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

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.

The zinc-binding nuclear protein HIPP3 acts as an upstream regulator of the salicylate-dependent plant immunity pathway and of flowering time in Arabidopsis thaliana.

Biotic and abiotic stress responses of plants are linked to developmental programs. Proteins involved in different signaling pathways are the molecular basis of this concerted interplay. In our study, we show that Arabidopsis thaliana HEAVY METAL-ASSOCIATED ISOPRENYLATED PLANT PROTEIN3 (HIPP3; At5g60800) acts as an upstream regulator of stress- and development-related regulatory networks. Localization, metal-binding and stress-responsive gene expression of HIPP3 were analyzed via microscopy, protein and inductively coupled plasma (ICP)-MS analyses and quantitative real-time PCR. In addition, transcriptome and phenotype analyses of plants overexpressing HIPP3 were used to unravel its function. Our data show that HIPP3 is a nuclear, zinc-binding protein. It is repressed during drought stress and abscisic acid (ABA) treatment and, similar to other pathogen-related genes, is induced after infection with Pseudomonas syringae pv. tomato. HIPP3 overexpression affects the regulation of > 400 genes. Strikingly, most of these genes are involved in pathogen response, especially in the salicylate pathway. In addition, many genes of abiotic stress responses and seed and flower development are affected by HIPP3 overexpression. Plants overexpressing HIPP3 show delayed flowering. We conclude that HIPP3 acts via its bound zinc as an upstream regulator of the salicylate-dependent pathway of pathogen response and is also involved in abiotic stress responses and seed and flower development.