Cytomegalovirus-Mediated Activation of Pyrimidine Biosynthesis Drives UDP–Sugar Synthesis to Support Viral Protein Glycosylation

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

Stefanie Renee DeVitoa, Emilio Ortiz-Riañob, Luis Martínez-Sobridob, and Joshua Mungera,1

Departments of aBiochemistry and Biophysics and bMicrobiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642

Edited by Thomas Shenk, Princeton University, Princeton, NJ, and approved November 13, 2014 (received for review August 18, 2014)

Human cytomegalovirus (HCMV) induces numerous changes to We have previously demonstrated that HCMV infection is the host metabolic network that are critical for high-titer viral responsible for numerous changes to the host cell metabolic replication. We find that HCMV infection substantially induces de network (14, 15). These changes include induction of many novo pyrimidine biosynthetic flux. This activation is important for branches of central carbon metabolism, including glycolysis and HCMV replication because inhibition of pyrimidine biosynthetic the tricarboxylic acid cycle (15). Additionally, HCMV infection enzymes substantially decreases the production of infectious results in an expansion of pyrimidine metabolite pools (14). De virus, which can be rescued through medium supplementation novo pyrimidine biosynthesis is the main source of pyrimidines with pyrimidine biosynthetic intermediates. Metabolomic analysis during cellular replication, whereas the pyrimidine salvage revealed that pyrimidine biosynthetic inhibition considerably pathway provides a smaller amount of pyrimidines to quiescent reduces the levels of various UDP–sugar metabolites in HCMV- cells and cells in G0 (16, 17). The de novo pathway is primarily infected, but not mock-infected, cells. Further, UDP–sugar biosyn- regulated through its rate-limiting enzyme, carbamoyl phosphate thesis, which provides the sugar substrates required for glycosyl- synthetase–aspartate transcarbamylase–dihydroorotase (CAD). ation reactions, was found to be induced during HCMV infection. CAD catalyzes the first three steps of the pathway, including the Pyrimidine biosynthetic inhibition also attenuated the glycosyla- first committed step (18, 19). CAD is a ∼250-kDa protein that

tion of the envelope glycoprotein B (gB). Both glycosylation of gB possesses three enzymatic activities and multimerizes in vivo (20, MICROBIOLOGY and viral growth were restored by medium supplementation with 21). CAD is heavily regulated by posttranslational modifications, either UDP–sugar metabolites or pyrimidine precursors. These which alter the sensitivity by which CAD is allosterically acti- results indicate that HCMV drives de novo-synthesized pyrimidines vated and inhibited and, in turn, induce or inhibit de novo py- to UDP–sugar biosynthesis to support virion protein glycosyla- rimidine biosynthesis, respectively (16, 22). tion. The importance of this link between pyrimidine biosynthe- De novo pyrimidine biosynthesis also provides pyrimidines for sis and UDP–sugars appears to be partially shared among diverse synthesis of UDP–sugars, which are widely used as substrates to virus families, because UDP–sugar metabolites rescued the growth feed cellular glycosylation reactions. The UDP–sugars, including attenuation associated with pyrimidine biosynthetic inhibition UDP–glucose and UDP–N-acetyl-glucosamine (UDP–GlcNAc), during influenza A and vesicular stomatitis virus infection, but along with GDP–mannose, are required for building the neces- not murine hepatitis virus infection. In total, our results indicate sary precursor oligosaccharide structure that forms immediately that viruses can specifically modulate pyrimidine metabolic flux to before N-linked glycosylation. Additionally, UDP–GlcNAc, but provide the glycosyl subunits required for protein glycosylation not UDP–glucose, is required for the formation of O-linked and production of high titers of infectious progeny. glycosyl groups (23). The HCMV viral envelope contains a number of glycoproteins that are critical for HCMV replication cytomegalovirus | pyrimidine | glycosylation | metabolism | UDP–sugar Significance variety of evolutionarily divergent viruses have been shown – Ato activate specific metabolic activities upon infection (1 3). Viruses use the host cell to provide the energy and molecular These virally induced metabolic activities can be targeted for subunits to assemble viral progeny. The progeny of a variety of antiviral therapy. The most common metabolic-based antivirals viral families possess envelope glycoproteins that are essential include those targeting divergent nucleotide metabolism and are for viral infection. The production of these functional glyco- used to treat hepatitis B virus, HIV, human cytomegalovirus proteins requires an ample supply of UDP–sugar subunits that (HCMV), and herpes simplex virus infections (4, 5). Increasing serve as the substrates for glycosylation reactions. Our results evidence has identified additional nonnucleotide metabolic ac- indicate that human cytomegalovirus induces a viral metabolic tivities that are both specifically induced by viral infection and program that activates pyrimidine biosynthesis to drive UDP– important for viral replication (6–9). Despite the importance of sugar biosynthesis. This metabolic activation is important for these activities, in most cases, little is known about how viruses viral protein glycosylation and high-titer viral replication. Fur- induce these activities or how they contribute to viral infection. ther, our results suggest that this metabolic link between pyrimi- HCMV, a member of the betaherpesvirus family, is a wide- dine and UDP–sugar biosynthesis is shared between evolutionarily spread pathogen that causes serious disease in immunosuppressed diverse viral families, which may provide novel avenues for anti- individuals, including cancer patients, transplant recipients, viral therapeutic intervention. and AIDS patients (10). Additionally, congenital HCMV infection occurs in 1–2% of all live births (11) and can result in multiple Author contributions: S.R.D., E.O.-R., L.M.-S., and J.M. designed research; S.R.D. and E.O.-R. system abnormalities, including central nervous system damage performed research; S.R.D., E.O.-R., L.M.-S., and J.M. analyzed data; and S.R.D., E.O.-R., L.M.-S., (12). HCMV is a double-stranded DNA virus that contains a ∼235-kb and J.M. wrote the paper. genome that encodes >200 ORFs. The genome is encapsulated in The authors declare no conflict of interest. a protein capsid that is surrounded by a tegument protein layer. This article is a PNAS Direct Submission. Collectively, this structure is enclosed in a phospholipid envelope, 1To whom correspondence should be addressed. Email: [email protected]. which contains a number of viral glycoproteins that mediate virus This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. attachment and entry (13). 1073/pnas.1415864111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1415864111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Inhibition of Pyrimidine Biosynthesis Attenuates the Production of HCMV Viral Progeny. Given that HCMV induces pyrimidine biosynthesis, we hypothesized that pyrimidine biosynthetic inhibition might attenuate viral replication. We chose to target de novo pyrimidine biosynthesis using N-phosphonacetyl-L- aspartate (PALA), a CAD inhibitor (Fig. 2A), and Leflunomide, a dihydroorotate dehydrogenase (DHODH) inhibitor (Fig. 2A), which has been shown to have antiviral effects (26). Human fibroblasts were infected with HCMV [multiplicity of infection (MOI) = 3] and treated with DMSO or various concentrations of Fig. 1. HCMV infection induces host de novo pyrimidine biosynthesis. (A) PALA (Fig. 2B) or Leflunomide (Fig. 2C). Samples were har- MRC5 cells were mock- or HCMV-infected (MOI = 3.0) and harvested at 48 vested at 96 hpi for quantification of viral progeny production. hpi for LC-MS/MS–based measurement of UTP concentrations (mean ± SEM, We found that both PALA (Fig. 2B) and Leflunomide (Fig. 2C) n = 6). *P < 0.05. (B) Schematic of UTP labeling from U–13C-glucose. 13C, red; treatment caused a decrease in the production of infectious viral 12C, black; N, blue; O, gray. Certain atoms were omitted for clarity. (C) MRC5 progeny. Both inhibitors also blocked the HCMV-induced ex- 13 cells infected as in A were labeled at 48 hpi for 0, 1, 2, or 6 h with U– C- pansion of UTP and CTP pools (Fig. S1C). Because Lefluno- 12 – glucose and analyzed by LC-MS/MS to measure the fraction of C UTP mide has been reported to possess additional activities unrelated 12 – present ( C UTP/total UTP). Values are representative of four biological to DHODH inhibition (27), we wanted to test whether the as- replicates. (D) UTP flux estimates based on the data in A and C were calcu- sociated viral attenuation resulted specifically from pyrimidine lated as indicated in SI Materials and Methods (mean ± SEM, n = 4). biosynthetic inhibition. Toward this end, we supplemented viral growth medium with uridine, which enables UMP formation through the salvage pathway downstream of the biosynthetic (24), but little is known about how HCMV infection may impact A the cellular glycosylation machinery. enzymes targeted (Fig. 2 ). Addition of exogenous uridine fully Here, we show that de novo pyrimidine biosynthetic flux is rescued viral replication during PALA treatment, providing evidence that PALA inhibition is specific for pyrimidine bio- induced upon HCMV infection and that inhibition of de novo synthesis (Fig. 2D). In contrast, the inhibitory effect of Leflu- pyrimidine biosynthesis reduces HCMV replication, indicating nomide treatment was only partially rescued by uridine that induction of pyrimidine biosynthesis is necessary for high- supplementation (Fig. 2E), indicating the possibility of off- titer viral replication. Further, we find that HCMV-infected cells target effects. To test the toxicity of PALA and Leflunomide – require pyrimidine biosynthesis to maintain UDP sugar pools on human cells, a live/dead assay was performed on human and proper glycosylation of the gB virion protein. This link be- fibroblasts treated with DMSO, either 100 μMPALAor100μM tween pyrimidine biosynthesis and the UDP–sugars is important Leflunomide, or 1% ethanol as a control. This assay stains to HCMV infection, because UDP–GlcNAc or UDP–glucose live cells green based on their esterase activity and dead cells red supplementation rescues viral growth in the face of pyrimidine biosynthetic inhibition. Further, the importance of this metabolic link was also observed during influenza A and vesicular stomatitis virus (VSV) infection, suggesting that it could be a common fea- ture of viral infection. Results HCMV Infection Induces Pyrimidine Biosynthetic Flux. To gain a more thorough understanding of how HCMV impacts pyrimidine biosynthesis, we used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to measure the concentration of pyrimidine pools in mock or HCMV-infected human fibroblasts. HCMV infection induced a ∼2.5-fold increase in UTP concentration at 48 hours postinfection (hpi) (Fig. 1A). Before 48 hpi, at 6 and 24 hpi, there was no change in UTP concentrations upon infection (Fig. S1A). The HCMV-mediated induction of UTP levels appears to be dependent on viral gene expression, but not viral DNA replication, because infection with UV-irradiated HCMV or treatment with cycloheximide blocked induction of UTP pools, as opposed to treatment with phosphonoacetic acid (PAA), an inhibitor of viral DNA synthesis, which did not (Fig. S1B). Molecular flux through metabolic pathways can be estimated through the analysis of pool turn over after a pulse with an Fig. 2. Impact of pyrimidine biosynthetic inhibition on HCMV replication. isotopically labeled metabolic tracer (15, 25). To determine (A) Schematic of de novo pyrimidine biosynthesis. PRPS1, phosphoribosyl pyrophosphate synthetase 1; UMPS, UMP synthetase; UK, uridine kinase. (B how HCMV impacts pyrimidine biosynthetic flux, mock and and C) MRC5 cells were HCMV-infected (MOI = 3.0), treated with DMSO, HCMV-infected fibroblasts were pulsed with medium con- PALA (B), or Leflunomide (C) at the indicated concentrations, and harvested at 13 taining a U– C–glucose isotope tracer, which resulted in label- 96 hpi for analysis of viral progeny (mean ± SEM, n = 4). *P < 0.05. (D)Cells ing of the UTP ribose moiety (Fig. 1B). HCMV infection sub- were infected as in B, treated with PALA (100 μM) and uridine at 0, 5, or 50 μM stantially induced the turnover of the unlabeled UTP pool upon as indicated, and harvested at 96 hpi for analysis of viral progeny (mean ± 13C-glucose labeling (Fig. 1C). Measurement of pool concen- SEM, n = 4). *P < 0.05. (E) Cells were infected as in B, treated with Leflunomide trations (Fig. 1A) and pool-labeling kinetics (Fig. 1C) enables (100 μM) and uridine at 0, 0.5, or 1 mM as indicated, and harvested at 96 hpi ± = estimation of UTP biosynthetic flux (25). HCMV infection in- for analysis of viral progeny production (mean SEM, n 4). (F)MRC5cells were treated with 100 μMPALA,100μM Leflunomide, DMSO, or 1% ethanol duced an approximately fourfold increase in UTP metabolic + D ( control) for 24 h. The toxicity of PALA and Leflunomide was measured by flux at 48 hpi (Fig. 1 ). These results indicate that HCMV in- using a live/dead assay, in which green fluorescence indicates the presence of fection strongly induces pyrimidine biosynthetic flux and in- esterase activity associated with viable cells, and red fluorescence indicates creases UTP levels upon infection. a loss of cellular membrane integrity associated with cell death.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1415864111 DeVito et al. Downloaded by guest on September 30, 2021 based on nucleic acid staining that is dependent on membrane integrity breakdown. Neither PALA nor Leflunomide induced significant toxicity at the tested concentrations (Fig. 2F). CAD is a rate-controlling enzyme of pyrimidine biosynthesis (20, 21). We analyzed CAD protein levels and found that HCMV had little impact on CAD protein abundance (Fig. 3A). Phosphorylation of CAD at Thr-456 results in increased sensitivity to allosteric activators, and decreased sensitivity to allosteric inhibitors (19, 28–30). HCMV infection increased Thr-456 phosphorylation, most notably at 48 hpi (Fig. 3A), which is consistent with increased CAD activity and consequent activation of pyrimidine biosynthesis. To further test the impact of pyrimidine biosynthetic inhibition on HCMV replication, we targeted CAD mRNA expression with siRNA. Transfection with the CAD siRNA, but not with a nonspecific control siRNA, successfully reduced CAD protein levels (Fig. 3B). Transfection with CAD-specific siRNA, but not control siRNA, attenuated the production of infectious viral progeny to levels comparable to PALA treatment (Fig. 3C). These results indicate that pyrimidine biosynthesis and CAD expression are important for high-titer HCMV replication.

The Impact of Pyrimidine Biosynthetic Inhibition on HCMV Infection. To explore the impact of pyrimidine biosynthetic inhibition on the viral life cycle, we examined the accumulation of viral proteins that represent the major kinetic classes of viral gene expression: IE1 for immediate early genes; UL26 and UL44 for early genes; and pp28 as a true late gene (13). PALA treatment

did not substantially impact the accumulation of IE1, UL26, MICROBIOLOGY UL44, or pp28 (Fig. 4A), indicating that PALA treatment did not Fig. 4. The impact of pyrimidine biosynthetic inhibition on HCMV infection. dramatically impact the coordinated expression of HCMV genes. = Analysis of viral DNA abundance indicated that PALA treat- 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 ment decreased viral DNA abundance by ∼3.3-fold (Fig. 4B). hpi for Western analysis of IE1, α-tubulin, UL26, UL44, or pp28 abundance. Surprisingly, the addition of exogenous uridine to PALA-treated Data are from a representative experiment of four. (B) Viral DNA was infected cells did not rescue the observed reduction in the viral extracted from cells at 72 hpi and analyzed by quantitative PCR (qPCR) for DNA pool (Fig. 4B). Given that uridine supplementation fully viral DNA abundance (mean ± SEM, n = 3). (C) Cells were harvested at 48 hpi rescues the growth defect associated with PALA treatment (Fig. for metabolite analysis by LC-MS/MS. LC-MS/MS–derived ion counts were 2D), this finding suggests that the reduced level of viral DNA normalized to mock-infected samples and plotted as a heat map relative to replication that occurs with PALA treatment is still sufficient to mock-infected cells (n = 6). enable the production of wild-type levels of viral progeny. This notion is supported by the results indicating that PALA treatment does not impact pp28 levels (Fig. 4A), because pp28 pyrimidine end products were substantially decreased by PALA is a true late gene whose production is dependent on viral treatment (Fig. 4C, black boxes). PALA, a CAD inhibitor, also DNA synthesis. completely abolished the HCMV-induced increases in N-carba- To investigate the impact of pyrimidine biosynthetic inhibition moyl-L-aspartate, the product of the CAD-catalyzed reaction on the host-cell metabolic network, we used LC-MS/MS to ex- (Fig. 4C, dashed black box). Additionally, our metabolomic amine the relative changes in metabolites between mock sam- analysis revealed that PALA treatment substantially reduced ples, HCMV-infected samples, and HCMV-infected samples UDP–sugar levels—both UDP–glucose and UDP–GlcNAc (Fig. treated with PALA. As expected, we found that a number of 4C, red boxes), which serve as the essential sugar donors for cellular glycosylation reactions. A more thorough analysis of these metabolites indicated that HCMV infection substantially expanded UDP–glucose and UDP–GlcNAc pools (Fig. 5 A and B, respectively). PALA treatment completely blocked these increases, reducing the size of these pools relative to mock- infected cells (Fig. 5 A and B). The UDP–sugar pools of mock- infected cells were completely resistant to PALA treatment, suggesting that the sensitivity of these pools to pyrimidine biosynthetic inhibition is HCMV-specific. Given the changes observed in UDP–sugar pools, we wanted to explore the impact of HCMV infection on UDP–sugar biosynthetic flux. Glucose carbon feeds UDP–sugar biosynthesis through both the UTP ribose and the conjugated sugar (Fig. 5C). To explore the impact of Fig. 3. siRNA-mediated targeting of CAD attenuates HCMV replication. (A) – = HCMV infection on UDP sugar biosynthesis, we pulsed cells MRC5 cells were mock- or HCMV-infected (MOI 3.0) and harvested at 6, 24, 13 – and 48 hpi for Western analysis of CAD, CAD phospho–Thr-456 (P-CAD), or with C-glucose and measured UDP sugar pool turnover to α-tubulin abundance (data are from a representative experiment of four). estimate metabolic flux. HCMV infection induced a modest increase in UDP–glucose flux and a larger increase in UDP– (B) Subconfluent MRC5 cells were transfected with a CAD-specific or control D E siRNA. At 24 h after transfection, cells were serum-starved for 24 h and GlcNAc flux (Fig. 5 and , respectively). These data indicate HCMV-infected (MOI = 3.0), before Western analysis at 24 and 96 hpi for the that HCMV specifically targets UDP–sugar metabolism, in- indicated antibodies (data are from a representative experiment of two). (C) ducing UDP–sugar biosynthesis and increasing UDP–sugar pool Cells transfected and infected as in B were harvested for viral titration at 96 sizes, which, in contrast to mock-infected cells, are dependent on hpi (mean ± SEM, n = 4). *P < 0.05. pyrimidine biosynthesis.

DeVito et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 CD44S, a glycosylated host protein, was not impacted by treatment with PALA for 72 h (Fig. 6D), whereas control treatment of these extracts with PNGase resulted in an electrophoretic mo- bility shift of CD44S, consistent with its deglycosylation (Fig. 6D). Although strong conclusions cannot be made after analysis of a single cellular protein, these results are suggestive that the link between pyrimidine biosynthetic activation and glycosylation may be specific for viral infection. It has been observed that inhibition of UDP–GlcNAc transferases upon tunicamycin treatment attenuates HCMV replication (33). Similarly, we found that tunicamycin treatment reduced the production of infectious virions by ∼100-fold (Fig. 6E), further highlighting the importance of glycosylation reactions for HCMV infection. Combined, our results indicate that HCMV infection requires pyrimidine biosynthesis to maintain high UDP–sugar levels and viral protein glycosylation, which are important for production of infectious viral progeny. If pyrimidine biosynthesis inhibits glycosylation through adecreaseinUDP–sugar levels, medium supplementation with UDP–sugars could be predicted to restore gB glycosylation. We tested this possibility and found that supplementation with UDP–GlcNAc, but not UDP–glucose, restored gB glycosylation to non–PALA-treated levels at 72 hpi (Fig. 6F). Interestingly, medium supplementation with UDP–glucose did restore gB 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)orUDP–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)or12C–UDP–GlcNAc (D). Flux estimates were calculated as indicated in SI Materials and Methods (mean ± SEM, n = 4).

Pyrimidine Biosynthetic Inhibition Attenuates Viral Protein Glycosylation. UTP is required for the formation of UDP–glucose and UDP– GlcNAc, which are the essential subunits that drive glycosylation reactions. It is known that HCMV encodes many viral proteins that are glycosylated and are necessary for tegument and envelope formation, and host cell entry (13). This viral dependence on glycosylated proteins suggests that HCMV may rely on increased pyrimidine biosynthesis to provide the glycosyl groups necessary for protein glycosylation. To address this possibility, we examined the accumulation of the gB protein, a highly abun- dant, essential HCMV glycoprotein. The gB protein goes through several posttranslational modifications. The nascent 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 protein ( 105 kDa) is first glycosylated, giving rise to an 170- μ μ kDa product, before being transported to the Golgi, where it is (100 M), or uridine (50 M) as indicated, and harvested at 72 hpi. The abundances of gB and GAPDH were measured by Western analysis. The data cleaved to form the mature protein, which consists of two frag- ∼ are from a representative experiment of three. The quantification of percent ments, one of 116 kDa and the other 55 kDa (31, 32). The two mature gB from these experiments is shown in B (glycosylated 116 -kDa cleavage products form a functional heterodimer (31, 32). The gB/total gB × 100; n = 3). (C) Cells were infected as in A, harvested at 24, 48, and different stages of gB processing are distinguishable through 72 hpi, and processed for mRNA analysis by qPCR using gB-specific primers differential SDS/PAGE migration (31, 32). We examined gB and normalization to GAPDH-specific primer amplification (mean ± SEM, n = accumulation at 72 hpi in the presence of DMSO, PALA, and 3). (D) MRC5 cells were infected as in A, treated with DMSO or 100 μM PALA, uridine and found that PALA treatment resulted in a decrease in and harvested at 72 hpi. Samples in Right were treated after harvest with the mature 116-kDa glycosylated form and an increase in the PNGase as indicated. The abundance of CD44S was measured by Western unglycosylated 105-kDa form (Fig. 6A). These PALA-induced analysis. (E) MRC5 cells were infected with HCMV (MOI = 3.0), treated with changes were reversed with uridine medium supplementation DMSO or tunicamycin (10 μg/mL), and harvested at 96 hpi for assessment of (Fig. 6A). Quantification of the different gB isoforms indi- viral titers (mean ± SE, n = 2 biological and 4 technical replicates). (F) MRC5 cated that the mature glycosylated isoform—i.e., the 116 kDa cells were HCMV-infected as in A and treated with PALA (100 μM) in the μ – μ – μ isoform—was substantially reduced relative to the total amount presence of uridine (50 M), UDP glucose (5 M), or UDP GlcNAc (100 M) as of gB upon PALA treatment, a decrease that was rescued with indicated. Cells were harvested at 72 and 144 dpi and processed for Western B analysis of gB and GAPDH abundance (n = 3). (G) Cells were HCMV-infected uridine supplementation (Fig. 6 ). The total amount of gB was = μ – – A (MOI 3.0), treated with DMSO, 100 M PALA, UDP glucose, or UDP GlcNAc also reduced with PALA treatment (Fig. 6 ), which likely as indicated, and harvested at 96 hpi for the quantification of viral progeny reflects the well-established necessity of protein glycosylation to production (mean ± SEM, n = 4). *P < 0.05. (H). Cells were HCMV-infected ensure the stability of freshly translated glycoproteins (23). (MOI = 1.0) and treated with DMSO or 100 μM PALA. Supernatant virions Consistent with this notion, PALA treatment had no effect on were partially purified at 144 hpi and analyzed for associated viral genomes the accumulation of gB mRNA levels, indicating that PALA by real-time PCR or pfus by plaque assay. The pfu/genome ratio is indicated does not impact gB transcription (Fig. 6C). The glycosylation of relative to wild-type infection (mean ± SEM).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1415864111 DeVito et al. Downloaded by guest on September 30, 2021 glycosylation when examined later during infection, at 144 hpi (Fig. 6F). To determine whether UDP–sugar supplementation also restored viral growth in the presence of pyrimidine biosynthetic inhibition, we treated cells with DMSO or PALA in presence or absence of UDP–GlcNAc or UDP–glucose. As shown in Fig. 6G, supplementation with either UDP–GlcNAc or UDP–glucose rescued PALA-mediated inhibition of HCMV replication. To explore how inhibition of pyrimidine biosynthesis impacts viral particles, we partially purified secreted virions and assessed their infectivity. As expected, PALA treatment substantially reduced the secretion of infectious virus (Fig. S2A). The amount of genomes per pfu was substantially higher upon PALA treatment (Fig. 6H), indicative of an increase in noninfectious particles. PALA treatment reduced the secretion of viral particles containing genomes by ∼2.5-fold, a small change in comparison Fig. 7. The link between pyrimidine and UDP–sugar metabolism is impor- with the larger reduction in secreted pfu upon PALA treatment tant 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), ( 2-log) (Fig. S2). Combined, our results indicate that the link μ – μ between pyrimidine biosynthesis and UDP–sugar production is orotate (2.5 mM), uridine (50 M), or UDP GlcNAc (100 M) as indicated. important for production of infectious HCMV virions. 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 – = μ – NS1 GFP (MOI 0.005). Cells were treated with A3 (2.5 M) in the presence The Metabolic Link Between Pyrimidine Biosynthesis and UDP GlcNAc of orotate (2.5 mM) or UDP–GlcNAc (100 μM) as indicated. At 48 hpi, the Is Important for the Replication of Diverse Viruses. Diverse envel- supernatants were collected for viral titration in MDCK cells (mean ± SEM). (C) oped viruses contain membrane-embedded glycoproteins that A549 cells were infected with rVSV–GFP (MOI = 0.0001). Cells were treated with are critical for their respective infections. The link between py- A3 (2.5 μM) in the presence of orotate (2.5 mM), or UDP–GlcNAc (100 μM) as rimidine biosynthesis and UDP–sugar production could there- indicated. At 48 hpi, the supernatants were collected for viral titration in Vero fore be important for the replication of evolutionarily disparate cells (mean ± SEM). (D)293T–mCEACAM1 cells were infected with rMHV–GFP viruses. To explore this possibility, we tested whether a pyrimi- (MOI = 0.0005). Cells were treated with A3 (2.5 μM) in the presence of orotate dine biosynthesis inhibitor, A3—which targets DHODH and has (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).

recently been shown to possess antiviral activity (34, 35) could MICROBIOLOGY attenuate infection of diverse viral families and whether the attenuation could be rescued with medium supplementation of various pyrimidine metabolites or UDP–GlcNAc. Similar to viral progeny. Our findings also indicate that pyrimidine bio- PALA, A3 attenuated HCMV replication. HCMV replication in synthetic inhibition dramatically reduced UDP–sugar pools in A3-treated cells could be rescued with the addition of uridine, infected, but not in uninfected, cells. This virally specific re- UDP–GlcNAc, or orotate (Fig. 7A). A3 treatment also reduced duction could reflect an increased utilization of UDP–sugars influenza A growth, which could be rescued with medium sup- during infection that drains these pools in the absence of an plemented with orotate or UDP–GlcNAc (Fig. 7B). Similarly, increased pyrimidine supply. Alternatively, these results could VSV infection was attenuated by A3 and was rescued with indicate that viral infection is specifically funneling pyrimidine orotate or UDP–GlcNAc (Fig. 7C). In contrast, orotate, but not end products into UDP–sugar biosynthesis. If so, this finding UDP–GlcNAc, rescued viral replication in A3-treated cells would join a recent and growing body of literature indicating that infected with mouse hepatitis virus (MHV). These results suggest viruses induce specific host-cell metabolic activities to facilitate that the importance of the link between pyrimidine biosynthesis infection (39–44). It was recently shown that HCMV infection and UDP–GlcNAc is maintained between divergent viral fami- induces the production of specific long-chain fatty acids for in- lies including HCMV, a herpesvirus; influenza A, an orthomyxo- corporation into the viral envelope, which was subsequently virus; and VSV, a rhabdovirus; but not MHV, a coronavirus. found to be important for high-titer HCMV replication (7). Results such as these suggest that viruses may require specific anabolic Discussion precursors and their associated synthetic enzymes for virion con- Our results indicate that HCMV infection induces pyrimidine struction. The metabolic requirements for these specialized pre- – biosynthesis, which is required for induction of UDP sugar cursors may reflect advantageous biophysical properties that they pools, proper viral protein glycosylation, and high titer infection. convey to newly produced virions. To the extent that these meta- This activation of pyrimidine biosynthesis is likely mediated in part bolic activities are specific for viral infection, they may represent by induction of CAD activity resulting from the phosphorylation of attractive targets for antiviral therapeutic intervention. An inherent CAD at Thr-456 (Fig. 3), which has been shown to induce CAD advantage of targeting virally induced host-cell metabolic enzymes, activity (30). CAD phosphoregulation is complex. MAPK kinase as opposed to viral factors, would be the higher evolutionary barrier activation has been shown to induce CAD Thr-456 phosphorylation that viruses would have to overcome to acquire drug resistance. (30), and the mTORC1–S6k complex has also been shown to ac- As a class, glycosylated envelope proteins negotiate the ini- tivate CAD through Ser-1859 phosphorylation (36). In contrast, PKA-mediated CAD phosphorylation has been reported to inhibit tial encounters of enveloped viruses with their hosts, including CAD activity (28). HCMV is known to modulate these pathways cellular binding and fusion. As such, glycosylated envelope (reviewed in refs. 37 and 38), raising the possibility that they play proteins are critical determinants of tropism and pathogenicity a role in HCMV-mediated CAD phosphorylation and activation (45). Virion glycan epitopes are also major determinants of and, consequently, HCMV-mediated induction of pyrimidine immune recognition and have been found to be critical viral biosynthesis. determinants that mediate escape from antibody neutralization Inhibition of pyrimidine biosynthesis lowered the levels of viral (46). Despite their importance, the mechanisms through which DNA accumulation. Surprisingly, uridine supplementation res- viruses modulate the metabolic processes responsible for specific cued viral growth without rescuing viral DNA abundance. These glycosyl linkages are not known. With further mechanistic eluci- results suggest that exogenously added uridine does not have dation of these processes, these areas may represent potential immediate access to purine deoxynucleotide pools. Further, intervention points to alter the outcome of viral infection. Our given that uridine rescues infectious virion production, these data indicate that the metabolic links between pyrimidines and results suggest that viral DNA is normally produced in sub- UDP–sugars are important to diverse viral families. These results stantial excess of what is required for the production of infectious highlight the possibility that the mechanisms through which

DeVito et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 viruses target this link may prove to be a vulnerability shared by Flux Analysis. Flux estimations were largely performed as described (25, 48), diverse viral types. with details indicated in SI Materials and Methods.

Materials and Methods Statistical Analysis. All statistical analysis was performed with Origin (Version Cell Culture and Biological Reagents. Cells and viruses were maintained under 8.0) as described in SI Materials and Methods. standard conditions specifically described in SI Materials and Methods. ACKNOWLEDGMENTS. This work was supported by National Institute of Protein and Nucleic Acid Analysis. Analysis of proteins and nucleic acids was Allergy and Infectious Diseases Grant R01AI081773 (to J.M.). S.R.D. was supported by NIH Ruth L. Kirschstein National Research Service Award performed by using standard procedures specifically described in SI Materials Training Grants GM068411 and AI049815-13. E.O.-R. is supported by and Methods. a postdoctoral fellowship for Diversity and Academic Excellence from the University of Rochester Office for Faculty Development and Diversity. Chemical Reagents. All chemical reagents are described in SI Materials Research in the L.M.-S. laboratory is supported by NIH Grants R01 and Methods. AI077719 and R03AI099681-01A1, National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Sur- LC-MS/MS Analysis. LC-MS/MS analysis was performed largely as reported (47), veillance Grant HHSN266200700008C, and University of Rochester Center with details as described in SI Materials and Methods. for Biodefense Immune Modeling Grant HHSN272201000055C.

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