https://orcid.org/0000-0001-5286-0924
Figures
Abstract
Viruses are obligate intracellular parasites that rely on host cell metabolism for successful replication. Thus, viruses rewire host cell pathways involved in central carbon metabolism to increase the availability of building blocks for successful propagation. However, the underlying mechanisms of virus-induced alterations to host metabolism are largely unknown. Noroviruses (NoVs) are highly prevalent pathogens that cause sporadic and epidemic viral gastroenteritis. In the present study, we uncovered several strain-specific and shared host cell metabolic requirements of three murine norovirus (MNV) strains, MNV-1, CR3, and CR6. While all three strains required glycolysis, glutaminolysis, and the pentose phosphate pathway for optimal infection of macrophages, only MNV-1 relied on host oxidative phosphorylation. Furthermore, the first metabolic flux analysis of NoV-infected cells revealed that both glycolysis and glutaminolysis are upregulated during MNV-1 infection of macrophages. Glutamine deprivation affected the viral lifecycle at the stage of genome replication, resulting in decreased non-structural and structural protein synthesis, viral assembly, and egress. Mechanistic studies further showed that MNV infection and overexpression of the non-structural protein NS1/2 increased the enzymatic activity of the rate-limiting enzyme glutaminase. In conclusion, the inaugural investigation of NoV-induced alterations to host glutaminolysis identified NS1/2 as the first viral molecule for RNA viruses that regulates glutaminolysis either directly or indirectly. This increases our fundamental understanding of virus-induced metabolic alterations and may lead to improvements in the cultivation of human NoVs.
Author summary
All viruses critically depend on the host cells they infect to provide the necessary machinery and building blocks for successful replication. Thus, viruses often alter host metabolic pathways to increase the availability of key metabolites they require. Human noroviruses (HNoVs) are a major cause of acute non-bacterial gastroenteritis, leading to significant morbidity and economic burdens. To date, no vaccines or antivirals are approved against NoVs, which demonstrates a need to better understand NoV biology, including the role host metabolism plays during infection. Using the murine norovirus (MNV) model, we show that host cell glutaminolysis is upregulated and required for optimal virus infection. Additional data point to a model whereby the viral non-structural protein NS1/2 upregulates the enzymatic activity of glutaminase, the rate-limiting enzyme in glutaminolysis. Insights gained through investigating the role host metabolism plays in MNV replication may assist with improving HNoV cultivation methods and development of novel therapies.
Citation: Hafner A, Meurs N, Garner A, Azar E, Kannan A, Passalacqua KD, et al. (2024) Norovirus NS1/2 protein increases glutaminolysis for efficient viral replication. PLoS Pathog 20(7): e1011909. https://doi.org/10.1371/journal.ppat.1011909
Editor: Caroline Tapparel, University of Geneva Faculty of Medicine: Universite de Geneve Faculte de Medecine, SWITZERLAND
Received: December 18, 2023; Accepted: June 24, 2024; Published: July 8, 2024
Copyright: © 2024 Hafner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are available within the manuscript and supplementary information.
Funding: These studies were funded by the University of Michigan Pandemic Relief fund to C.E.W. D.N. and N.M. are supported by NCI grant nos. R01CA227622 and R01CA204969. D.N. is also supported by grants from the Rogel Cancer Center and the Forbes Institute for Cancer Discovery. A.H. was supported by the Molecular Mechanisms of Microbial Pathogenesis Training Grant (5T32AI007528). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Viruses are metabolically inert and must rely on host cell metabolic events to generate the necessary building blocks to multiply[1]. Historically, host metabolism has been thought to play only host-specific roles in cellular homeostasis, the immune response, and autophagy [2–3]. However, recent studies have shown that pathogens such as parasites, bacteria, and viruses influence host cell metabolism [4–6] to create a more favorable environment to ensure their optimal propagation [7]. Many investigations within the past decade have examined how viruses alter the host cellular metabolic profile and identified some of the metabolic pathways important during virus infection. These studies have shown that a common consequence of viral infection is induction of high glucose metabolism, which can lead to aerobic glycolysis, or the Warburg effect [8]. In addition, other pathways such as glutaminolysis, the pentose phosphate pathway (PPP), fatty acid synthesis, and tricarboxylic acid cycle (TCA) activity may also be altered, thus highlighting that central carbon metabolism is significantly perturbed during many viral infections [8]. Viruses often hijack these pathways to divert the production of nucleotides, lipids, amino acids, and other metabolites away from host processes toward virus particle construction. Virus-induced alterations to host metabolism can be shared among different viruses but are usually context dependent and variable between specific virus families or infected host cell types. For example, glucose deprivation significantly decreases dengue virus replication, while lack of glutamine does not [9]. In contrast, glutamine deprivation significantly reduces vaccinia virus replication, while glucose deprivation has no effect [10]. Thus, dengue virus and vaccinia virus show opposite dependencies on host glycolysis and glutaminolysis during infection. Other examples of global virus-induced changes to central carbon metabolism come from adenovirus, human cytomegalovirus, chikungunya virus, Zika virus, SARS-CoV-2, rhinovirus, lytic gammaherpesvirus-68, both latent and lytic Kaposi sarcoma-associated herpesvirus and hepatitis C virus [11–21]. While multiple studies have reported that metabolic pathways are altered during virus infection, the mechanistic details of how viruses achieve these changes remain elusive. Increased investigation into how viruses reprogram and usurp host metabolic pathways with an emphasis on mechanistic insights may reveal innovative therapeutic targets and provide a deeper understanding of specific viral replicative cycles.
Noroviruses (NoVs) are positive-sense single-stranded RNA viruses and the leading cause of acute non-bacterial gastroenteritis worldwide [22]. Globally, human NoV (HNoV) infections are extremely common, with estimated cases reaching ~685 million per year. Annually, HNoV infections result in ~200,000 fatalities, mostly in infants but also in immunocompromised individuals and in older adults [23]. Additionally, HNoV infections result in serious annual economic burdens, with global economic costs surpassing US$60 billion [24]. In the United States alone, HNoV infections cause ~21 million cases of gastroenteritis and are the leading cause of death in older adults with viral gastroenteritis [25–26]. Although HNoV infections are self-limiting in most individuals, the intense vomiting, diarrhea, and abdominal pain associated with this infection can be debilitating. However, despite the devastating public health and economic burdens caused by HNoV, no approved vaccines or antivirals against this virus exist [27], and development of anti-NoV therapeutics has been hampered by the lack of a cell culture model for HNoV. Although human intestinal enteroids (HIEs), non-transformed epithelium-only organoids derived from stem cells isolated from biopsies of the small intestine, and human B cells support varying degrees of infection, a cell culture–derived HNoV stock is still not available [28–30]. To overcome the limitations inherent to HNoV research, murine NoV (MNV) is used as a model system in the field to study general NoV biology because MNV readily replicates in cell culture, is genetically similar to HNoV, and has a genetically tractable small animal model and infectious clones available [31]. MNV strains, although genetically closely related, fall into two groups based on their in vivo phenotype. The acute strain, MNV-1, is cleared from infected mice within one week, while persistent strains, including MNV-CR6 (CR6) and MNV-CR3 (CR3), are shed for months [32]. The strains also differ in their in vivo (although not in vitro) tropism, in which CR6 infects tuft cells while MNV-1 infects immune cells (macrophages, dendritic cells, and lymphocytes) [33–34].
We previously performed a metabolomic screen of MNV-1–infected macrophages, which revealed that metabolites in many pathways were significantly upregulated, including those integral to central carbon metabolism [35]. Our screen identified glycolysis, nucleotide biosynthesis via the PPP, and oxidative phosphorylation (OXPHOS) as being required for optimal MNV-1 replication in murine macrophages based on experiments using common metabolic inhibitors [35]. We further determined that glycolysis is important for the replication step in the MNV lifecycle since treatment with the hexokinase inhibitor 2-deoxyglucose (2DG) led to a decrease in viral protein and RNA synthesis [35]. However, the requirement for glycolysis was independent of the host antiviral type I interferon response, and the underlying mechanisms behind NoV-induced upregulation of host metabolism and the role that host metabolic pathways plays in persistent MNV replication are not known. Thus, the goals of this current study were to further define the role of host metabolism in NoV replication, explore the role of host metabolism for persistent MNV strains, and begin to uncover the underlying mechanisms of NoV-induced metabolic alterations. Untangling the process of virus-induced metabolic alterations may enable development of more efficient HNoV cultivation systems and identify innovative metabolic therapeutic targets aimed at reducing chronic HNoV infections.
With these goals in mind, we investigated the dependence of CR3 and CR6 strains on host cell glycolysis, the PPP, and OXPHOS. While MNV-1, CR3, and CR6 all relied on glycolysis and nucleotide biosynthesis, OXHPOS was not required for replication of CR3 and CR6. We also performed metabolic flux analysis of MNV-1–infected macrophages, which revealed increased glycolysis and glutaminolysis. Reducing host glutaminolysis via pharmacological inhibition with the inhibitor CB839 and via glutamine deprivation showed that all MNV strains tested rely on glutamine metabolism, in particular for viral genome replication, which has repercussions for later steps in the viral life cycle. Early mechanistic investigations revealed that the observed increase in glutaminolysis during MNV infection is driven by the viral non-structural protein NS1/2 that caused increased glutaminase activity, the rate limiting enzyme within the glutamine catabolic pathway [36]. Overall, our findings highlight the importance of pathways in central carbon metabolism in MNV infection, albeit with strain-specific differences, and show that glutaminolysis is universally required for optimal MNV replication. Our finding that glutaminolysis is modulated by NS1/2 provides a foundation for future mechanistic studies, which may reveal novel chokepoints for therapeutic intervention.
Results
MNV strains CR3 and CR6 rely on glycolysis and nucleotide biosynthesis, but not OXPHOS, for optimal replication
We previously performed a metabolomics screen of MNV-1-infected macrophages, which identified increased metabolites from glycolysis, PPP, and OXPHOS in infected cells [35]. Inhibition of these pathways resulted in significantly lower MNV titers, ranging from an 0.5 to 2-log10 reduction [35]. However, whether other MNV strains within the same genogroup GV.1 also rely on these important metabolic pathways for optimal replication was not known. Thus, we chose the widely used CR6 and CR3 strains for our analysis. While CR6 and MNV-1 nucleotide sequences are 86% similar, CR3 and MNV-1 have an 88% similar sequence. To investigate whether these MNV strains have a common dependence on host cell metabolism, RAW 264.7 (RAW) cells were inoculated with MNV-1, CR3, and CR6 at an MOI of 5 for 1 hour. Medium containing the glycolysis inhibitor 2DG, the PPP inhibitor 6-Aminonicotinamide (6AN), or the OXPHOS inhibitor oligomycin-A was then added after inoculation, and cells were incubated for 8 hours, corresponding to approximately one round of viral replication. Non-toxic concentrations of 2DG and 6AN were previously determined [35], and cell viability assays were performed to ensure the concentration of oligomycin-A used would maintain >80% cell viability (S1A Fig). Infectious titers were measured after 8 hours via plaque assay. A significant (>2 log10) decrease was observed in the number of infectious MNV-1, CR3, and CR6 titers in 2DG-treated cells (Fig 1A). Treatment with 6AN also resulted in significantly decreased MNV-1, CR3, and CR6 titers; however, only a 1 log10 decrease in infectious particles was observed (Fig 1B). Additionally, the 1 versus 2 log10 decrease in viral titers observed after 6AN and 2DG treatment, respectively, suggested that all three MNV strains depend more on glycolysis than the PPP for optimal reproduction.
RAW 264.7 cells were infected for 1 hour at an MOI of 5 with either MNV-1, CR3, or CR6. Virus inoculum was removed and replaced with medium containing (A) 10 mM 2-deoxuglucose (2DG), (B) 500 μM 6-aminonicotinamide (6AN), (C) 1 μM oligomycin-A (Oligo), or vehicle control (DMSO). Infected cells were incubated for 8 hours and infectious MNV titers were measured via plaque assay. Experiments represent combined data from at least three independent experiments. Statistical analysis was performed using Two-tailed Students-t tests. ***, P<0.001; **, P <0.01; *, P<0.05; ns, not significant.
Because active viral replication requires large amounts of host energy, we also investigated whether CR3 and CR6 require OXPHOS for optimal replication. Surprisingly, we observed that CR3 and CR6 infection did not depend on OXPHOS because viral titers remained similar between oligomycin-A treated and untreated cells. This is in contrast to MNV-1, which showed an 0.5-log10 titer decrease. Lack of a significant reduction in viral titers of CR3 and CR6 during oligomycin-A treatment suggests that glycolysis-derived ATP is sufficient to meet the energetic requirements for sustaining optimal replication. These data highlight strain-specific dependencies on individual metabolic pathways for efficient MNV virion production.
Taken together, these data demonstrate that like MNV-1, strains CR3 and CR6 require host glycolysis and nucleotide biosynthesis for optimal replication; however, unlike MNV-1, OXPHOS is dispensable for the other two strains.
MNV-1 infection upregulates glycolysis and glutaminolysis
Our previous static metabolomic screen [35] analyzed the intracellular concentrations of metabolites but did not measure metabolite flux or metabolite turnover. To this end, we performed a metabolic flux analysis, which is an assay that uses uniformly labeled metabolites measured via gas chromatography mass spectrometry (GC-MS) to track incorporation of molecules into various metabolic pathways. Because glucose and glutamine are the two leading carbon sources used by mammalian cells [37], we analyzed their incorporation during MNV-1 infection to determine whether infection mediates an increase in their catabolism (Fig 2). RAW cells were infected for 1 hour with MNV-1 or mock lysate at an MOI of 5. After a 1-hour incubation, the virus inoculum was replaced with medium containing either 13C5-glucose or 13C5-glutamine. Samples were collected and analyzed after an 8-hour incubation. Through analysis of the enrichment of uniformly labeled glucose, we observed higher glucose metabolism in MNV-1–infected cells than in mock-infected cells as seen by increased incorporation of glucose into lactate, a common glycolytic byproduct, and into citrate, a downstream metabolite within the TCA cycle that can be generated from the final glycolytic product pyruvate through acetyl co-enzyme A (Fig 2A). These findings are consistent with our previous metabolic screen that showed higher concentrations of several glycolytic intermediates such as 2- and 3-phosphoglycerate and fructose-bisphosphate in infected cells [35] and confirmed that MNV-1 induces host glucose metabolism during its replicative cycle. Additionally, we further observed increased glutamine metabolism in MNV-1–infected cells relative to mock-infected cells. Glutamine undergoes a deaminase reaction to produce glutamate followed by a second deaminase reaction to produce alpha-ketoglutarate (aKG), an intermediate that can enter the TCA cycle (Fig 2B). In MNV-1–infected cells, higher production of both metabolites was observed after 8 hours, thus showing increased glutamine metabolism (Fig 2B). Given this finding, we revisited our previous metabolomic screen and investigated whether the concentrations of glutamate or aKG were significantly altered during MNV-1 infection. While aKG was not included in the screen, glutamate levels were significantly higher during infection [35]. Taken together, our previous metabolomic screen [35] and current analysis provide strong evidence that glutamine metabolism is upregulated during MNV infection. As a control to ensure that the presence of uniformly labeled glucose and glutamine did not negatively affect virus replication, we titered MNV-infected RAW cells in the presence of both labeled metabolites and measured viral replication via plaque assay (Fig 2C). We observed no negative effects from the uniformly labeled glucose or glutamine on virus replication, with a ~6.5 log10 growth after 8 hours (Fig 2C), which is similar to titers obtained in unlabeled medium (Fig 1).
RAW 264.7 cells were mock-infected or infected with MNV-1 for 1 hour at an MOI of 5. The virus inoculum was removed and replaced with medium containing (A, C) 13C5-glucose or (B-E) 13C5-glutamine for 8 hours. After 8 hrs, intracellular metabolites were extracted with ice-cold methanol and measured by mass spectrometry. (C) RAW 264.7 cells were infected as before, and MNV-1 titers measured via plaque assay. Experiments represent combined data from at least two independent experiments with at least two technical replicates. Statistical analysis was performed by multiple unpaired t-tests. ****, P<0.0001; ***, P<0.001; **, P <0.01; *, P<0.05; ns, not significant.
Activated macrophages can dramatically upregulate immunoresponsive gene 1 (IRG1) expression leading to itaconate production from cis-aconitate in the TCA cycle [38–40]. Furthermore, itaconate can play diverse roles in the immune response, including inhibition of succinate dehydrogenase in the TCA cycle [41]. Consistent with macrophage host defense responses, we measured approximately two-fold higher itaconate and succinate abundances in MNV-1 infected cells (Fig 2D) with a statistically larger fraction being glutamine-derived (Figs 2E and S2A). To determine how itaconate production might affect mitochondrial metabolism in macrophages, we analyzed the utilization of reductive carboxylation in MNV-1 infected cells. Reductive carboxylation is a glutamine-dependent metabolism favored by cells when the oxidative mitochondrial metabolism is dysfunctional [42]. We reasoned that production of itaconate during viral infection may reduce reliance on oxidative metabolism. Indeed, we measured a decrease in the ratio of oxidative to reductive metabolism in MNV-1 infected cells as measured by the ratio of oxidative-derived M4 citrate, M4 fumarate, and M4 malate to reductive-derived M5 citrate, M3 fumarate, and M3 malate (Fig 2F).
Overall, metabolite flux analysis of MNV-1 infected cells demonstrates upregulation of glucose and glutamine metabolism, and production of itaconate coupled with reductive TCA cycle activity, which are all hallmarks of virus-induced metabolic reprograming of infected cells [43].
Inhibition of glutaminolysis significantly reduces MNV replication
Glutaminolysis catabolizes glutamine for anaplerosis, which is the process by which metabolites from additional pathways, including glutaminolysis, replenish the TCA cycle [44]. Glutamine breakdown also provides a nitrogen source to fuel nucleotide and amino acid biosynthesis, key building blocks required for viral propagation [44]. The rate-limiting enzyme within the pathway is glutaminase (GLS), which catalyzes the first deaminase reaction [36]. Since we uncovered higher incorporation of labeled glutamine in MNV-1 infected cells (Fig 2), we hypothesized that this pathway would be required for optimal MNV replication. To test this, we infected RAW cells and primary bone marrow-derived macrophages (BMDMs) with MNV-1, CR3, and CR6 at an MOI of 5 for 1 hour. Medium containing CB839, a non-competitive GLS inhibitor, was added after infection and infectious titers were measured after 8 hours by plaque assay. The concentrations of CB839 used in both RAW cells and primary BMDMs were non-toxic and maintained >80% cell viability (S1B and S1C Fig). Cells treated with CB839 had significantly lower MNV titers (by ~1.5-log10) than cells that were treated with vehicle control (Fig 3A). RAW cells are transformed macrophages, and transformed cells can have altered metabolic processes [45]. Thus, to confirm the phenotype observed in RAW cells, we repeated infections in primary BMDMs. MNV-infected primary BMDMs treated with CB839 harbored significantly lower MNV titers (by >1.0-log10) than vehicle control (DMSO) cells for all strains despite using a slightly higher non-toxic concentration of CB839 (Fig 3B). The results in BMDMs confirmed data in RAW cells and showed that glutaminolysis is required for optimal replication of all MNV strains tested.
(A) RAW 264.7 cells or (B) primary bone marrow-derived macrophages (BMDMs) were infected for 1 hour at an MOI of 5 with either MNV-1, CR3, or CR6. Virus inoculum was removed and replaced with medium containing (A) 10 μM or (B) 15 μM CB839 or vehicle control (DMSO). (C) RAW 264.7 cells were infected as before but infection was performed with glutamine-free or -replete medium. After an 8 hr incubation, MNV titers were measured via plaque assay. Experiments represent combined data from at least three independent experiments. Statistical analysis was performed using Two-tailed Students-t tests. ***, P<0.001; **, P <0.01; *, P<0.05; ns, not significant.
Pharmacologic inhibitors can have off-target effects. Given the essential nature of GLS, instead of genetic ablation, short-term glutamine deprivation is the commonly used complementary approach in the field [9–10,19,21,46]. Hence, we repeated infections in RAW cells with medium lacking glutamine. Infections were performed as before, and viral titers were measured by plaque assay at 8 hours postinfection (hpi). While glutamine deprivation has been reported to negatively affect cell viability after 48 hours in numerous cell types [47–48], we confirmed that an 8-hour incubation without extracellular glutamine did not negatively affect RAW cell viability (> 80% viability) (S1D Fig). Glutamine deprivation resulted in significantly lower (by 2–2.5-log10) MNV titers for all strains tested (Fig 3C).
Taken together, these results demonstrate that MNV strains have a dependence on glutaminolysis for optimal replication, similar to findings with latent and lytic Kaposi’s Sarcoma-associated herpesvirus, adenovirus, lytic gammaherpesvirus-68, enterovirus A71, poliovirus, and human cytomegalovirus [11–12,18–19,21,46,49].
MNV genome replication is the stage in the viral life cycle most dependent upon glutaminolysis
Typical of a positive-sense, single-stranded virus, the MNV life cycle involves the following steps: host cell uptake of viral particles, uncoating of the positive-strand viral RNA genome, direct translation of the positive-sense viral RNA to produce nonstructural proteins, and synthesis of viral negative-sense RNA strand for eventual production of new positive-strand viral RNA, translation of structural proteins, followed by progeny virion assembly, maturation, and finally egress. To identify the stage within the MNV lifecycle that is most dependent upon glutaminolysis, we continued investigating infection under glutamine-starved conditions to avoid potential off-target effects of CB839. Since glutamine can be used as a nitrogen source for nucleotide biosynthesis [44] and for amino acid synthesis [50], we first checked in our metabolite analysis the intracellular amino acid levels in MNV-1–infected vs mock-infected cells. However, no significant differences were observed (S2B Fig). Thus, we reasoned that overall protein content, including receptor levels and viral entry by extension, were unlikely to be significantly altered. Instead, we focused on the role of glutaminolysis during viral genome replication, given the critical role of glutamine for nucleotide production. To test this, RAW cells were infected with MNV-1, CR3, or CR6 for 1 hour at an MOI of 5. After 1 hour, the virus inoculum was replaced with glutamine-free medium, and cells were incubated for 8 hours. After the incubation period, we extracted RNA and assessed viral genome levels via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Glutamine-deprived cells had significantly fewer genome copies for all three strains, a 1.8–2.0-log10 decrease (Fig 4A).
(A) RAW 264.7 cells were infected for 1 hour at an MOI of 5 with either MNV-1, CR3, or CR6. Virus inoculum was removed and replaced with glutamine-free or -replete medium. Infected cells were incubated for 8 hours. RNA was extracted and MNV genome levels were assessed via qRT-PCR. (B) RAW 264.7 cells were infected as above, and western blot analysis was performed for MNV viral proteins NS1/2 and VP1 capsid. β-actin was used as a loading control. Data shown are representative western blots from 3 independent experiments. Numbers below blots indicate densitometry measurement of protein level relative to MNV-infected cells receiving replete medium. (C) RAW 264.7 cells were infected as before. Supernatants and cell-associated virus were measured separately via plaque assay. Experiments represent combined data from at least three independent experiments. Statistical analysis was performed using Two-tailed Students-t tests and One-Way ANOVA. ***, P<0.001; **, P <0.01; *, P<0.05; ns, not significant.
Since viral genomes serve as templates for viral protein synthesis, we next investigated whether MNV protein synthesis is dependent on host glutaminolysis. RAW cells were infected as before, and after the 8 hr incubation period, levels of the non-structural protein NS1/2 and the capsid protein were measured via western blot (Fig 4B). NS1/2 protein levels were low in samples from infections with glutamine-containing medium, but not detectable in protein samples from infections with glutamine-free media (Fig 4B left panel). Quantification of NS1/2 protein signals from three independent replicates indicated a >90% decrease for all strains tested when grown in glutamine-free medium (Fig 4B middle panel), indicating that glutaminolysis is required for NS1/2 synthesis. Quantification of the capsid protein, which is translated from the subgenomic RNA, also showed significantly lower levels of this protein during glutamine starvation (Fig 4B left panel). For MNV-1 and CR6 infected cells starved for glutamine, we observed a ~60% reduction in capsid protein levels compared to infections in replete media, while a ~40% reduction was observed for CR3-infected glutamine-starved cells (Fig 4B right panel). Additionally, there were no significant changes in capsid levels between the different MNV strains in glutamine-free media (Fig 4B right panel). These data suggested that glutaminolysis is important for MNV viral protein synthesis (Fig 4B).
Last, we investigated viral assembly and egress, the end stages of infection. RAW cells were infected with MNV-1, CR3, or CR6 as before in replete and glutamine-starved media. After the 8 hr incubation period, supernatants and cell monolayers were collected separately to measure viral titers in each fraction and calculate the released virus. In the cell-associated fraction, about a 2.0-log10 decrease in viral titers was observed during glutamine starvation vs. replete media for all three strains tested (Fig 4C left panel). This was similar to the results obtained for total MNV titers, which combined cell-associated (i.e., intracellular) and extracellular (i.e., MNV released in the supernatant) titers (Fig 3C). The significant decrease in cell-associated MNV titers during glutamine starvation suggests that glutaminolysis is required for MNV assembly in all three strains. However, analysis of extracellular MNV showed a significant decrease of MNV titers in glutamine-depleted media only for CR3 and CR6 (Fig 4C middle panel). Specifically, we observed a 0.75-log10 decrease in extracellular CR3 and CR6 titers but no significant decrease for MNV-1 titers (Fig 4C middle panel), highlighting strain-specific dependencies on glutaminolysis. Additionally, we calculated the ratio of released-to-total viral titers to investigate whether glutamine deprivation affects MNV release efficiency. Surprisingly, glutamine deprivation led to increased release efficiency in all strains, with the highest increase in release efficiency observed in MNV-1 infected cells (Fig 4C bottom panel).
In summary, because glutamine can be used for nucleotide synthesis and amino acid synthesis, but no change in the intracellular amino acid pool was detected in MNV-1–infected cells in our metabolite analysis (S2B Fig), we conclude that genome replication is the stage of the MNV lifecycle that most imminently relies on host glutaminolysis. All other phenotypes observed during later stages of the viral life cycle are most likely a consequence of this initial effect.
Glutaminase (GLS) activity is upregulated during MNV infection
Our previous data indicated that glutaminolysis is upregulated and required for optimal MNV replication. Therefore, we were interested in whether MNV infection increases glutaminolysis through changes in GLS expression. We first directed our attention to GLS transcript and protein levels, since HCMV and HIV have previously been shown to increase GLS protein levels and mRNA expression, respectively [12, 51]. To test whether MNV infection modulates GLS expression, we infected RAW cells with MNV-1, CR3, and CR6 for 1 hour at an MOI of 5. After 8 hours, we assessed GLS transcript and protein levels via RT-qPCR and western blot, respectively. We observed that GLS transcript levels were significantly higher in MNV-infected cells compared to mock-infected cells (Fig 5A). Using the housekeeping gene beta-actin as a measure of baseline transcription, we observed some strain-specific differences, with MNV-1 infection leading to a 3-fold increase in GLS transcript levels and CR3 and CR6 leading to a 0.5-1-fold increase (Fig 5A). Western blot analysis of GLS protein levels resulted in no observable difference between MNV and mock-infected cells (Fig 5B, left). The two bands present in the immunoblot potentially represent the two isoforms of GLS, K-type glutaminase (KGA) and glutaminase C (GAC), which are identical in all aspects except the C-terminal domain [52]. Surprisingly, quantification of GLS protein levels revealed a small but significant decrease (5–7%) in GLS protein levels in MNV-infected relative to mock-infected cells (Fig 5B). This discordance observed between transcription and translation is not uncommon and could be caused by differential rates of transcription and translation [53]. From our data, we conclude that the upregulation of glutamine metabolism during MNV infection is not due to increased GLS mRNA or protein expression.
(A) RAW 264.7 cells were infected for 1 hour at an MOI of 5 with either MNV-1, CR3, or CR6. Virus inoculum was removed and replaced with replete medium. Infected cells were incubated for 8 hours. RNA was extracted and glutaminase transcripts were assessed via qRT-PCR. (B) RAW 264.7 cells were infected as before. western blot analysis was then performed for glutaminase protein levels. β-actin was used as a loading control. A representative Western blot is shown on the left and quantification from 3 independent experiments on the right. (C) RAW 264.7 cells were infected as before. Glutaminase activity was analyzed utilizing the Cohesion Biosciences Glutaminase Microassay kit. Experiments represent combined data from at least three independent experiments. Statistical analysis was performed using Two-tailed Students-t tests. ***, P<0.001; **, P <0.01; *, P<0.05; ns, not significant.
We next investigated whether GLS enzymatic activity was increased during MNV infection, a phenotype observed during HCMV infections [12]. Increased GLS activity would also be consistent with our metabolite analysis showing increased glutamine catabolism during MNV infection. RAW cells were infected with MNV-1, CR3, and CR6 for 8 hours as before and GLS enzymatic activity was analyzed with a commercially available kit that measures ammonia, the byproduct of the reaction that GLS catalyzes [52]. We observed higher levels of GLS enzyme activity in MNV-infected cells than in mock-infected cells (Fig 5C). When analyzing the fold change in GLS activity over mock-infected cells, an approximately 1.75-fold increase was detected for all three MNV strains, with each strain increasing GLS activity to a similar extent (Fig 5C).
Overall, we conclude that increased rates of glutaminolysis during MNV infection in macrophages are the result of increased host cell GLS enzymatic activity, but not due to changes in GLS transcript or protein levels.
NS1/2 is a viral mediator of increased GLS activity
Viral proteins can mediate changes to host metabolism to ensure optimal infection. For example, dengue virus NS1 interacts with glyceraldehyde-3-phosphate dehydrogenase to upregulate glycolysis [54]. Therefore, we investigated whether increased GLS activity in MNV-infected cells is mediated by a viral protein. To test this, we overexpressed individual MNV non-structural proteins in Huh-7 cells expressing the viral receptor CD300lf and measured GLS activity as before. We chose these cells, not only because they have been engineered to express the MNV receptor, but they also show robust MNV replication, have high transfection efficiency, and are utilized throughout the field [55]. As a control, we first tested whether MNV infection of CD300lf-expressing Huh-7 cells would be sensitive to glutaminolysis inhibition. Cell viability studies determined the concentration of CB839 at which >80% cell viability is maintained to be 5 μM (S1E Fig). We then infected the cells with MNV-1, CR3, and CR6 for 1 hour at an MOI of 5 before adding medium containing 5 μM CB839 or vehicle control (DMSO) for 8 hrs. Viral titers were measured via plaque assay. We observed a 0.5-1-log10 decrease in MNV titers when glutaminolysis was inhibited, confirming that similar to infected macrophages, CD300lf-expressing Huh-7 cells are sensitive to glutaminolysis inhibition (Fig 6A) and provide an efficient cell line for protein overexpression studies.
(A) Huh-7 cells expressing the viral receptor CD300lf were infected for 1 hour at an MOI of 5 with either MNV-1, CR3, or CR6. Virus inoculum was removed and replaced with medium containing 5 μM CB839 or vehicle control (DMSO). Infected cells were incubated for 8 hours and MNV titers were measured via plaque assay. (B) Huh-7 CD300lf cells were transfected with plasmids encoding the indicated MNV-1 non-structural protein or green fluorescent protein (GFP). Transfected cells were incubated for 24–48 hours. Glutaminase activity was analyzed utilizing the Cohesion Biosciences Glutaminase Microassay kit. Experiments represent combined data from at least three independent experiments. Statistical analysis was performed using Two-tailed Students-t tests. **, P <0.01; *, P<0.05; ns, not significant.
Having confirmed the importance of glutaminolysis during MNV infection in CD300lf-expressing Huh-7 cells, we investigated whether the expression of an individual viral protein would alter GLS activity. To this end, we transfected these cells with plasmids for the expression of 7 MNV-1 non-structural proteins (NS1/2 to NS7) or green fluorescent protein (GFP) as a negative control. Transfected cells were incubated for 24–48 hours, and cell lysates were first tested for successful protein expression via western blot (S3 Fig). After confirming expression of the proteins of interest, cell lysates were analyzed for GLS activity. We observed increased GLS activity in cells expressing NS1/2 (Fig 6B left panel), with an approximately 1.5-fold change in GLS activity (right panel), over cells expressing GFP. This is slightly less than the 1.75-fold increase in GLS activity observed during MNV infection (Fig 5C). Our observed 1.5-fold increase in GLS activity following viral protein overexpression (Fig 6B) and our 1.75-fold increase in GLS activity during MNV infections (Fig 5C) is consistent with previously published results showing 1.25–1.5-fold increases in metabolic enzyme activity during viral infections [54,56–57].
Taken together, these data demonstrate that the MNV structural protein NS1/2 mediates an increase in GLS activity and thus the viral factor upregulating glutaminolysis.
Uncleaved NS1/2 increases glutaminase activity
MNV NS1/2 remains as an uncleaved precursor throughout most of the lifecycle and is only cleaved by host caspase 3 to produce NS1 and NS2 late in infection (at 18–22 hours post infection) [58]. NS1 can then be secreted out of the cell to subvert the antiviral type III interferon response [59]. To determine whether caspase cleavage of NS1/2 mediated increased GLS activity, we measured GLS activity during infection with recombinant MNV mutants. NS1/2 can be cleaved at residues D121 and/or D131 [58]. Single mutant D121G and D131G, and the double mutant of D121/131G in NS1/2 in the CR6 background and D131G in the MNV-1 background were tested based on availability. To this end, we infected RAW cells with either WT MNV or MNV NS1/2 mutants at an MOI of 5. Virus inoculum was removed, infected cells were incubated for 8 hours, and GLS activity was analyzed. We observed at least a 0.25-fold increase in GLS activity in cells infected with mutant NS1/2 viruses compared to those infected with WT NS1/2 virus (Fig 7A). We also noted that viral titers are 0.5-1-log10 lower in macrophages infected with D131G mutants compared to WT MNV (Fig 7B, left panel). Therefore, we normalized the activity to the respective MNV NS1/2 WT titers. After normalization, we still observe increased GLS activity during infection with all NS1/2 mutant viruses (Fig 7B, right panel).
Caspase cleavage site mutations in NS1/2 increases GLS activity: (A) RAW 264.7 cells were infected for 1 hour at an MOI of 5 with wild-type MNV-1, CR6, or indicated MNV mutants. Virus inoculum was removed, and fresh medium was added. After an 8 hr incubation, glutaminase activity was analyzed utilizing the Cohesion Biosciences Glutaminase Microassay kit. (B) RAW 264.7 cells were infected as before and incubated for 8 hrs. MNV titers were measured via plaque assay. Experiments represent combined data from at least three independent experiments. Statistical analysis was performed using Two-tailed Students-tests or one-way ANOVA with multiple comparisons. ***, P<0.001; **, P <0.01; *, P<0.05; ns, not significant.
Collectively, these data suggest the precursor form of NS1/2 increases GLS activity and the D131 cleavage site is important for efficient MNV replication. Host caspases cleave NS1/2 late during infection but glutaminolysis is upregulated to sustain genome replication (Fig 4) early in infection, thus taken together these data suggest that the uncleaved precursor NS1/2 is the predominant form mediating the increase in GLS activity.
Discussion
Viruses have evolved numerous mechanisms for manipulating host cellular metabolism to create a more favorable intracellular environment to support optimal replication. Our previous study showed that MNV-1 infection significantly alters numerous host metabolic pathways, including glycolysis, the PPP, and OXHPOS, thereby supporting the energetic and biosynthetic needs for optimal virion production [35]. In our present study, we extended our investigation to include two persistent MNV strains, CR3 and CR6, and observed strain-dependent differences compared to MNV-1 in that while these strains also required host glycolysis and the PPP for optimal replication, they did not require OXPHOS. To support the previous static metabolomic analysis, we performed metabolic flux analysis to measure the incorporation of labeled carbon from glucose and glutamine. These data showed significantly higher glucose and glutamine catabolism during MNV-1 infection, thus supporting the observation that MNV infection upregulates both metabolic pathways concurrently. Having previously investigated the role of glycolysis during MNV infection, we focused on the role of glutaminolysis during MNV infection in this study. Glutamine deprivation and pharmacological inhibition of glutamine catabolism resulted in significantly lower MNV-1, CR3, and CR6 viral titers in multiple cell types, thus revealing that glutaminolysis is required for optimal MNV replication. Our results also showed that MNV genome replication is the first step in the viral life cycle that depends on glutaminolysis. Additionally, our mechanistic studies point to NS1/2 as the viral protein that mediates the upregulation of GLS activity, the key enzyme in glutaminolysis. Thus, in addition to glycolysis, glutaminolysis is another intrinsic host metabolic factor that contributes to optimal MNV replication. Collectively, our investigation has revealed both shared and strain-specific metabolic dependencies that may underly the different pathogenic phenotypes of various MNV strains.
Glycolysis and glutaminolysis are the catabolic pathways for glucose and glutamine, respectively, and these molecules are the main carbon sources used by mammalian cells to perform a myriad of cellular processes. Importantly, these pathways are often concurrently rewired by viruses, since metabolites from the glycolytic pathway can not only be used for energy production via OXPHOS, but also can be used within the PPP for nucleotide synthesis, molecules that viruses need for genome replication. Additionally, glycolytic intermediates can be used in lipid biosynthesis, and when glycolytic intermediates are used more for lipid biosynthesis or lactic acid assembly rather than energy production, alpha-ketogluterate, a glutaminolysis product, can be shuttled into the TCA cycle to ensure continuous downstream ATP production via anaplerosis. This phenotype is observed in HCMV infections [12]. Glutamine catabolism also provides nitrogen-containing metabolites for amino acid and nucleotide biosynthesis [44]. Together, glycolysis and glutaminolysis provide the necessary building blocks and energetic needs for optimal progeny virion production. Hence, viruses may target both pathways to promote optimal replication. In the present study we observed increased glycolysis and glutaminolysis during MNV-1 replication in murine macrophages. Glutamine deprivation and treatment with the pharmacological inhibitor CB839 significantly decreased virion production of MNV-1, CR3, and CR6 through reduced genome replication, which resulted in lower levels of non-structural and structural protein synthesis, viral assembly, and release. Diverse viruses such as HIV-1, white-spot syndrome virus, hepatitis C virus, influenza virus, and adenovirus also upregulate both glycolysis and glutaminolysis during infection [11,56–57,60–64]. However, the molecular mechanisms underlying the upregulation of these two key metabolic pathways and how this metabolic rewiring affects virus replication vary by virus and host cell type. Uncovering these mechanisms may reveal shared metabolic dependencies and therapeutic chokepoints.
As obligate intracellular parasites, viruses rely on the metabolic products of host cells and have evolved capabilities to hijack metabolic resources and stimulate specific metabolic pathways required for replication. However, the mechanisms and viral proteins responsible for metabolic control are mostly unknown. In this study, we identified the NoV non-structural protein NS1/2 as being involved in host cell metabolic modulation. This protein is released from the viral polyprotein precursor via proteolytic activity of the viral protease NS6 [65]. Our results strongly suggest that upon release from the polyprotein one function of the NS1/2 protein is to enhance GLS enzymatic activity, leading to increased glutaminolysis. Other viral proteins known to mediate changes to host metabolism come from diverse virus families. For example, three different DNA viruses use non-structural proteins to modulate host metabolism. Epstein-Barr virus increases fatty acid synthase expression during lytic replication through the immediate-early non-structural protein BRLF1, which works in a p38 stress mitogen-activated protein kinase-dependent manner to increase fatty acid production [66]. Hepatitis B virus uses viral protein X to reprogram liver glucose metabolism through increased expression of key gluconeogenic enzymes [67]. And adenoviruses use the E4ORF1 gene product through a direct interaction with c-Myc to increase anabolic glucose metabolism and glutaminolysis [11,56]. Enterovirus A71, on the other hand, affects host cell metabolism through its structural protein VP1, which directly binds to trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase to promote increased pyrimidine synthesis [46]. These examples highlight that both non-structural and structural viral proteins from diverse viral families can contribute to altering host metabolism during viral infection. However, our work on NoV NS1/2 increasing GLS activity provides the first example of an RNA virus that directly or indirectly upregulates glutaminolysis through a specific non-structural viral protein. Future investigations into the mechanism of NS1/2-mediated increase in GLS enzymatic activity are needed but have the potential to reveal fundamental insights into NoV-host interactions and pathogenesis.
Macrophages are highly plastic immune cells that adapt to different physiological microenvironments. These cells are often parsed into two major categories: pro-inflammatory (M1) and anti-inflammatory/pro-resolving (M2) macrophages [68]. Importantly, these two macrophage phenotypes are associated with distinct metabolic profiles. Hallmarks of M1 macrophages include high rates of glycolysis, fatty acid synthesis, and pentose phosphate activity. In contrast, hallmarks of M2 macrophages include high rates of glutaminolysis, fatty acid oxidation, and OXPHOS [68]. Our previous [35] and current metabolomic analyses revealed significant upregulation of central carbon metabolism and increased carbon flow through glycolysis and glutaminolysis during MNV infection of macrophages. Since upregulation of glycolysis, the PPP, and increased succinate production are hallmarks of M1 macrophages, while upregulation of glutaminolysis and OXPHOS are hallmarks of an M2 macrophage, MNV-infected macrophages display a hybrid metabolic profile during infection. Intriguingly, the underlying metabolic program is crucial for macrophage function [68]. However, how the metabolic alterations induced by MNV infection impact macrophage function remains unknown. Like MNV, bacteria also rewire macrophage metabolism to grow and evade innate immunity. Legionella pneumophila, Brucella abortus, and Listeria monocytogenes rewire macrophages towards aerobic glycolysis, and L. pneumophila enhances glycolysis by a yet-to-be-determined mechanism [69]. L. monocytogenes uses a bacterial toxin to induce mitochondrial fragmentation and takes advantage of increased glycolysis in M1 macrophages to efficiently proliferate [70]. While chronic B. abortus infection preferentially occurs in M2 macrophages, it requires PPARγ to increase glucose availability [71]. Parasites can also alter macrophage metabolism during intracellular infection. For example, Leishmania spp. are protozoan parasites that infect macrophages and activate HIF-1α to upregulate HIF-1α target genes, including glucose transporters and glycolytic enzymes, resulting in increased glucose uptake, glycolysis, and activation of the PPP [72]. These examples suggest that while MNV infection increases the availability of resources for optimal infection, rewired macrophage metabolism may also promote changes to the host immune response. While activation of the innate immune response increases glycolytic and TCA cycle flux in macrophages [73], our previous work has shown that MNV-induced upregulation of glycolysis is independent of the type I interferon response [35]. We posit this is similarly true for MNV-induced upregulation of glutaminolysis since an extensive search of the literature did not uncover a link between glutaminolysis and the type I interferon response. While host immune signaling can play a role in altering host metabolism, disentangling which metabolic pathways are directly altered by MNV, and which are consequences of macrophage host defenses is an important area for future investigations.
As with any study, there are limitations to the current study. Host metabolism is an intricate interdependent web of interactions and manipulation of one point has repercussions throughout the entire web. For example, if cancerous cells are deprived of glucose; glutamine and other carbon sources such as amino acids are used to sustain cellular proliferation [74–75]. In contrast, if cancerous cells are deprived of glutamine, several additional pathways are activated such as increasing glycolytic products and TCA intermediates to sustain prolonged growth [74–75]. These examples show the flexibility of host metabolism in response to the external environment and highlight that it is not possible to truly study one pathway in isolation. This complex metabolic plasticity of cells is being manipulated by viruses to ensure successful replication. Understanding this detailed interplay between the various pathways during virus infections and downstream consequences requires further investigations. Another limitation is that metabolic manipulations and virus infections, individually or in combination, are significant stressors of cells, which trigger stress-response programs in cells. The consequences of which are not always obvious. For example, we observed an increase in MNV release during glutamine-starved conditions (Fig 4C). This observed increase in release efficiency could be due to increased extracellular vesicle release, which occurs in states of cellular stress [76]. Given that MNV release is largely via extracellular vesicles at our 8-hour timepoint [77], MNV may hijack the increased extracellular vesicle production/release process in an attempt to leave this stressed environment. Interestingly, MNV-1 release was increased to a greater extent than CR3 and CR6 during glutamine deprivation (Fig 4C), while the opposite was observed during inhibition of the Akt serine/threonine kinase (i.e., CR3 and CR6 release was increased to a greater extent that MNV-1) [78]. The underlying mechanism of these divergent egress phenotypes are unknown. Future investigations into NoV egress mechanisms during various cellular stresses may enhance our understanding of viral pathogenesis and spread. Other limitations are that the current study focused primarily on one cell type (i.e., macrophages), one timepoint (i.e., 8 hrs), and only MNV, albeit multiple strains. However, different cell types, such as tuft cells, may respond to MNV infection in a different way, and MNV-induced metabolic alterations may exhibit a temporal dynamic that was not investigated given our focus on one timepoint capturing a single round of infection. In support of this, infection of macrophages with the plaque isolate MNV-1.CW1 for 10 hrs at an MOI of 10 resulted in amino acid depletion, in contrast to our results with MNV-1.CW3 for 8 hrs at an MOI of 5 (see S2B Fig) [79]. It is also conceivable that human NoV, which infects mature enterocytes and enteroendocrine cells [80–81], may manipulate the metabolic program of these infected cell types differently or that these cell types respond to virus infection in a different manner. For example, infection of human intestinal organoids with a human NoV downregulated transcripts related to glycolysis [82]. Furthermore, we have shown that recombinant human NoV NS1 treatment of B cells alters metabolism [30], suggesting NoVs may not only modulate the metabolism of infected but also bystander cells. Thus, future investigations of different cell types infected with MNV or human NoVs, or physiologically relevant bystander cell types, are warranted.
In conclusion, we have shown that glutaminolysis, in addition to glycolysis, is an intrinsic host factor promoting optimal replication of MNV. Our data are consistent with a model whereby MNV uses the NS1/2 protein to upregulate GLS activity during infection of macrophages, which increases glutamine catabolism and promotes viral propagation. Our previous and current findings reveal that central carbon metabolism plays an important role in NoV replication, and these findings suggest novel chokepoints for therapeutic intervention and may provide new avenues for improving HNoV cultivation.
Methods
Compounds and reagents
2-Deoxyglucose (2DG) (Sigma #D8375) was solubilized fresh for each experiment in cell culture medium to 100 mM and added to the culture medium at a final concentration of 10 mM. CB839 (Cayman Chemical #22038) was solubilized in DMSO at 10 mM and used at final concentrations of 5, 10, or 15 μM. 6-Aminonicotinamide (6AN) (Cayman #10009315) was solubilized in DMSO at 500 mM and used at 500 or 750 μM. Oligomycin A (Cayman #11342) was solubilized in DMSO at 5 mM and used at 1 μM. Glutamine-free media was prepared fresh for each experiment using DMEM-10 medium (Gibco DMEM medium #11995–044 with 4.5 g/L D-Glucose, 10% dialyzed fetal bovine serum (Thermo Fischer Scientific #A3382001), and 1% HEPES buffer (1M, Gibco #15630–080). MNV-1 NS1/2, NS3, and NS5 plasmids were a kind gift from Dr. Jason Mackenzie (University of Melbourne, AUS) and previously described [83]. Flag-tagged MNV-1 NS4, NS6, and NS7 plasmids were a kind gift from Dr. Ian Goodfellow (University of Cambridge, UK) and previously described [84].
Cell culture and virus strains
The RAW 264.7 macrophage cell line (referred to herein as RAW cells) (ATCC TIB-71) and CD300lf-expressing Huh-7 cells were maintained in DMEM-10 medium (Gibco DMEM medium #11995–065 with 4.5 g/L D-Glucose and 110 mg/L Sodium Pyruvate, 10% Fetal Bovine Serum [HyClone #SH30396.03], 1% HEPES buffer [1M, Gibco #15630–080], 1% Non-Essential Amino Acids [100X, Gibco #11140–050] and 1% L-Glutamine [200 mM, Gibco #25030–081]) in treated tissue culture flasks at 37°C/5% CO2. CD300lf-expressing Huh-7 cells were a gift from Dr. Stefan Taube (University of Lübeck, Germany) and were previously described [55]. Primary bone marrow-derived macrophages (BMDM) were differentiated from male Balb/C mouse femur and tibia bone marrow in 20% L929 medium (Gibco DMEM medium, 20% FBS [HyClone #SH30396.03], 30% L9 supernatant, 1% L-Glutamine, 1% Sodium Pyruvate, 0.25 mL β-mercaptoethanol/L and 2% Penicillin/Streptomycin). All experiments using primary cells were performed with 10% L929 working medium (same as 20% L929 medium but with 10% L929 supernatant). The plaque purified MNV-1 clone (2002/USA) MNV-1.CW3 (referred herein as MNV-1) was used at passage 6 in all experiments. CR3 (GV/CR3/2005/USA) and CR6 (GV/CR6/2005/USA) were also used at passage 6 in all experiments. Plasmids encoding the caspase cleavage mutants were a kind gift from Drs. Timothy J. Nice (Oregon Health and Science University) and Craig Wilen (Yale University). Recombinant viruses were generated as previously described [85].
Virus infections and plaque assay
All MNV infections were performed in the RAW 264.7 cell line, Balb/C primary bone marrow-derived macrophages (BMDM), or CD300lf-expressing Huh-7 cells. Cells were grown in 12-well tissue culture plates seeded at 5x105 cells/well. Infections involving CB839 and glutamine free media had a 30-minute pretreatment prior to addition of virus. At the time of infection, the medium was replaced with 1 mL of media containing MNV-1, CR3, or CR6 at the indicated MOI. Plates were rocked for 1 hour on ice. Then, cells were washed 3X with cold DPBS++ (+Calcium and +Magnesium Chloride—Gibco #14040), fresh medium was added containing metabolic inhibitors at the indicated concentrations, vehicle control, or glutamine-free media. Cells were incubated for indicated times. Cells were then frozen at -80°C and freeze-thawed two times before lysates were analyzed by plaque assay as previously described [86]. Vehicle control experiments were performed using DMSO in a v/v match to the volume of metabolic inhibitors. Primary cell infections were done the same as RAW infections except in medium containing 10% L929 supernatant.
RNA extraction and RT-qPCR
Experiments to quantify MNV genome copies and glutaminase expression were performed on MNV- or mock-infected RAW cells as indicated above. At time of RNA extraction, cells were washed 1X with cold DPBS++ and then 500 μL of Zymo Research TriReagent (#R2050-1) was added. Extraction was performed per manufacturer’s directions using the Zymo Research Direct-zol RNA MiniPrep Plus (#R2072) and then used for One-Step TaqMan Assay. Primers used to measure murine glutaminase transcript and MNV genome levels were previously described [87–88].
Protein extraction, SDS-PAGE, and immunoblotting
Experiments were performed as described above in 6-well tissue culture plates seeded at 8x105 cells/well the day prior. At time of harvest, cells were washed 2X with cold DPBS++ and RIPA buffer (Pierce #89900) containing complete EDTA-free protease inhibitor cocktail (Roche #11873580001) was added to wells. Cells were scraped, moved to Eppendorf tubes, and incubated on ice for 15 minutes. Cells were then spun at 4°C at 14,000 x g for 15 minutes. Lysates were moved to fresh tubes, and Laemmli buffer with β-mercaptoethanol was added at 3:1 lysate to buffer ratio before freezing the sample until analysis. SDS-PAGE was performed with BioRad 4–20% Mini-Protean TGX gels (BioRad #456–1096) per standard SDS-PAGE procedures [89]. Gels were transferred to Immobilon-FL transfer membranes (#IPFL00010, pore size 0.45 μm) using a Semi-Dry transfer at 10V for 60 minutes. Membranes were blocked in PBS+0.05% Tween + 1% low-fat milk for 1 hour at room temp, then primary antibodies were added in the same buffer and membranes were rocked at 4°C overnight. Membranes were washed 3X with 1X PBS, then secondary LI-COR fluorescent antibodies were added for 1 hour at room temp and then visualized on the LI-COR Odyssey Imager. Western blots were quantified by densitometry using ImageJ and normalizing bands to β-actin. Antibodies used: mouse mAb β–Actin (clone 8H10D10, Cell Signaling #3700) at 1:10,000 dilution; rabbit mAb β-Actin (clone 13E5, Cell Signaling #8457) at 1:10,000 dilution; anti-rabbit polyclonal glutaminase (Proteintech #12855-1-AP) at 1:1000 dilution; anti-mouse monoclonal FLAG (Sigma #F1804) at 1:3000 dilution. The rabbit polyclonal anti-MNV-1 capsid antibody (used at 1:500 dilution) was described previously [35]. The mouse monoclonal anti-NS1/2 and anti-NS5 antibodies (both used at 1:3000 dilution) were a kind gift from Dr. Vernon Ward (University of Otago, New Zealand) and previously described [90–91].
Cell viability assay
Cell viability was tested with the WST-1 Cell Proliferation Reagent (Sigma #5015944001) or Resazurin Cell Viability Assay Kit (Biotium #30025–1). Briefly, RAW cells, primary BMDMS, or CD300lf-expressing Huh-7 cells were plated at 2x105 per well of a 24well plate. After overnight growth at 37°C/5% CO2, medium was replaced with DMEM-10 medium containing a specific pharmacological inhibitor. Treated cells were then placed back at 37°C/5% CO2 for a 24-hour incubation period. The following day, cell viability was calculated according to the manufacturer’s recommendations.
To measure the viability of RAW cells in glutamine-free media, cells were plated at 5x105 per well in a 6-well plate. After overnight growth at 37°C/5% CO2, media was replaced with glutamine-free DMEM-10 medium for 8 hours. After the incubation, cells were scrapped with a cell scrapper and cell viability was measured using trypan blue staining on a Life Technologies Countess 3 automated cell counter assay platform. Cell viability was calculated as the percent of live cells in glutamine-free media treated vs. untreated controls.
Metabolic flux analysis
5x105 RAW cells were plated in 6-well plates and infected with MNV-1 or mock-infected as described above. Following the removal of the virus inoculum, fresh medium was added containing uniformly labeled 13C5 glucose or glutamine and incubated at 37°C/5% CO2 for 8 hours. Following the 8-hour incubation, cells were washed 2x DPBS (+Calcium and +Magnesium Chloride, Gibco #14040) and 300 μL of ice-cold methanol was added. Wells were scraped with a cell lifter and the volume was transferred to a fresh Eppendorf tube where 300 μL of water containing 1μg of norvaline internal standard was added to each tube. Next, 600μL of high-performance liquid-chromatography grade chloroform was added to each tube to isolate nonpolar lipid content from the sample matrix. Tubes were then vortexed at 4°C for 30 minutes and centrifuged at 17,000 x g for 15 minutes at 4°C to separate contents into an upper aqueous layer and lower chloroform layer. The upper phase was collected into new tubes which were then dried by vacuum centrifugation in a SpeedVac for 5 hours at room temperature. After drying, samples were stored at -80°C until GC-MS analysis.
For polar metabolite analysis, dried samples were derivatized with 30μL of 2% methoxyamine hydrochloride in pyridine at 45°C for 1 hour under constant shaking. Then 30 μL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MBTSTFA) + 1% tertbutyldimetheylchlorosilane (TBDMCS) was added, and samples were further incubated at 45°C for 30 min. Derivatized samples were then transferred to GC vials with glass inserts and loaded for autosampler injection. GC-MS analysis was performed using an Agilent 7890 GC equipped with a 30m DB-35MS UI capillary column connected to an Agilent 5977B MS. Samples were run with 1 mL/min helium flow with the following heating cycle for the GC oven: 100°C for 1 minute, ramp of 3.5°C/min to 255°C, ramp of 15°C to 320°C, then held at 320°C for 3 min to a total run time of 52.6 min. MS source was held at 230°C and quadrupole at 150°C. Data was acquired in scan mode (70–600 m/z). The relative abundance of metabolites was calculated from the integrated signal of all potentially labeled ions for each metabolite fragment. Metabolite levels were normalized to the norvaline internal standard and quantified using 10-point calibration with external standards for 36 polar metabolites. Mass Isotopomer Distributions (MIDs) were corrected for natural isotope abundances and tracer purity using IsoCor.
Glutaminase activity assay
Glutaminase enzymatic activity was assessed with the commercially available Cohesion Biosciences Microplate Assay Kit (#CAK1065). Briefly, RAW or CD300lf-expressing Huh-7 cells were either mock- or MNV-infected as described above. After 8 hours of incubation at 37°C/5% CO2, cells were sonicated for 10 seconds 30x and kit contents added per the manufacturer’s instructions. Samples were transferred to a 96-wellplate and absorbance at 620 nm was measured in a Synergy H1 plate reader. Glutaminase activity was calculated following the manufacturer’s instructions.
Overexpression of viral proteins
A total of 2.0 μg of plasmid DNA harboring sequences for individual MNV non-structural proteins or green fluorescent protein (GFP) was added to 100 μL of Opti-MEM media (Thermo Fischer Scientific #11058–02 with L-Glutamine and HEPES). Then, 8 μL of FuGENE HD Transfection reagent (FuGENE #0000553572) was added to the Opti-MEM plasmid mix and centrifuged for 10 s at 8000 x g. Plasmid mix was then incubated for 15 minutes at room temperature. After the incubation, the plasmid mix was added to a separate Eppendorf tube containing 1.6x106 CD300lf-expressing Huh-7 cells and incubated for 10 minutes at room temperature. After the incubation, 500 μL of the cell suspension was plated per well in a 6-well plate and incubated at 37°C/5% CO2 for 24–48 hours. After the incubation period, two of the wells were used to confirm successful expression of the viral protein via western blot analysis as described above. The remaining well was used to analyze glutaminase activity with the commercially available Cohesion Biosciences Microplate Assay Kit as described above.
Supporting information
S1 Fig. Cell viability assays of indicated cell lines.
(A-B) RAW 264.7 cells were treated with indicated concentrations of (A) Oligomycin-A, (B) CB839, or vehicle control (DMSO) for either 8 or 24 hours, respectively. Cell viability was measured using Resazurin or WST-1 reagent. (C) Primary bone marrow-derived macrophages (BMDMs) were treated with CB839 or vehicle control at the indicated concentrations for 24 hours. Cell viability was measured using WST-1 reagent. (D) RAW 264.7 cells were incubated with glutamine free or replete medium for 8 hours. Cell viability was measured using trypan blue staining on a Life Technologies Countess 3 automated cell counter assay platform. (E) Huh-7 CD300lf cells were treated with indicated concentrations of CB839 for 24 hrs. Cell viability was measured using WST-1 reagent. Experiments represent combined data from at least two independent experiments with two technical replicates each.
https://doi.org/10.1371/journal.ppat.1011909.s001
(TIF)
S2 Fig. MNV-1 infection does not alter the intracellular amino acid pool.
(A-B) RAW 264.7 cells were either mock-infected or infected with MNV-1 for 1 hour at an MOI of 5. The virus inoculum was removed and replaced with medium containing 13C5-glutamine for 8 hours. Intracellular metabolites and amino acids were extracted with ice-cold methanol and measured by mass spectrometry. Experiments represent combined data from two independent experiments with four technical repeats. Statistical analysis was performed using Two-tailed Students-tests. ***, P<0.001; *, P<0.05.
https://doi.org/10.1371/journal.ppat.1011909.s002
(TIF)
S3 Fig. Validation of MNV-1 nonstructural protein expression.
(A-E) Huh-7 CD300lf cells were transfected with plasmids encoding the indicated MNV-1 nonstructural protein or green fluorescent protein (GFP). Transfected cells were incubated for 24–48 hours. Western blot analysis was performed to confirm successful expression. β-actin was used as a loading control. Data shows representative western blots from three independent experiments.
https://doi.org/10.1371/journal.ppat.1011909.s003
(TIF)
Acknowledgments
We thank past and present members of the Wobus lab for helpful discussions, and Drs. Ian Goodfellow, Vernon Ward, Stefan Taube, Jason Mackenzie, Timothy J. Nice, and Craig Wilen for the indicated reagents.
References
- 1. Sumbria D, Berber E, Mathayan M, Rouse BT. Virus Infections and Host Metabolism—Can We Manage the Interactions? Front Immunol. 2021 Feb 3;11:594963. pmid:33613518
- 2. Olenchock BA, Rathmell JC, Vander Heiden MG. Biochemical Underpinnings of Immune Cell Metabolic Phenotypes. Immunity. 2017 May 16;46(5):703–13. pmid:28514672
- 3. Kaur J, Debnath J. Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol. 2015 Aug;16(8):461–72. pmid:26177004
- 4. Goodwin CM, Xu S, Munger J. Stealing the Keys to the Kitchen: Viral Manipulation of the Host Cell Metabolic Network. Trends Microbiol. 2015 Dec;23(12):789–98. pmid:26439298
- 5. Caradonna KL, Engel JC, Jacobi D, Lee CH, Burleigh BA. Host metabolism regulates intracellular growth of Trypanosoma cruzi. Cell Host Microbe. 2013 Jan 16;13(1):108–17. pmid:23332160
- 6. Bravo-Santano N, Ellis JK, Mateos LM, Calle Y, Keun HC, Behrends V, et al. Intracellular Staphylococcus aureus Modulates Host Central Carbon Metabolism To Activate Autophagy. mSphere. 2018 Aug 8;3(4):e00374–18. pmid:30089650
- 7. Thaker SK, Ch’ng J, Christofk HR. Viral hijacking of cellular metabolism. BMC Biol. 2019 Jul 18;17(1):59. pmid:31319842
- 8. Sanchez EL, Lagunoff M. Viral activation of cellular metabolism. Virology. 2015 May;479–480:609–18. pmid:25812764
- 9. Fontaine KA, Sanchez EL, Camarda R, Lagunoff M. Dengue virus induces and requires glycolysis for optimal replication. J Virol. 2015 Feb;89(4):2358–66. pmid:25505078
- 10. Fontaine KA, Camarda R, Lagunoff M. Vaccinia virus requires glutamine but not glucose for efficient replication. J Virol. 2014 Apr;88(8):4366–74. pmid:24501408
- 11. Thai M, Thaker SK, Feng J, Du Y, Hu H, Ting Wu T, et al. MYC-induced reprogramming of glutamine catabolism supports optimal virus replication. Nat Commun. 2015 Nov 12;6:8873. pmid:26561297
- 12. Chambers JW, Maguire TG, Alwine JC. Glutamine Metabolism Is Essential for Human Cytomegalovirus Infection. J Virol. 2010 Feb;84(4):1867–73. pmid:19939921
- 13. Thaker SK, Chapa T, Garcia G, Gong D, Schmid EW, Arumugaswami V, et al. Differential metabolic reprogramming by Zika virus promotes cell death in human versus mosquito cells. Cell Metab. 2019 May 7;29(5):1206–1216.e4. pmid:30827860
- 14. Dias SSG, Cunha-Fernandes T, Souza-Moreira L, Soares VC, Lima GB, Azevedo-Quintanilha IG, et al. Metabolic reprogramming and lipid droplets are involved in Zika virus replication in neural cells. J Neuroinflammation. 2023 Mar 8;20:61. pmid:36882750
- 15. Shrinet J, Shastri JS, Gaind R, Bhavesh NS, Sunil S. Serum metabolomics analysis of patients with chikungunya and dengue mono/co-infections reveals distinct metabolite signatures in the three disease conditions. Sci Rep. 2016 Nov 15;6:36833. pmid:27845374
- 16. Syed GH, Amako Y, Siddiqui A. Hepatitis C Virus Hijacks Host Lipid Metabolism. Trends Endocrinol Metab. 2010 Jan;21(1):33. pmid:19854061
- 17. Gualdoni GA, Mayer KA, Kapsch AM, Kreuzberg K, Puck A, Kienzl P, et al. Rhinovirus induces an anabolic reprogramming in host cell metabolism essential for viral replication. Proc Natl Acad Sci U S A. 2018 Jul 24;115(30):E7158–65. pmid:29987044
- 18. Sanchez EL, Carroll PA, Thalhofer AB, Lagunoff M. Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival. PLoS Pathog. 2015 Jul;11(7):e1005052. pmid:26197457
- 19. Sanchez EL, Pulliam TH, Dimaio TA, Thalhofer AB, Delgado T, Lagunoff M. Glycolysis, Glutaminolysis, and Fatty Acid Synthesis Are Required for Distinct Stages of Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication. J Virol. 2017 May 15;91(10):e02237–16. pmid:28275189
- 20. Mullen PJ, Garcia G, Purkayastha A, Matulionis N, Schmid EW, Momcilovic M, et al. SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition. Nat Commun. 2021 Mar 25;12(1):1876. pmid:33767183
- 21. Clark SA, Vazquez A, Furiya K, Splattstoesser MK, Bashmail AK, Schwartz H, et al. Rewiring of the Host Cell Metabolome and Lipidome during Lytic Gammaherpesvirus Infection Is Essential for Infectious-Virus Production. J Virol. 2023 Jun 29;97(6):e0050623. pmid:37191529
- 22. Campillay-Véliz CP, Carvajal JJ, Avellaneda AM, Escobar D, Covián C, Kalergis AM, et al. Human Norovirus Proteins: Implications in the Replicative Cycle, Pathogenesis, and the Host Immune Response. Front Immunol. 2020 Jun 16;11:961. pmid:32612600
- 23. Ahmed SM, Hall AJ, Robinson AE, Verhoef L, Premkumar P, Parashar UD, et al. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect Dis. 2014 Aug;14(8):725–30. pmid:24981041
- 24. Bartsch SM, Lopman BA, Ozawa S, Hall AJ, Lee BY. Global Economic Burden of Norovirus Gastroenteritis. PLoS One. 2016 Apr 26;11(4):e0151219. pmid:27115736
- 25. de Graaf M, van Beek J, Koopmans MPG. Human norovirus transmission and evolution in a changing world. Nat Rev Microbiol. 2016 Jul;14(7):421–33. pmid:27211790
- 26. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, et al. Foodborne Illness Acquired in the United States—Major Pathogens. Emerg Infect Dis. 2011 Jan;17(1):7–15. pmid:21192848
- 27. Netzler NE, Enosi Tuipulotu D, White PA. Norovirus antivirals: Where are we now? Med Res Rev. 2019 May;39(3):860–86. pmid:30584800
- 28. Ettayebi K, Crawford SE, Murakami K, Broughman JR, Karandikar U, Tenge VR, et al. Replication of human noroviruses in stem cell-derived human enteroids. Science. 2016 Sep 23;353(6306):1387–93. pmid:27562956
- 29. Estes MK, Ettayebi K, Tenge VR, Murakami K, Karandikar U, Lin SC, et al. Human Norovirus Cultivation in Nontransformed Stem Cell-Derived Human Intestinal Enteroid Cultures: Success and Challenges. Viruses. 2019 Jul 11;11(7):638. pmid:31336765
- 30. Mirabelli C, Jones MK, Young VL, Kolawole AO, Owusu I, Shan M, et al. Human Norovirus Triggers Primary B Cell Immune Activation In Vitro. mBio. 2022 Apr 26;13(2):e0017522. pmid:35404121
- 31. Wobus CE, Thackray LB, Virgin HW. Murine Norovirus: a Model System To Study Norovirus Biology and Pathogenesis. J Virol. 2006 Jun;80(11):5104–12. pmid:16698991
- 32. Thackray LB, Wobus CE, Chachu KA, Liu B, Alegre ER, Henderson KS, et al. Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence. J Virol. 2007 Oct;81(19):10460–73. pmid:17652401
- 33. Ingle H, Makimaa H, Aggarwal S, Deng H, Foster L, Li Y, et al. IFN-λ derived from nonsusceptible enterocytes acts on tuft cells to limit persistent norovirus. Sci Adv. 2023 Sep 15;9(37):eadi2562.
- 34. Wobus CE. The Dual Tropism of Noroviruses. J Virol. 2018 Jul 31;92(16):e01010–17.
- 35. Passalacqua KD, Lu J, Goodfellow I, Kolawole AO, Arche JR, Maddox RJ, et al. Glycolysis Is an Intrinsic Factor for Optimal Replication of a Norovirus. mBio. 2019 Mar 12;10(2):e02175–18. pmid:30862747
- 36. Katt WP, Cerione RA. Glutaminase regulation in cancer cells: a druggable chain of events. Drug Discov Today. 2014 Apr;19(4):450–7. pmid:24140288
- 37. Wang L, Li JJ, Guo LY, Li P, Zhao Z, Zhou H, et al. Molecular link between glucose and glutamine consumption in cancer cells mediated by CtBP and SIRT4. Oncogenesis. 2018 Mar 13;7(3):26. pmid:29540733
- 38. Strelko CL, Lu W, Dufort FJ, Seyfried TN, Chiles TC, Rabinowitz JD, et al. Itaconic acid is a mammalian metabolite induced during macrophage activation. J Am Chem Soc. 2011 Oct 19;133(41):16386–9. pmid:21919507
- 39. Michelucci A, Cordes T, Ghelfi J, Pailot A, Reiling N, Goldmann O, et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7820–5. pmid:23610393
- 40. O’Neill LAJ, Artyomov MN. Itaconate: the poster child of metabolic reprogramming in macrophage function. Nat Rev Immunol. 2019 May;19(5):273–81. pmid:30705422
- 41. Cordes T, Wallace M, Michelucci A, Divakaruni AS, Sapcariu SC, Sousa C, et al. Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to Modulate Intracellular Succinate Levels. J Biol Chem. 2016 Jul 1;291(27):14274–84. pmid:27189937
- 42. Mullen AR, Wheaton WW, Jin ES, Chen PH, Sullivan LB, Cheng T, et al. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature. 2011 Nov 20;481(7381):385–8. pmid:22101431
- 43. Allen CNS, Arjona SP, Santerre M, Sawaya BE. Hallmarks of Metabolic Reprogramming and Their Role in Viral Pathogenesis. Viruses. 2022 Mar 14;14(3):602. pmid:35337009
- 44. Walker MC, van der Donk WA. The Many Roles of Glutamate in Metabolism. J Ind Microbiol Biotechnol. 2016 Mar;43(0):419–30. pmid:26323613
- 45. Taciak B, Białasek M, Braniewska A, Sas Z, Sawicka P, Kiraga Ł, et al. Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages. PLoS One. 2018;13(6):e0198943. pmid:29889899
- 46. Cheng ML, Chien KY, Lai CH, Li GJ, Lin JF, Ho HY. Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection. Cells. 2020 Feb 18;9(2):473. pmid:32085644
- 47. Rubin H. Deprivation of glutamine in cell culture reveals its potential for treating cancer. Proc Natl Acad Sci U S A. 2019 Apr 2;116(14):6964–8. pmid:30877243
- 48. Gwangwa MV, Joubert AM, Visagie MH. Effects of glutamine deprivation on oxidative stress and cell survival in breast cell lines. Biol Res. 2019 Mar 27;52(1):15. pmid:30917872
- 49. Darnell JE, Eagle H. Glucose and glutamine in poliovirus production by HeLa cells. Virology. 1958 pmid:13593191
- 50. Matés JM, Segura JA, Campos-Sandoval JA, Lobo C, Alonso L, Alonso FJ, et al. Glutamine homeostasis and mitochondrial dynamics. The International Journal of Biochemistry & Cell Biology. 2009 Oct 1;41(10):2051–61. pmid:19703661
- 51. Zhao L, Huang Y, Tian C, Taylor L, Curthoys N, Wang Y, et al. Interferon-α regulates glutaminase 1 promoter through STAT1 phosphorylation: relevance to HIV-1 associated neurocognitive disorders. PLoS One. 2012;7(3):e32995.
- 52. Katt WP, Lukey MJ, Cerione RA. A tale of two glutaminases: homologous enzymes with distinct roles in tumorigenesis. Future Med Chem. 2017 Jan;9(2):223–43. pmid:28111979
- 53. Komili S, Silver PA. Coupling and coordination in gene expression processes: a systems biology view. Nat Rev Genet. 2008 Jan;9(1):38–48. pmid:18071322
- 54. Allonso D, Andrade IS, Conde JN, Coelho DR, Rocha DCP, da Silva ML, et al. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity. J Virol. 2015 Dec;89(23):11871–83.
- 55. Graziano VR, Walker FC, Kennedy EA, Wei J, Ettayebi K, Strine MS, et al. CD300lf is the primary physiologic receptor of murine norovirus but not human norovirus. PLoS Pathog. 2020 Apr 6;16(4):e1008242. pmid:32251490
- 56. Thai M, Graham NA, Braas D, Nehil M, Komisopoulou E, Kurdistani SK, et al. Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication. Cell Metab. 2014 Apr 1;19(4):694–701. pmid:24703700
- 57. Lévy PL, Duponchel S, Eischeid H, Molle J, Michelet M, Diserens G, et al. Hepatitis C virus infection triggers a tumor-like glutamine metabolism. Hepatology. 2017 Mar;65(3):789–803. pmid:27863447
- 58. Robinson BA, Van Winkle JA, McCune BT, Peters AM, Nice TJ. Caspase-mediated cleavage of murine norovirus NS1/2 potentiates apoptosis and is required for persistent infection of intestinal epithelial cells. PLoS Pathog. 2019 Jul;15(7):e1007940. pmid:31329638
- 59. Lee S, Liu H, Wilen CB, Sychev ZE, Desai C, Hykes BL, et al. A Secreted Viral Nonstructural Protein Determines Intestinal Norovirus Pathogenesis. Cell Host Microbe. 2019 Jun 12;25(6):845–857.e5. pmid:31130511
- 60. Mikaeloff F, Svensson Akusjärvi S, Ikomey GM, Krishnan S, Sperk M, Gupta S, et al. Trans cohort metabolic reprogramming towards glutaminolysis in long-term successfully treated HIV-infection. Commun Biol. 2022 Jan 11;5(1):27. pmid:35017663
- 61. Ripoli M, D’Aprile A, Quarato G, Sarasin-Filipowicz M, Gouttenoire J, Scrima R, et al. Hepatitis C virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1 alpha-mediated glycolytic adaptation. J Virol. 2010 Jan;84(1):647–60. pmid:19846525
- 62. He ST, Lee DY, Tung CY, Li CY, Wang HC. Glutamine Metabolism in Both the Oxidative and Reductive Directions Is Triggered in Shrimp Immune Cells (Hemocytes) at the WSSV Genome Replication Stage to Benefit Virus Replication. Front Immunol. 2019;10:2102. pmid:31555294
- 63. Chen IT, Lee DY, Huang YT, Kou GH, Wang HC, Chang GD, et al. Six Hours after Infection, the Metabolic Changes Induced by WSSV Neutralize the Host’s Oxidative Stress Defenses. Sci Rep. 2016 Jun 9;6:27732. pmid:27279169
- 64. Keshavarz M, Solaymani-Mohammadi F, Namdari H, Arjeini Y, Mousavi MJ, Rezaei F. Metabolic host response and therapeutic approaches to influenza infection. Cell Mol Biol Lett. 2020;25:15. pmid:32161622
- 65. Sosnovtsev SV, Belliot G, Chang KO, Prikhodko VG, Thackray LB, Wobus CE, et al. Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells. J Virol. 2006 Aug;80(16):7816–31. pmid:16873239
- 66. Li Y, Webster-Cyriaque J, Tomlinson CC, Yohe M, Kenney S. Fatty acid synthase expression is induced by the Epstein-Barr virus immediate-early protein BRLF1 and is required for lytic viral gene expression. J Virol. 2004 Apr;78(8):4197–206. pmid:15047835
- 67. Shin HJ, Park YH, Kim SU, Moon HB, Park DS, Han YH, et al. Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase. J Biol Chem. 2011 Aug 26;286(34):29872–81. pmid:21690090
- 68. Viola A, Munari F, Sánchez-Rodríguez R, Scolaro T, Castegna A. The Metabolic Signature of Macrophage Responses. Front Immunol. 2019;10:1462. pmid:31333642
- 69. Escoll P, Song OR, Viana F, Steiner B, Lagache T, Olivo-Marin JC, et al. Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages. Cell Host Microbe. 2017 Sep 13;22(3):302–316.e7. pmid:28867389
- 70. Stavru F, Bouillaud F, Sartori A, Ricquier D, Cossart P. Listeria monocytogenes transiently alters mitochondrial dynamics during infection. Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3612–7. pmid:21321208
- 71. Xavier MN, Winter MG, Spees AM, den Hartigh AB, Nguyen K, Roux CM, et al. PPARγ-mediated increase in glucose availability sustains chronic Brucella abortus infection in alternatively activated macrophages. Cell Host Microbe. 2013 Aug 14;14(2):159–70.
- 72. Bichiou H, Bouabid C, Rabhi I, Guizani-Tabbane L. Transcription Factors Interplay Orchestrates the Immune-Metabolic Response of Leishmania Infected Macrophages. Front Cell Infect Microbiol. 2021;11:660415. pmid:33898331
- 73. Wu D, Sanin DE, Everts B, Chen Q, Qiu J, Buck MD, et al. Type 1 Interferons Induce Changes in Core Metabolism that Are Critical for Immune Function. Immunity. 2016 Jun 21;44(6):1325–36. pmid:27332732
- 74. Yoo HC, Yu YC, Sung Y, Han JM. Glutamine reliance in cell metabolism. Exp Mol Med. 2020 Sep;52(9):1496–516. pmid:32943735
- 75. Hosios AM, Hecht VC, Danai LV, Johnson MO, Rathmell JC, Steinhauser ML, et al. Amino Acids Rather than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells. Dev Cell. 2016 Mar 7;36(5):540–9. pmid:26954548
- 76. O’Neill CP, Gilligan KE, Dwyer RM. Role of Extracellular Vesicles (EVs) in Cell Stress Response and Resistance to Cancer Therapy. Cancers (Basel). 2019 Jan 24;11(2):136. pmid:30682793
- 77. Santiana M, Ghosh S, Ho BA, Rajasekaran V, Du WL, Mutsafi Y, et al. Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission. Cell Host Microbe. 2018 Aug 8;24(2):208–220.e8. pmid:30092198
- 78. Owusu IA, Passalacqua KD, Mirabelli C, Lu J, Young VL, Hosmillo M, et al. Akt Plays Differential Roles during the Life Cycles of Acute and Persistent Murine Norovirus Strains in Macrophages. J Virol. 2022 Feb 9;96(3):e0192321. pmid:34787460
- 79. Brocard M, Lu J, Hall B, Borah K, Moller-Levet C, Georgana I, et al. Murine Norovirus Infection Results in Anti-inflammatory Response Downstream of Amino Acid Depletion in Macrophages. J Virol. 2021 Sep 27;95(20):e0113421. pmid:34346771
- 80. Karandikar UC, Crawford SE, Ajami NJ, Murakami K, Kou B, Ettayebi K, et al. Detection of human norovirus in intestinal biopsies from immunocompromised transplant patients. J Gen Virol. 2016 Sep;97(9):2291–300. pmid:27412790
- 81. Green KY, Kaufman SS, Nagata BM, Chaimongkol N, Kim DY, Levenson EA, et al. Human norovirus targets enteroendocrine epithelial cells in the small intestine. Nat Commun. 2020 Jun 2;11(1):2759. pmid:32488028
- 82. Mirabelli C, Santos-Ferreira N, Gillilland MG, Cieza RJ, Colacino JA, Sexton JZ, et al. Human Norovirus Efficiently Replicates in Differentiated 3D-Human Intestinal Enteroids. Journal of Virology. 2022 Nov 7;96(22):e00855–22. pmid:36342297
- 83. Hyde JL, Mackenzie JM. Subcellular localization of the MNV-1 ORF1 proteins and their potential roles in the formation of the MNV-1 replication complex. Virology. 2010 Oct 10;406(1):138–48. pmid:20674956
- 84. Jahun AS, Sorgeloos F, Chaudhry Y, Arthur SE, Hosmillo M, Georgana I, et al. Leaked genomic and mitochondrial DNA contribute to the host response to noroviruses in a STING-dependent manner. Cell Rep. 2023 Mar 28;42(3):112179. pmid:36943868
- 85. Hwang S, Alhatlani B, Arias A, Caddy SL, Christodoulou C, Cunha JB, et al. Murine norovirus: propagation, quantification, and genetic manipulation. Curr Protoc Microbiol. 2014 May 1;33:15K.2.1–61. pmid:24789596
- 86. Gonzalez-Hernandez MB, Bragazzi Cunha J, Wobus CE. Plaque assay for murine norovirus. J Vis Exp. 2012 Aug 22;(66):e4297. pmid:22951568
- 87. Chatot CL, Lawry JR, Germain B, Ziomek CA. Analysis of glutaminase activity and RNA expression in preimplantation mouse embryos. Mol Reprod Dev. 1997 Jul;47(3):248–54. pmid:9170104
- 88. Wobus CE, Peiper AM, McSweeney AM, Young VL, Chaika M, Lane MS, et al. Murine Norovirus: Additional Protocols for Basic and Antiviral Studies. Curr Protoc. 2023 Jul;3(7):e828. pmid:37478303
- 89. Sambrook J, Russell DW. SDS-Polyacrylamide Gel Electrophoresis of Proteins. CSH Protoc. 2006 Sep 1;2006(4):pdb.prot4540. pmid:22485921
- 90. Davies C, Ward VK. Expression of the NS5 (VPg) Protein of Murine Norovirus Induces aG1/S Phase Arrest. PLoS One. 2016;11(8):e0161582. pmid:27556406
- 91. Baker ES, Luckner SR, Krause KL, Lambden PR, Clarke IN, Ward VK. Inherent structural disorder and dimerisation of murine norovirus NS1-2 protein. PLoS One. 2012;7(2):e30534. pmid:22347381