enhancing the glycolytic pathway further promoted H1N1 replication. Furthermore, the change of H1N1 replication upon glycolysis inhibition or enhancement was independent of interferon signaling. Taken together, these findings suggest that influenza A virus induces the glycolytic pathway and thus facilitates efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat influenza A virus infection in the future.

Keywords: Glycolysis; H1N1; Hypoxia-inducible factor 1 (HIF-1); Interferon; Replication.

Fig. 1

H1N1 infection induces glycolysis in vivo and in vitro. A A549 cells were mock infected or infected with H1N1 at an MOI of 1, and cells were harvested at 8, 16, and 24 h p.i.. The expression of HK2, PKM2, and PDK3 was analyzed by Western blotting. B A549 cells were mock infected or infected with H1N1 at an MOI of 1. Cells were harvested at 24 h p.i., and intracellular ATP levels were measured. C–E Mice were intranasally administered H1N1 (A/PR/8/34) 800 PFU/mouse or mock infected as controls (n = 3 mice/group). At 3 d p.i., the expression of HK2, PKM2, and PDK3 in mouse lung tissues was measured by western blotting (C) (the lower bands in PDK3 are nonspecific). Serum lactate (D) and lactate in lung tissue homogenate (E) were measured by a lactate assay kit. M, mock infection. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2

Glycolytic inhibitors impair H1N1 replication and alleviate virus-induced cell injury. A Schematic overview of the glucose metabolism and the functional targets of glycolytic inhibitors (2DG, oxamate, and DCA) and enhancer (PS48) used in this study. B–L A549 cells were infected with H1N1 at an MOI of 1, with or without glycolytic inhibitors treatment at the same time. B–D Cells were harvested at 24 h p.i., and intracellular NP levels were measured by Western blotting. E–G Cells were harvested at 24 h p.i., and intracellular viral RNA (M) levels were measured by qRT-PCR. β-actin expression was used as an internal control. H–J Cell supernatant were harvested at 24 h p.i., and viral titer levels were measured by plaque forming unit assay. K, L The morphological changes of A549 cells at 24 h p.i. under a phase contrast microscope were shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

H1N1 replication is impaired by knocking down HIF-1 pathway. A Cell viability of shcontrol and shHIF-1α A549 cells was measured by a CCK-8 assay. B, C shcontrol and shHIF-1α A549 cells were infected with H1N1 at an MOI of 1, and cells and supernatants were harvested at 24 h p.i.. HIF-1α mRNA level was measured by qRT-PCR (B). Lactate in cell supernatant was measured by a lactate assay kit (C). D–F shcontrol and shHIF-1α A549 cells were mock infected or infected with H1N1 at an MOI of 1, and cells and supernatant were harvested at 24 h p.i.. The expression of HIF-1α and NP was analyzed by western blotting (D). Intracellular viral RNA (M gene) in infected groups was measured by qRT-PCR (E), β-actin expression was used as an internal control. Virus titer in cell supernatant in infected groups was measured by plaque forming unit assay (F). G Cell viability of shcontrol and shHIF-1β A549 cells was measured by a CCK-8 assay. H shcontrol and shHIF-1β A549 cells were mock infected or infected with H1N1 at an MOI of 1, and cells were harvested at 24 h p.i.. The expression of HIF-1β and NP was analyzed by western blotting. ns, not significant. **P < 0.01, ***P < 0.001.

Fig. 4

Pharmacologically enhancing the glycolytic pathway further promotes H1N1 replication. A A549 cells were infected with H1N1 at an MOI of 1, with or without PS48 (5 or 10 mmol/L) treatment at the same time. Cells were harvested at 24 h p.i., and intracellular NP level was measured by Western blotting. B, C A549 cells were infected with H1N1 at an MOI of 1, with or without PS48 (10 mmol/L) treatment at the same time. Cells were harvested at 24 h p.i., and intracellular viral RNA (M) was measured by qRT-PCR (B), β-actin expression was used as an internal control. Cell supernatants were harvested at 24 h p.i., and viral titer levels were measured by plaque forming unit assay (C). D A549 cells were infected with H1N1 at an MOI of 1, with or without PS48 (10 mmol/L) treatment at the same time. The morphological changes of A549 cells at 24 h p.i. under a phase contrast microscope were shown. **P < 0.01, ***P < 0.001.

Fig. 5

The effect of glycolysis inhibitors or enhancer on interferon induction. A–D A549 cells were mock infected or infected with H1N1 at an MOI of 1, with or without 50 mmol/L 2DG or 100 mmol/L oxamate treatment at the same time. Cells were harvested at 24 h p.i., and IFN-α (A), IFITM1 (B), ISG56 (C), and MxA (D) mRNA levels were measured by qRT-PCR. β-actin expression was used as an internal control. E–H A549 cells were mock infected or infected with H1N1 at an MOI of 1, with or without 10 mmol/L PS48 treatment at the same time. Cells were harvested at 24 h p.i., and IFN-α (E), IFITM1 (F), ISG56 (G), and MxA (H) mRNA levels were measured by qRT-PCR. β-actin expression was used as an internal control. **P < 0.01, ***P < 0.001.

Fig. 6

Schematic of the proposed model for glycolysis involvement in H1N1 replication. H1N1 activates HIF-1 pathway and then induces glycolysis to promote efficient viral replication.