Cytomegalovirus-mediated activation of pyrimidine biosynthesis drives UDP-sugar synthesis to support viral protein glycosylation

Abstract

Human cytomegalovirus (HCMV) induces numerous changes to the host metabolic network that are critical for high-titer viral replication. We find that HCMV infection substantially induces de novo pyrimidine biosynthetic flux. This activation is important for HCMV replication because inhibition of pyrimidine biosynthetic enzymes substantially decreases the production of infectious virus, which can be rescued through medium supplementation with pyrimidine biosynthetic intermediates. Metabolomic analysis revealed that pyrimidine biosynthetic inhibition considerably reduces the levels of various UDP-sugar metabolites in HCMV-infected, but not mock-infected, cells. Further, UDP-sugar biosynthesis, which provides the sugar substrates required for glycosylation reactions, was found to be induced during HCMV infection. Pyrimidine biosynthetic inhibition also attenuated the glycosylation of the envelope glycoprotein B (gB). Both glycosylation of gB and viral growth were restored by medium supplementation with either UDP-sugar metabolites or pyrimidine precursors. These results indicate that HCMV drives de novo-synthesized pyrimidines to UDP-sugar biosynthesis to support virion protein glycosylation. The importance of this link between pyrimidine biosynthesis and UDP-sugars appears to be partially shared among diverse virus families, because UDP-sugar metabolites rescued the growth attenuation associated with pyrimidine biosynthetic inhibition during influenza A and vesicular stomatitis virus infection, but not murine hepatitis virus infection. In total, our results indicate that viruses can specifically modulate pyrimidine metabolic flux to provide the glycosyl subunits required for protein glycosylation and production of high titers of infectious progeny.

Keywords: UDP–sugar; cytomegalovirus; glycosylation; metabolism; pyrimidine.

Fig. 1.

HCMV infection induces host de novo pyrimidine biosynthesis. (A) MRC5 cells were mock- or HCMV-infected (MOI = 3.0) and harvested at 48 hpi for LC-MS/MS–based measurement of UTP concentrations (mean ± SEM, n = 6). *P < 0.05. (B) Schematic of UTP labeling from U–¹³C-glucose. ¹³C, red; ¹²C, black; N, blue; O, gray. Certain atoms were omitted for clarity. (C) MRC5 cells infected as in A were labeled at 48 hpi for 0, 1, 2, or 6 h with U–¹³C-glucose and analyzed by LC-MS/MS to measure the fraction of ¹²C–UTP present (¹²C–UTP/total UTP). Values are representative of four biological replicates. (D) UTP flux estimates based on the data in A and C were calculated as indicated in SI Materials and Methods (mean ± SEM, n = 4).

Fig. 2.

Impact of pyrimidine biosynthetic inhibition on HCMV replication. (A) Schematic of de novo pyrimidine biosynthesis. PRPS1, phosphoribosyl pyrophosphate synthetase 1; UMPS, UMP synthetase; UK, uridine kinase. (B and C) MRC5 cells were HCMV-infected (MOI = 3.0), treated with DMSO, PALA (B), or Leflunomide (C) at the indicated concentrations, and harvested at 96 hpi for analysis of viral progeny (mean ± SEM, n = 4). *P < 0.05. (D) Cells were infected as in B, treated with PALA (100 µM) and uridine at 0, 5, or 50 µM as indicated, and harvested at 96 hpi for analysis of viral progeny (mean ± SEM, n = 4). *P < 0.05. (E) Cells were infected as in B, treated with Leflunomide (100 µM) and uridine at 0, 0.5, or 1 mM as indicated, and harvested at 96 hpi for analysis of viral progeny production (mean ± SEM, n = 4). (F) MRC5 cells were treated with 100 µM PALA, 100 µM Leflunomide, DMSO, or 1% ethanol (+ control) for 24 h. The toxicity of PALA and Leflunomide was measured by using a live/dead assay, in which green fluorescence indicates the presence of esterase activity associated with viable cells, and red fluorescence indicates a loss of cellular membrane integrity associated with cell death.

Fig. 3.

siRNA-mediated targeting of CAD attenuates HCMV replication. (A) MRC5 cells were mock- or HCMV-infected (MOI = 3.0) and harvested at 6, 24, and 48 hpi for Western analysis of CAD, CAD phospho–Thr-456 (P-CAD), or α-tubulin abundance (data are from a representative experiment of four). (B) Subconfluent MRC5 cells were transfected with a CAD-specific or control siRNA. At 24 h after transfection, cells were serum-starved for 24 h and HCMV-infected (MOI = 3.0), before Western analysis at 24 and 96 hpi for the indicated antibodies (data are from a representative experiment of two). (C) Cells transfected and infected as in B were harvested for viral titration at 96 hpi (mean ± SEM, n = 4). *P < 0.05.

Fig. 4.

The impact of pyrimidine biosynthetic inhibition on HCMV infection. MRC5 cells were HCMV-infected (MOI = 3.0) and treated with DMSO, PALA (100 µM), or 50 µM uridine as indicated. (A) Cells were harvested at 24 and 72 hpi for Western analysis of IE1, α-tubulin, U_L26, U_L44, or pp28 abundance. Data are from a representative experiment of four. (B) Viral DNA was extracted from cells at 72 hpi and analyzed by quantitative PCR (qPCR) for viral DNA abundance (mean ± SEM, n = 3). (C) Cells were harvested at 48 hpi for metabolite analysis by LC-MS/MS. LC-MS/MS–derived ion counts were normalized to mock-infected samples and plotted as a heat map relative to mock-infected cells (n = 6).

Fig. 5.

HCMV increases UDP–sugar biosynthesis and pool sizes. (A and B) MRC5 cells were mock- or HCMV-infected (MOI = 3.0) and treated with DMSO or 100 µM PALA. At 48 hpi, cell were harvested and processed for LC-MS/MS analysis to quantify UDP–glucose (A) or UDP–GlcNAc (B) (means ± SEM, n = 6). *P < 0.05. (C) Schematic of ¹³C–glucose labeling of UDP–sugars (¹³C–glucose, red). Some atoms were omitted for clarity. (D and E) MRC5 cells were infected as in A. At 48 hpi, cells were labeled with ¹³C–glucose for 0, 1, 2, or 6 h and processed for LC-MS/MS to measure the fraction of ¹²C–UDP–glucose (C) or ¹²C–UDP–GlcNAc (D). Flux estimates were calculated as indicated in SI Materials and Methods (mean ± SEM, n = 4).

Fig. 6.

Pyrimidine biosynthesis is important for glycosylation of gB. (A and B) MRC5 cells were HCMV-infected (MOI = 3.0), treated with DMSO, PALA (100 µM), or uridine (50 µM) as indicated, and harvested at 72 hpi. The abundances of gB and GAPDH were measured by Western analysis. The data are from a representative experiment of three. The quantification of percent mature gB from these experiments is shown in B (glycosylated 116 -kDa gB/total gB × 100; n = 3). (C) Cells were infected as in A, harvested at 24, 48, and 72 hpi, and processed for mRNA analysis by qPCR using gB-specific primers and normalization to GAPDH-specific primer amplification (mean ± SEM, n = 3). (D) MRC5 cells were infected as in A, treated with DMSO or 100 µM PALA, and harvested at 72 hpi. Samples in Right were treated after harvest with PNGase as indicated. The abundance of CD44S was measured by Western analysis. (E) MRC5 cells were infected with HCMV (MOI = 3.0), treated with DMSO or tunicamycin (10 µg/mL), and harvested at 96 hpi for assessment of viral titers (mean ± SE, n = 2 biological and 4 technical replicates). (F) MRC5 cells were HCMV-infected as in A and treated with PALA (100 µM) in the presence of uridine (50 µM), UDP–glucose (5 µM), or UDP–GlcNAc (100 μM) as indicated. Cells were harvested at 72 and 144 dpi and processed for Western analysis of gB and GAPDH abundance (n = 3). (G) Cells were HCMV-infected (MOI = 3.0), treated with DMSO, 100 µM PALA, UDP–glucose, or UDP–GlcNAc as indicated, and harvested at 96 hpi for the quantification of viral progeny production (mean ± SEM, n = 4). *P < 0.05. (H). Cells were HCMV-infected (MOI = 1.0) and treated with DMSO or 100 µM PALA. Supernatant virions were partially purified at 144 hpi and analyzed for associated viral genomes by real-time PCR or pfus by plaque assay. The pfu/genome ratio is indicated relative to wild-type infection (mean ± SEM).

Fig. 7.

The link between pyrimidine and UDP–sugar metabolism is important for the replication of evolutionarily diverse viruses. (A) MRC5 cells were infected with HCMV (MOI = 3.0) and treated with DMSO, A3 (2.5 µM), orotate (2.5 mM), uridine (50 µM), or UDP–GlcNAc (100 µM) as indicated. Cells were harvested at 96 hpi for analysis of the production of viral progeny (mean ± SEM). (B) A549 cells were infected with influenza A/Puerto Rico/8/34 NS1–GFP (MOI = 0.005). Cells were treated with A3 (2.5 μM) in the presence of orotate (2.5 mM) or UDP–GlcNAc (100 μM) as indicated. At 48 hpi, the supernatants were collected for viral titration in MDCK cells (mean ± SEM). (C) A549 cells were infected with rVSV–GFP (MOI = 0.0001). Cells were treated with A3 (2.5 μM) in the presence of orotate (2.5 mM), or UDP–GlcNAc (100 μM) as indicated. At 48 hpi, the supernatants were collected for viral titration in Vero cells (mean ± SEM). (D) 293T–mCEACAM1 cells were infected with rMHV–GFP (MOI = 0.0005). Cells were treated with A3 (2.5 μM) in the presence of orotate (2.5 mM) or UDP–GlcNAc (100 μM) as indicated. At 48 hpi, the supernatants were collected for viral titration in L929 cells (mean ± SEM).