Viruses have evolved sophisticated strategies to exploit host cell function for their benefit. Here we show that under physiologically normal oxygen levels (normoxia) vaccinia virus (VACV) infection leads to a rapid stabilization of hypoxia-inducible factor (HIF)-1α, its translocation into the nucleus and the activation of HIF-responsive genes, such as vascular endothelial growth factor (VEGF), glucose transporter-1, and pyruvate dehydrogenase kinase-1. HIF-1α stabilization is mediated by VACV protein C16 that binds the human oxygen sensing enzyme prolyl-hydroxylase domain containing protein (PHD)2 and thereby inhibits PHD2-dependent hydroxylation of HIF-1α. The binding between C16 and PHD2 is direct and specific, and ectopic expression of C16 alone induces transcription of HIF-1α responsive genes. Conversely, a VACV strain lacking the gene for C16, C16L, is unable to induce HIF-1α stabilization. Interestingly, the N-terminal region of C16 is predicted to have a PHD2-like structural fold but lacks the catalytic active site residues of PHDs. The induction of a hypoxic response by VACV is reminiscent of the biochemical consequences of solid tumor formation, and illustrates a poxvirus strategy for manipulation of cellular gene expression and biochemistry.Orthopoxvirus | variola virus | Warburg effect | glucose metabolism V accinia virus (VACV) is the live vaccine used against smallpox, an extinct human disease caused by variola virus (VARV) (1). VACV and VARV belong to the Orthopoxvirus genus of the Poxviridae, and each have large dsDNA genomes and replicate in the cell cytoplasm (2). VACV can be engineered to express foreign genes and these recombinant viruses have potential as live vaccines against other diseases (3) and are useful laboratory tools, for instance in identifying the target antigens of CD8+ cytotoxic T lymphocytes (4, 5). VACV encodes many genes that are nonessential for virus replication but are important in vivo for defense against the host innate immune system, such as the VACV steroid biosynthetic enzyme 3-β-hydroxysteroid dehydrogenase (6, 7), or for modulation of host cell biochemistry, such as the VACV enzymes thymidine kinase (8), thymidylate kinase (9), and ribonuceotide reductase (10).VACV strain Western Reserve (WR) gene C16L encodes a 37-kDa intracellular protein that contributes to virulence by an unknown mechanism (11). C16 is nonessential for VACV replication, yet orthologs of C16 are conserved in several poxviruses, suggesting an important function (11). VACV also encodes a related protein, C4, with 43% amino acid identity to C16 and that inhibits activation of NF-κB (12).Here a direct interaction is shown between the N-terminal region of C16 and the human oxygen sensor prolyl-hydroxylase domain containing protein (PHD)2. During normoxia, PHD2 is the major enzyme that hydroxylates hypoxia-inducible factor (HIF)-1α on either of two proline residues within the oxygendependent degradation domain (ODD), P402 and P564 (13, 14). After hydroxylation, HIF-1α is ubiquitinated by the von Hippe...