Divergent Effects of Human Cytomegalovirus and Herpes Simplex Virus-1 on Cellular Metabolism
Figure 7
HSV-1 replication is inhibited by reducing flux from glucose toward pyrimidine nucleotide synthesis.
(A) Schematic diagram of glucose flux to pyrimidine nucleotide biosynthesis. Red lines mark siRNA-targeted reactions catalyzed by pyruvate carboxylase (PC) and aspartate transaminase 2 (GOT2). (OAA: oxaloacetate, AKG: oxoglutarate, Gln: glutamine). (B) RNA interference knockdown of pyruvate carboxylase (marked by arrow) in MRC5 cells. Cells were transfected with non-targeting siRNAs (NT) or siRNAs targeting pyruvate carboxylase (PC) and harvested at indicated time points after transfection. Pyruvate carboxylase levels in the cells were detected by western blot using specific antibodies. Beta-actin was employed as a loading control. (C) Buildup of 13C3-labeled malate after switching MRC5 cells to uniformly 13C-labeled glucose medium for 2 hours at 10 hpi of HSV-1 (F) infection. The cells have been transfected with a universal non-targeting siRNA (NT) or an siRNA targeting pyruvate carboxylase (PC) 120 h prior to infection (significance: p = 0.007). Symbols indicate experimental data points ±1 s.d.; n = 3; values are given in arbitrary units. (D) Production of infectious HSV-1 (F) and HCMV (AD169) virions in cells transfected with siRNAs against pyruvate carboxylase (PC), aspartate transaminase 2 (GOT2), or a universal negative control (NT). The transfection and infection of MRC5 cells were performed as described in Materials and Methods. Values are expressed relative to non-targeting control (±1 s.d.; n = 3). Conditions resulting in significantly altered virus production (p≤0.05) compared to treatment with the universal negative control are marked with a star.