Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity.

Kidney cancer, one of the ten most prevalent malignancies in the world, has exhibited increased incidence over the last decade. The most common subtype is "clear cell" renal cell carcinoma (ccRCC), which features consistent metabolic abnormalities, such as highly elevated glycogen and lipid deposition. By integrating metabolomics, genomic, and transcriptomic data, we determined that enzymes in multiple metabolic pathways are universally depleted in human ccRCC tumors, which are otherwise genetically heterogeneous. Notably, the expression of key urea cycle enzymes, including arginase 2 (ARG2) and argininosuccinate synthase 1 (ASS1), is strongly repressed in ccRCC. Reduced ARG2 activity promotes ccRCC tumor growth through at least two distinct mechanisms: conserving the critical biosynthetic cofactor pyridoxal phosphate and avoiding toxic polyamine accumulation. Pharmacological approaches to restore urea cycle enzyme expression would greatly expand treatment strategies for ccRCC patients, where current therapies only benefit a subset of those afflicted with renal cancer.

ChREBP, a glucose-responsive transcriptional factor, enhances glucose metabolism to support biosynthesis in human cytomegalovirus-infected cells.

Carbohydrate-response element binding protein (ChREBP) plays a key role in regulating glucose metabolism and de novo lipogenesis in metabolic tissues and cancer cells. Here we report that ChREBP is also a critical regulator of the metabolic alterations induced during human cytomegalovirus (HCMV) infection. The expression of both ChREBP-α and ChREBP-ß is robustly induced in HCMV-infected human fibroblasts; this induction is required for efficient HCMV infection. Depletion of ChREBP in HCMV-infected cells results in reduction of HCMV-induced glucose transporter 4 and glucose transporter 2 expression, leading to inhibition of glucose uptake, lactate production, nucleotide biosynthesis, and NADPH generation. We previously reported that HCMV infection induces lipogenesis through the activation of sterol regulatory element binding protein 1, which is mediated by the induction of PKR-like endoplasmic reticulum kinase. Data from the present study show that HCMV-induced lipogenesis is also controlled by the induction of ChREBP, in a second mechanism involved in the regulation of HCMV-induced de novo lipogenesis. These results suggest that ChREBP plays a key role in reprogramming glucose and lipid metabolism in HCMV infection.

PKR-like endoplasmic reticulum kinase is necessary for lipogenic activation during HCMV infection.

PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α) to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1), resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV) infection in human fibroblasts (HF) induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA), resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1) protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP complexes for SREBP1 processing.

Human cytomegalovirus infection induces adipocyte-like lipogenesis through activation of sterol regulatory element binding protein 1.

Sterol regulatory element binding proteins (SREBPs) are essential transcriptional factors that control expression of lipogenic genes and adipocyte differentiation. Human cytomegalovirus (HCMV) infection has been shown to require the induction of lipogenesis. Here we show that the induction of lipogenesis and expression of key lipogenic enzymes in human fibroblasts occurs by 24 h post-HCMV infection. This activation correlates with increased cleavage of the SREBP1 precursors to form the mature active transcription factors that enter the nucleus to transcriptionally activate lipogenic genes. SREBP1 cleavage is normally inhibited by increased sterol levels; however, our data show that this level of control is overridden in infected cells to allow constitutive activation of lipogenesis. This process requires viral protein synthesis, since UV-irradiated HCMV cannot activate SREBP cleavage. The cleavage of SREBP1 requires it to be in complex with SREBP cleavage activation protein (SCAP). Depleting SCAP using short hairpin RNA (shRNA) showed that SREBP1 cleavage and the induction of lipogenic genes and lipid synthesis are all inhibited in HCMV-infected cells. As a result, production of infectious virions is reduced in SCAP-depleted cells. Thus, the SCAP-mediated mechanism for SREBP cleavage is utilized by HCMV during infection. Our studies suggest that HCMV induces adipocyte-like lipogenesis and overrides normal sterol feedback controls in order to maintain high levels of constitutive lipid synthesis during infection.

Human cytomegalovirus infection maintains mTOR activity and its perinuclear localization during amino acid deprivation.

The mammalian target of rapamycin (mTOR) kinase is present in 2 functionally distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). Active mTORC1 mediates phosphorylation of eIF4E-binding protein (4E-BP) and p70 S6 kinase (S6K), which is important for maintaining translation. During human cytomegalovirus (HCMV) infection, cellular stress responses are activated that normally inhibit mTORC1; however, previous data show that HCMV infection circumvents stress responses and maintains mTOR kinase activity. Amino acid deprivation is a stress response that normally inhibits mTORC1 activity. Amino acids can signal to mTORC1 through the Rag proteins, which promote the colocalization of mTORC1 with its activator Rheb-GTP in a perinuclear region, thereby inducing 4E-BP and S6K phosphorylation. As expected, our results show that amino acid depletion in mock-infected cells caused loss of mTORC1 activity and loss of the perinuclear localization; however, there was no loss of activity or perinuclear localization in HCMV-infected cells where the perinuclear localization of Rheb-GTP and mTOR coincided with the perinuclear assembly compartment (AC). This suggested that HCMV infection bypasses normal Rag-dependent amino acid signaling. This was demonstrated by short hairpin RNA (shRNA) depletion of Rag proteins, which had little effect on mTORC1 activity in infected cells but inhibited activity in mock-infected cells. Our data show that HCMV maintains mTORC1 activity in an amino acid- and Rag-independent manner through the colocalization of mTOR and Rheb-GTP, which occurs in association with the formation of the AC, thus bypassing inhibition that may result from lowered amino acid levels.

The changing role of mTOR kinase in the maintenance of protein synthesis during human cytomegalovirus infection.

The mammalian target of rapamycin (mTOR) kinase occurs in mTOR complex 1 (mTORC1) and complex 2 (mTORC2), primarily differing by the substrate specificity factors raptor (in mTORC1) and rictor (in mTORC2). Both complexes are activated during human cytomegalovirus (HCMV) infection. mTORC1 phosphorylates eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) and p70S6 kinase (S6K) in uninfected cells, and this activity is lost upon raptor depletion. In infected cells, 4E-BP1 and S6K phosphorylation is maintained when raptor or rictor is depleted, suggesting that either mTOR complex can phosphorylate 4E-BP1 and S6K. Studies using the mTOR inhibitor Torin1 show that phosphorylation of 4E-BP1 and S6K in infected cells depends on mTOR kinase. The total levels of 4E-BP1 and viral proteins representative of all temporal classes were lowered by Torin1 treatment and by raptor, but not rictor, depletion, suggesting that mTORC1 is involved in the production of all classes of HCMV proteins. We also show that Torin1 inhibition of mTOR kinase is rapid and most deleterious at early times of infection. While Torin1 treatment from the beginning of infection significantly inhibited translation of viral proteins, its addition at later time points had far less effect. Thus, with respect to mTOR's role in translational control, HCMV depends on it early in infection but can bypass it at later times of infection. Depletion of 4E-BP1 by use of short hairpin RNAs (shRNAs) did not rescue HCMV growth in Torin1-treated human fibroblasts as it has been shown to in murine cytomegalovirus (MCMV)-infected 4E-BP1(-/-) mouse embryo fibroblasts (MEFs), suggesting that during HCMV infection mTOR kinase has additional roles other than phosphorylating and inactivating 4E-BP1. Overall, our data suggest a dynamic relationship between HCMV and mTOR kinase which changes during the course of infection.

Human cytomegalovirus activates glucose transporter 4 expression to increase glucose uptake during infection.

Glucose transport into mammalian cells is mediated by a group of glucose transporters (GLUTs) on the plasma membrane. Human cytomegalovirus (HCMV)-infected human fibroblasts (HFs) demonstrate significantly increased glucose consumption compared to mock-infected cells, suggesting a possible alteration in glucose transport during infection. Inhibition of GLUTs by using cytochalasin B indicated that infected cells utilize GLUT4, whereas normal HFs use GLUT1. Quantitative reverse transcription-PCR and Western analysis confirmed that GLUT4 levels are greatly increased in infected cells. In contrast, GLUT1 was eliminated by a mechanism involving the HCMV major immediate-early protein IE72. The HCMV-mediated induction of GLUT4 circumvents characterized controls of GLUT4 expression that involve serum stimulation, glucose concentration, and nuclear functions of ATP-citrate lyase (ACL). In infected cells the well-characterized Akt-mediated translocation of GLUT4 to the cell surface is also circumvented; GLUT4 localized on the surface of infected cells that were serum starved and had Akt activity inhibited. The significance of GLUT4 induction for the success of HCMV infection was indicated using indinavir, a drug that specifically inhibits glucose uptake by GLUT4. The addition of the drug inhibited glucose uptake in infected cells as well as viral production. Our data show that HCMV-specific mechanisms are used to replace GLUT1, the normal HF GLUT, with GLUT4, the major glucose transporter in adipose tissue, which has a 3-fold-higher glucose transport capacity.

Role of the endoplasmic reticulum chaperone BiP, SUN domain proteins, and dynein in altering nuclear morphology during human cytomegalovirus infection.

The process of assembly and egress of human cytomegalovirus (HCMV) virions requires significant morphological alterations of the nuclear and cytoplasmic architecture. In the studies presented we show that the nuclear periphery is dramatically altered, especially near the cytoplasmic assembly compartment, where the nuclear lamina is specifically rearranged, the outer nuclear membrane is altered, and the nucleus becomes permeable to large molecules. In addition, the tethering of the inner and outer nuclear membranes is lost during infection due to a decrease in levels of the SUN domain proteins. We previously demonstrated that the endoplasmic reticulum protein BiP functions as a component of the assembly compartment and disruption of BiP causes the loss of assembly compartment integrity. In this study we show that the depletion of BiP, and the loss of assembly compartment integrity, results in the loss of virally induced lamina rearrangement and morphology of the nucleus that is characteristic of HCMV infection. BiP functions in lamina rearrangement through its ability to affect lamin phosphorylation. Depletion of BiP and disruption of the assembly compartment result in the loss of lamin phosphorylation. The dependency of lamin phosphorylation on BiP correlates with an interaction between BiP and UL50. Finally, we confirm previous data (S. V. Indran, M. E. Ballestas, and W. J. Britt, J. Virol. 84:3162-3177, 2010) suggesting an involvement of dynein in assembly compartment formation and extend this observation by showing that when dynein is inhibited, the nuclear morphology characteristic of an HCMV infection is lost. Our data suggest a highly integrated assembly-egress continuum.

Glutamine metabolism is essential for human cytomegalovirus infection.

Human fibroblasts infected with human cytomegalovirus (HCMV) were more viable than uninfected cells during glucose starvation, suggesting that an alternate carbon source was used. We have determined that infected cells require glutamine for ATP production, whereas uninfected cells do not. This suggested that during infection, glutamine is used to fill the tricarboxylic acid (TCA) cycle (anaplerosis). In agreement with this, levels of glutamine uptake and ammonia production increased in infected cells, as did the activities of glutaminase and glutamate dehydrogenase, the enzymes needed to convert glutamine to alpha-ketoglutarate to enter the TCA cycle. Infected cells starved for glutamine beginning 24 h postinfection failed to produce infectious virions. Both ATP and viral production could be rescued in glutamine-starved cells by the TCA intermediates alpha-ketoglutarate, oxaloacetate, and pyruvate, confirming that in infected cells, a program allowing glutamine to be used anaplerotically is induced. Thus, HCMV infection activates the mechanisms needed to switch the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically.

The endoplasmic reticulum chaperone BiP/GRP78 is important in the structure and function of the human cytomegalovirus assembly compartment.

We previously demonstrated that the endoplasmic reticulum (ER) chaperone BiP functions in human cytomegalovirus (HCMV) assembly and egress. Here, we show that BiP localizes in two cytoplasmic structures in infected cells. Antibodies to the extreme C terminus, which includes BiP's KDEL ER localization sequence, detect BiP in regions of condensed ER near the periphery of the cell. Antibodies to the full length, N terminus, or larger portion of the C terminus detect BiP in the assembly compartment. This inability of C-terminal antibodies to detect BiP in the assembly compartment suggests that BiP's KDEL sequence is occluded in the assembly compartment. Depletion of BiP causes the condensed ER and assembly compartments to dissociate, indicating that BiP is important for their integrity. BiP and pp28 are in association in the assembly compartment, since antibodies that detect BiP in the assembly compartment coimmunoprecipitate pp28 and vice versa. In addition, BiP and pp28 copurify with other assembly compartment components on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous data show that cells infected with a TRS1-deficient virus have cytoplasmic and assembly compartment defects like those seen when BiP is depleted. We show that a fraction of TRS1 purifies with the assembly compartment. These findings suggest that BiP and TRS1 share a function in assembly compartment maintenance. In summary, BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.

Human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone BiP/GRP78, which is required for virion assembly.

The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress.

AMPK-mediated inhibition of mTOR kinase is circumvented during immediate-early times of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) infection increases synthetic rates in infected cells. The resulting increase in energy utilization could potentially increase the AMP:ATP ratio, causing activation of 5'-AMP-activated protein kinase (AMPK). Activated AMPK promotes inhibition of mammalian target of rapamycin (mTOR) kinase, which could be deleterious to the viral infection. Using the AMPK-activating drug 5-amino-4-imidazolecarboxamide ribose (AICAR), we showed that, by 12 h post-HCMV infection, inhibition of mTOR by AMPK is circumvented. However, growth curves showed that progeny virion production is inhibited when AICAR is added, suggesting other inhibitory effects of AICAR or activated AMPK.

Human cytomegalovirus infection alters the substrate specificities and rapamycin sensitivities of raptor- and rictor-containing complexes.

Signaling mediated by the mammalian target of rapamycin kinase (mTOR) is activated during human cytomegalovirus (HCMV) infection. mTOR is found in two complexes differing by the binding partner, rictor or raptor. Activated mTOR-raptor promotes cap-dependent translation through the hyperphosphorylation of the eIF4E-binding protein (4E-BP). This activity of the raptor complex is normally inhibited by cell stress responses or the drug rapamycin. However, we previously showed that this inhibition of mTOR signaling can be circumvented during HCMV infection such that hyperphosphorylation of 4E-BP is maintained. Here we show that HCMV infection also activates the rictor complex, as indicated by increased phosphorylation of Akt S473; this phosphorylation is insensitive to rapamycin but sensitive to caffeine in both uninfected and infected cells. By using short-hairpin RNAs to deplete rictor and raptor, we find that rictor is more significant than raptor for the viral infection. Surprisingly, the inhibitory effects of rapamycin on viral growth are primarily due to the presence of rictor, not raptor. Raptor and rictor depletion experiments show that in HCMV-infected cells, both raptor- and rictor-containing complexes can mediate the hyperphosphorylation of 4E-BP and the phosphorylation of p70S6 kinase. Under these conditions, the rictor complex is rapamycin-sensitive for the hyperphosphorylation of 4E-BP, but the raptor complex is not. These data suggest that, during HCMV infection, the rictor- and raptor-containing complexes are modified such that their substrate specificities and rapamycin sensitivities are altered. Our data also suggest that the present understanding of rapamycin's inhibitory effects is incomplete.

Human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mTOR kinase.

Signaling mediated by the cellular kinase mammalian target of rapamycin (mTOR) activates cap-dependent translation under normal (nonstressed) conditions. However, translation is inhibited by cellular stress responses or rapamycin treatment, which inhibit mTOR kinase activity. We show that during human cytomegalovirus (HCMV) infection, viral protein synthesis and virus production proceed relatively normally when mTOR kinase activity is inhibited due to hypoxic stress or rapamycin treatment. Using rapamycin inhibition of mTOR, we show that HCMV infection induces phosphorylation of two mTOR effectors, eucaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) and eIF4G. The virally induced phosphorylation of eIF4G is both mTOR and phosphatidylinositol 3-kinase (PI3K) independent, whereas the phosphorylation of 4E-BP is mTOR independent, but PI3K dependent. HCMV infection does not induce mTOR-independent phosphorylation of a third mTOR effector, p70S6 kinase (p70S6K). We show that the HCMV-induced phosphorylation of eIF4G and 4E-BP correlates with the association of eIF4E, the cap binding protein, with eIF4G in the eIF4F translation initiation complex. Thus, HCMV induces mechanisms to maintain the integrity of the eIF4F complex even when mTOR signaling is inhibited.