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Regulation and function of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 in innate immune response to viral infection

  • Author(s): Gold, Sarah
  • Advisor(s): Cheng, Genhong
  • et al.

In response to viral infection innate immune cells dramatically increase expression of cy- tokines, interferons, and antiviral genes in a mobilization effort that can include major metabolic reprogramming. How these cells control metabolism and how metabolic pathways themselves serve as generators of immune signals are major questions in immunometabolism. PFKFB3 encodes the metabolic regulatory enzyme phosphofructokinase-2/fructose 2,6- bis- phosphatase (PFK-2/FBPase), is induced in response to a variety of stress and inflammatory signals through distinct pathways and has recently been identified as an interferon stimulated gene (ISG). However, the role of interferon in the regulation of PFKFB3 expression has not been clearly established, and PFKFB3’s potential functions in antiviral response remain al- most entirely unexplored in the literature. We demonstrate here that PFKFB3 is induced in response to either viral infection or viral nucleic acid analogs in a type I interferon-dependent manner. We also found that PFKFB3 induction in response to LPS stimulation similarly requires type I interferon signaling, and that it is potentiated by IL-10 but not Hif1αin nor- moxia. PFKFB3 has two major isoforms, A-C-G and A-C-D, the second of which has an alternative C-terminal peptide sequence due to inclusion of a frameshift-generating 23 nt exon D at the 3’ end. We designed qPCR primers to quantify induction of PFKFB3’s C-D and C-G exon-exon junctions and found that BMDM, MDM and A549 cells all responded to either viral infection or viral nucleic acid analogs by preferentially inducing PFKFB3 A-C-G but not A-C-D or by switching from A-C-D to A-C-G expression. Overexpression of A-C-G or the truncation mutant A-C, but not A-C-D, inhibited VSV infection in 293T cells; this viral inhibitory effect also depended on PFKFB3 kinase activity and could be conferred via conditioned media but did not depend on an intact nuclear localization signal. Metabolic footprinting by LC/MS indicated increased choline and phosphocholine in the media of 293T cells overexpressing PFKFB3 A-C-G, and addition of choline to cell culture media inhibited VSV infection in a dose-dependent manner. Metabolic profiling by LC/MS of 293T cells overexpressing PFKFB3 isoforms or the A-C truncation mutant demonstrated the expected, increased glycolytic flux phenotype in the A-C-G and A-C but not A-C-D condition, indi- cating a disabling role for exon D consistent with its lack of viral inhibition. CDP-choline was unexpectedly elevated in tandem with increased glycolysis, and homocysteine levels were increased nearly 100 fold, indicating a severe inhibition or dysfunction of one-carbon metabolism in cells expressing either the fully-active A-C-G isoform or truncation mutant. Finally, we found that PFKFB3 directly binds to and co-precipitates with the outer mito- chondrial membrane protein and glycolytic potentiator hexokinase 2 (HK2), in yet another localization for PFKFB3, which is also found in the nucleus and in association with the outer lysosomal membrane.

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