Reprogramming of glucose metabolism in virus infected cells

Glucose metabolism in a host cell is necessary for some viruses to replicate. Energy is required for its survival and for the synthesis of biomolecules required for virus replication. The energy for all these events comes through the alteration of host cell glycolysis and other metabolic pathways. Thus, inhibition of glycolysis blocks the replication of majority of viruses. The carbon metabolic alterations could either be a cellular response to virus infection or triggered by the virus itself to complete its life cycle. The shift in host cellular metabolism is required to cope up with the imbalance caused by virus infection. One of the reasons for increased glycolysis during virus infection could be apoptosis, as cell death during virus replication induces disruption of mitochondrial membrane resulting in the inhibition of cellular respiration. To compensate this condition, glycolysis and other cellular metabolisms may be increased [31]. There are several types of inhibitors that are used to inhibit glycolysis which in turn inhibit viral life cycle. Glycolytic inhibitors generally result in mitochondrial pathway-induced apoptosis in cancerous cell [32, 33] which is similar to the condition observed in virus infected cells. Glycolytic enzyme inhibitors, glucose transporter inhibitors, ATP by allosteric inhibition, etc., are used to inhibit glycolysis. Reduction of viral RNA synthesis of Norovirus [15], and Dengue virus [34] following inhibition of glycolysis has been demonstrated using glycolysis inhibitors such as 2DG and Oxamate. Thus, glycolysis is an intrinsic host factor that is required for optimal replication of many viruses. Most of the above viruses had shown to be regulating a common strategy; that is increased glycolysis is always linked with increased glucose uptake and the increased expression of glucose transporters. However, some variations between oncogenic and non-oncogenic viruses were reported in the literature. Various viruses that are known to modulate host cell glycolysis and their possible regulatory mechanism are listed in Table 1.

Table 1 Modulation of glucose metabolism by different virusesOncogenic viruses

Oncogenic viruses account for 11.9% of all human cancers, and are known to regulate various host metabolic pathways to maintain the oncogenic phenotype of transformed cells [35]. Cancer cells are mainly dependent on aerobic glycolysis for their high energy demand which is considered as the hallmark of cancer [36, 37] Likewise, virus replication requires loads of energy, and resources from the host cell, therefore Warburg effect is essential in case of oncogenic virus infected cells for their survival [28]. Some cancer cells do utilize glutamine, amino acid or fatty acid metabolism for their proliferation and survival [38, 39]. As both the virus and the cancer cell have the ability to modify the rate of energy metabolism for the endless proliferation, it is likely that the mechanisms regulated by both have originated from a common mechanism [40]. Oncogenic viral proteins stimulate various oncogenic signaling pathways associated with energy metabolism and cell growth to promotes angiogenesis, and metastasis of viral infected/transformed cells [41]. GLUT1 had reported to be the main glucose transporter, transported to the membrane for the survival of cancerous cells [42]. Translocation of GLUT1 to the plasma membrane, and binding of PI3 kinase to middle T-pp60c−src leads to the increased uptake of glucose in polyoma virus transformed cells [43]. In HCV infected cells, the direct correlation between virus encoded NS5A protein, and the increased expression/activation of cellular hexokinase-2 result in intensification of glycolytic rate by increasing glucose uptake and lactate efflux [44]. Whereas in KSHV infected endothelial cells, mainly Akt and HIF signaling pathways appear to be playing a major role in Warburg effect [28]. However, induction of Warburg effect by KSHV is not universal but limited to endothelial cells [28]; while glycolytic inhibitors induce apoptosis in KSHV infected endothelial cells. Another study reported hyperactivation of PI3-K/Akt pathway, and GLUT1 translocation to the plasma membrane increases the oncogenic potential in KHSV infected THP-1 cells [45]. These viral infected cells are more potent to death by glucose inhibitor, 2-DG in combination with bortezomib, an anti-cancer drug [45]. Under nutrient stress conditions, KSHV encoded miRNA and vFLIP genes promote cellular transformation, and suppresses aerobic glycolysis by activating NF-κβ signaling pathway to downregulate GLUT1 and GLUT3 transporter [19]. However, this paradox in KSHV infections is an important aspect to be investigated in detail.

Oncogenic viral proteins of Human Papilloma Virus, Murine Sarcoma Virus reported to be playing a regulatory role in inducing Warburg Effect, but the underlying mechanism is yet to be understood [18, 46]. In NPC cells, stabilization of transcriptional factor, c-Myc, and the transcriptional activation of Hexokinase-2 by EBV encoded LMP-1 mediated signal pathway causes upregulation of glycolysis and the proliferation of cancerous cells [16]. LMP-1 also upregulates the transcription of GLUT1 which enhances the aerobic glycolysis and the malignancy of the infected cells through mTORC1/NF-κB signaling pathway [17]. Studies on modulated glucose metabolism mentioned above by oncogenic viruses approve that oncogenic virus infected cells develop different mechanisms such as activation of various signaling pathways, increased cellular transporters, and increased nutrient uptake to sustain their high demand for energy. No studies have shown that cells are transformed due to increased glycolysis induced by oncogenic viruses. However, following transformation of cells by oncogenic viruses, viral proteins modulate glycolysis to meet the energy needs of proliferating cells.

Non-oncogenic viruses

It is obvious that oncogenic virus infections lead to metabolic alterations in the host cell because of their high energy demand. Interestingly, in non-transforming virus infected cells as well, glucose metabolism is altered. In case of Mayaro virus infected cells, increased glycolytic flux had been reported in association with increased glucose consumption and lactate production by a significant increase in 6-phosphofructo-1- kinase enzyme activity in infected cell [27].

MDCK cells infected with H1N1 strain of influenza A virus show differential regulation of several enzyme activities of key metabolic pathways to compensate the metabolic imbalance caused by infection [47]. Influenza virus does modulate glycolysis but the exact mechanism is not clear. It our study, rate of glycolysis was observed to be increased in influenza A virus infected cells through increased expression of glucose transporters 1 and 4. Besides, there was an interplay between alpha enolase and pyruvate kinase activity with viral gene expression was also noted (unpublished data). Influenza virus replication is dependent on host cell glucose, and is in dose-dependent manner; treatment of infected cells with glycolytic inhibitors reduces virus replication [48]. While higher level of glucose increases the assembly, and the proton transport activity of Vacuolar type ATPase within the cells increase the viral replication [48]. Enhanced intracellular metabolite concentration of the upper part of glycolysis was reported in influenza virus infected cells following increased glucose uptake and lactate export [31]. One recent study reported upregulation of Hexokinase-2, PKM2 and PDK3 enzymes of glycolytic pathway in A549 cells, and the mouse lung tissue following infection with H1N1 strain of influenza A virus. Earlier we reported, interaction of influenza A virus structural proteins M1 and NP with glycolytic enzymes; alpha enolase and pyruvate kinase [49]; however, the effect of this interaction on glycolysis in infected cells needs to be investigated. HIF-1 pathway, another pathway had shown to be critical for the transcriptional activation of enzymes involved in glycolysis to support virus infection through increased glycolysis. Virus replication gets inhibited upon targeting HIF-1 pathway. This study also showed that the change of H1N1 replication upon glycolysis inhibition or enhancement is independent of interferon signaling [50].

Dengue virus, another non-oncogenic virus alters glycolysis through increased glucose uptake by upregulation of GLUT1 transporter and the first enzyme of glycolysis i.e., hexokinase-2. While the inhibition of glycolytic pathway using glycolytic inhibitors halts the virus progeny production [34]. Adenovirus, although does not induce tumors in its natural host, encodes proteins with an ability to transform normal cells into cancerous in vitro. Adenovirus also reported to be modulating glucose uptake by increased expression of GLUT1, GLUT4 transporters, and the translocation of GLUT4 to the plasma membrane via Ras activated PI3Kinase pathway in an insulin-independent manner [14]. Adenovirus infected primary cultures of cardiac myocytes, and H9c2 cells show upregulation of HIF-1α when subjected to hypoxia in the absence of glucose. On the contrary, addition of extracellular glucose to the medium resulted in decreased HIF-1α levels by almost 50% [51]. In consensus with the above results, adenovirus-induced overexpression of GLUT1 in cardiac myocytes followed by hypoxia reduced the level of HIF-1α [51]. Another study reported the activation of transcriptional factor Myc, followed by Myc-dependent expression of glycolytic enzymes, Hexokinase-2 and PFKM by adenovirus encoded E4Orf1 protein promoting glucose uptake and increased glycolysis in infected cells [40].

Herpes Simplex Virus, also non-oncogenic in nature causes an increased glucose uptake, lactate secretion, and ATP content by elevating the expression and the activity of PFK-1enzyme. Its phosphorylation at serine residue was reported to be viral MOI dependent [23]. A prototype of beta-herpesvirus, HCMV also upregulates the level of metabolic components involved in glycolysis, TCA cycle, and pyrimidine biosynthesis in fibroblasts [52]. HCMV encoded immediate early protein IE72 mediates the inhibition of GLUT1 level in infected cells [53], and the inhibition of GLUT1 results in Akt mediated translocation of GLUT4 onto the cell surface which leads to increased glucose uptake, subsequently increased glycolysis [54].

The ongoing pandemic virus, SARS CoV-2 infected patients had elevated blood glucose levels during the life cycle of the virus which might be providing optimal conditions for the virus to replicate, and evade the host immune system [55]. One recent report showed that the increased glucose level, and the glycolysis promotes Monocytes infecting SARS-CoV-2 replication through HIF-1α-dependent pathway, while the treatment of cells with 2-Deoxy-d-glucose (2-DG), a glycolytic inhibitor blocked viral replication [56]. Thus, the drug 2-DG was used as an anti-viral and anti-inflammatory drug to combat the cytokine storm in COVID-19 patients [57]. All the above reports collectively suggest that most of the viruses irrespective of their genome nature, and oncogenic potential, modulate glucose uptake and glycolysis for successful replication in a host.

Considering different cells types, viruses have been reported to be regulating glucose metabolic pathways in different cell types such as immune cells, glioma cells, fibroblasts as well. The modulation of glycolysis by viruses in these cells are more or less similar to cancer cells; either by increasing proliferative pathways or increased expression and activity of glycolytic enzymes. Elevated level of Glut1 expression in CD4 + T cells contributes to increased glucose transport and increased glycolysis in HIV infected cells [58]. HHV-6 infection was found to promote glucose metabolism in T cells leading to increased glucose uptake, glucose consumption and lactate secretion through increased expression of major glucose transporters and glycolytic enzymes. Activated AKT-mTORC1 signaling was also reported in HHV-6A infected cells, while inhibition of mTORC1 signal pathway blocked HHV-6A mediated glycolytic pathway subsequently viral DNA replication, protein synthesis and progeny production which suggests an interplay between above mentioned signal pathways with glycolysis in HHV-6A infected immune cells [59]. Stimulation of glycolysis in glioma cell lines was reported through upregulation of key glycolytic enzymes hexokinase, GAPDH and alpha enolase by HIV glycoprotein gp120. It led to increased activity of pyruvate kinase and pyruvate synthesis [60]. Overview of glucose uptake and glycolysis regulation in oncogenic and non-oncogenic viruses is shown in Fig. 1.

Fig. 1figure 1

Overview of glucose uptake and glycolysis in virus infected cells. Figure shows various viruses and their proteins involved in induction of increased glycolysis in infected cells by modulating mechanisms such as deregulation of signal pathways to induce increased expression of glucose transporters and specific glycolytic enzymes. Created with Biorender.com

Oncolytic viruses

Cancer cells are rapidly dividing cells, and depend more on glucose than the normal cells do for ATP generation via glycolysis. An aerobic glycolysis is the principal metabolic pathway in cancerous cells, targeting it is the main approach to inhibit cancer cell progression. Viral demand of macromolecule synthesis is similar to cancerous cells. In most viral infected, normal and cancerous cells, glycolysis and uptake of glucose get intensified the use of glycolytic inhibitors results in oncolysis by many viruses [28, 61,62,63].

As cancer is a complex disease, it demands an effective way of treatment. In the recent past, trials on developing a targeted therapy to lyse cancer cells using virus gaining much interest in the field of cancer therapeutics. Oncolytic virus has an ability to selectively kill cancer cells leaving the normal cells unharmed. Oncolytic viruses work as cancer therapeutics by two major mechanisms; direct lysis or by triggering anti-cancerous immune response. Energy metabolic pathway plays a main role in both the cases to report the outcome of oncolytic virus mediated cell lysis. Oncolytic viruses hijack the host cellular metabolic pathways that are necessary for viral replication which results in oncolysis [64]; it is also reported that by targeting the glycolytic pathway of cancer cell through glycolytic inhibitors may enhance the oncolytic virotherapy activity in cancer cells [65].

New Castle Disease virus (NDV), a natural tumor tropic virus with oncolytic ability downregulates glycolytic enzyme PGK [66]. Glucose analog, 2-DG, an anti-metabolite of cancer cell inhibits the glucose metabolism of cancer cells more effectively in combination with oncolytic NDV to inhibit the tumor growth/ increased cytotoxicity in breast cancer cells through GAPDH downregulation as compared to monotherapy [63]. Another study showed that downregulation of hexokinase via d-Mannoheptulose, a non- metabolize analog of glucose [67, 68], and the use of hexokinase inhibitor combined with NDV infection inhibits glycolysis which in turn induces apoptosis in breast cancer cell line efficiently in comparison to either of the agents administered alone [69]. Dichloroacetate which is a mitochondrial pyruvate dehydrogenase kinase inhibitor reported to increase the NDV-mediated viro-immunotherapy in Hepatocellular carcinoma by enhancing anti-tumor immune response, and viral replication [70]. Lytic potential of M1 virus, a novel oncolytic virus shows its dependence on glycolysis. It had been shown that increased viral replication, and oncolysis is independent of Hexokinase-2 using lonidamine, a hexokinase inhibitor. However, enhanced viral replication, and oncolysis is mediated by downregulation of Myc, an antiviral immune response factor, and by upregulation of ER stress mediated apoptosis. On the contrary, glycolytic inhibitor, 2-DG (glucose analog) in combination with M1 virus not only inhibited the virus replication, but the oncolysis as well [71]. From the above studies, it is evident that virus replication, and cancer cell destruction by therapeutic viruses do require modulation of glycolysis. However, oncolytic virus associated host cell glycolysis needs to be elucidated in detail for developing anti-tumor drugs targeting Warburg effect in combination with oncolytic viro-therapeutics. The first Chinese SFDA approved Oncolytic-based therapy, Oncorine, a recombinant human adenovirus type 5 was approved for clinical use in 2005 against NPC [72]. In 2015, a modified Herpes Simplex Virus, talimogene laherparepvec (T-Vec), the first FDA approved oncolytic virus in United States and Europe were acclaimed for clinical use against metastatic melanoma [73]. Oncolytic virotherapy combined with metabolic interventions that work together may enhance the potential of virus-based cancer therapeutics.

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