HCMV Targets the Metabolic Stress Response through Activation of AMPK Whose Activity Is Important for Viral Replication

Human Cytomegalovirus (HCMV) infection induces several metabolic activities that have been found to be important for viral replication. The cellular AMP-activated protein kinase (AMPK) is a metabolic stress response kinase that regulates both energy-producing catabolic processes and energy-consuming anabolic processes. Here we explore the role AMPK plays in generating an environment conducive to HCMV replication. We find that HCMV infection induces AMPK activity, resulting in the phosphorylation and increased abundance of several targets downstream of activated AMPK. Pharmacological and RNA-based inhibition of AMPK blocked the glycolytic activation induced by HCMV-infection, but had little impact on the glycolytic pathway of uninfected cells. Furthermore, inhibition of AMPK severely attenuated HCMV replication suggesting that AMPK is an important cellular factor for HCMV replication. Inhibition of AMPK attenuated early and late gene expression as well as viral DNA synthesis, but had no detectable impact on immediate-early gene expression, suggesting that AMPK activity is important at the immediate early to early transition of viral gene expression. Lastly, we find that inhibition of the Ca²⁺-calmodulin-dependent kinase kinase (CaMKK), a kinase known to activate AMPK, blocks HCMV-mediated AMPK activation. The combined data suggest a model in which HCMV activates AMPK through CaMKK, and depends on their activation for high titer replication, likely through induction of a metabolic environment conducive to viral replication.     

Herpes simplex type 1 activates glycolysis through engagement of the enzyme 6-phosphofructo-1-kinase (PFK-1)

Viruses such as HIV, HCV, Mayaro and HCMV affect cellular metabolic pathways, including glycolysis. Although some studies have suggested that the inhibition of glycolysis affects HSV-1 replication and that HSV-1-infected eyes have increased lactate production, the mechanisms by which HSV-1 induces glycolysis have never been investigated in detail. In this study, we observed an increase in glucose uptake, lactate efflux and ATP content in HSV-1-infected cells. HSV-1 triggered a MOI-dependent increase in the activity of phosphofructokinase-1 (PFK-1), a key rate-limiting enzyme of the glycolytic pathway. After HSV-1 infection, we observed increased PFK-1 expression, which increased PFK-1 total activity, and the phosphorylation of this enzyme at serine residues. HSV-1-induced glycolysis was associated with increased ATP content, and these events were critical for viral replication. In summary, our results suggest that HSV-1 triggers glycolysis through a different mechanism than other herpesviruses, such as HCMV. Thus, this study contributes to a better understanding of HSV-1 pathogenesis and provides insights into novel targets for antiviral therapy. HIGHLIGHTS: ?HSV-1 activates glycolysis by PFK-1 activation. ?In HSV-1-infected cells PFK-1 synthesis is up-regulated and phosphorylated at serine residues. ?PFK-1 knockdown impairs HSV-1 replication. ?HSV-1-mediated glycolysis activation increases ATP content.     

Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38

Recent evidence indicates activated mitogen-activated protein kinase (MAPK) p38 has a critical function in human cytomegalovirus (HCMV) viral DNA replication in infected human fibroblasts. To elucidate the mechanism of HCMV-mediated p38 activation, we have performed a detailed analysis of p38 activation and the kinases associated with this activation at different times postinfection. We demonstrate that p38 kinase activity is strongly increased following viral infection. Inhibition of this activity significantly inhibited HCMV-induced hyperphosphorylation of pRb and phosphorylation of heat shock protein 27, suggesting that p38 activation is involved in virus-mediated changes in host cell metabolism throughout the course of infection. We then provide evidence that p38 activation is mediated by different mechanisms at early times versus later times of infection. At early times of infection (8 to 14 h postinfection [hpi]), when p38 activation is first observed, no significant activation of the three kinases which can directly phosphorylate p38 (namely, MKK3, MKK6, and MKK4) is detected. Using vectors which express dominant negative proteins, we demonstrate that basal MKK6 kinase activity is necessary for HCMV-mediated p38 activation at these early times of infection (12 hpi). Then, we use ATP depletion to show that at 12 hpi, HCMV inhibits dephosphorylation of activated p38. These two experiments suggest that HCMV activates p38 by inhibition of dephosphorylation of p38. In contrast to early times of infection, at later times of infection (48 to 72 hpi), increased MKK3/6, but not MKK4, activity is observed. These results indicate that at early times of HCMV infection, increased steady-state levels of activated p38 is mediated at least in part by inhibition of dephosphorylation of p38, while at later times of infection p38 activation is due to increased activity of the upstream kinases MKK3 and MKK6. These findings indicate that HCMV has developed multiple mechanisms to ensure activation of the MAPK p38, a kinase critical to viral infection.     

Polyamine metabolism in MRC5 cells infected with different herpesviruses.

Both methyglyoxal bis(guanylhydrazone), an inhibitor of S-adenosyl-L-methionine decarboxylase (EC.4.1.1.50) and DL-α-methylornithine, an inhibitor of ornithine decarboxylase (EC.4.1.1.17), are shown to be potent inhibitors of the replication of human cytomegalovirus (HCMV) in MRC-5 cells. These compounds, both inhibitors of polyamine biosynthesis, do not affect the replication of either herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). This difference in antiviral effect is shown to be related to the stimulation of spermidine and spermine synthesis in host cells following HCMV infection and the inhibition of polyamine metabolism in HSV-1 or HSV-2-infected cells. Inhibition of HCMV replication by the inhibitors of polyamine biosynthesis is accompanied by a marked decrease in the formation of intranuclear, DNA-containing inclusions characteristic of HCMV infection. These results suggest significant differences in the mechanisms of replication of different herpesviruses.     

Human cytomegalovirus TRS1 and IRS1 gene products block the double-stranded-RNA-activated host protein shutoff response induced by herpes simplex virus type 1 infection

Human cytomegalovirus (HCMV) attachment and entry stimulates the expression of cellular interferon-inducible genes, many of which target important cellular functions necessary for viral replication. Double-stranded RNA-dependent host protein kinase (PKR) is an interferon-inducible gene product that limits viral replication by inhibiting protein translation in the infected cell. It was anticipated that HCMV encodes gene products that facilitate the evasion of this PKR-mediated antiviral response. Using a deltagamma1 34.5 herpes simplex virus type 1 (HSV-1) recombinant that triggers PKR-mediated protein synthesis shutoff, experiments identified an HCMV gene product expressed in the initial hours of infection that allows continued protein synthesis in the infected cell. Recombinant HSV-1 viruses expressing either the HCMV TRS1 or IRS1 protein demonstrate that either of these HCMV gene products allows the deltagamma1 34.5 recombinant viruses to evade PKR-mediated protein shutoff and maintain late viral protein synthesis.     

Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism

Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV.     

Inhibition of the Ca2+/calmodulin-dependent protein kinase I cascade by cAMP-dependent protein kinase

Several recent studies have shown that Ca2+/calmodulin-dependent protein kinase I (CaMKI) is phosphorylated and activated by a protein kinase (CaMKK) that is itself subject to regulation by Ca2+/calmodulin. In the present study, we demonstrate that this enzyme cascade is regulated by cAMP-mediated activation of cAMP-dependent protein kinase (PKA). In vitro, CaMKK is phosphorylated by PKA and this is associated with inhibition of enzyme activity. The major site of phosphorylation is threonine 108, although additional sites are phosphorylated with lower efficiency. In vitro, CaMKK is also phosphorylated by CaMKI at the same sites as PKA, suggesting that this regulatory phosphorylation might play a role as a negative-feedback mechanism. In intact PC12 cells, activation of PKA with forskolin resulted in a rapid inhibition of both CaMKK and CaMKI activity. In hippocampal slices CaMKK was phosphorylated under basal conditions, and activation of PKA led to an increase in phosphorylation. Two-dimensional phosphopeptide mapping indicated that activation of PKA led to increased phosphorylation of multiple sites including threonine 108. These results indicate that in vitro and in intact cells the CaMKK/CaMKI cascade is subject to inhibition by PKA-mediated phosphorylation of CaMKK. The phosphorylation and inhibition of CaMKK by PKA is likely to be involved in modulating the balance between cAMP- and Ca2+-dependent signal transduction pathways.     

Suppression of extracellular signal-regulated kinase activity in herpes simplex virus 1-infected cells by the Us3 protein kinase

Host mitogen-activated protein kinases (MAPKs) are deregulated by herpes simplex virus 1 (HSV-1). Unlike p38 MAPK and Jun N-terminal protein kinase (JNK), which require ICP27 for their activation early in infection, extracellular signal-regulated kinase (ERK) activity is suppressed by an unknown mechanism. Here, we establish that HSV-1-induced suppression of ERK activity requires viral gene expression, occurs with delayed-early kinetics, and requires the functional virus-encoded Us3 Ser/Thr protein kinase. Finally, Us3 expression in uninfected cells was necessary and sufficient to suppress ERK activity in the absence of any other virus-encoded gene products. This demonstrates that inhibition of ERK activity in HSV-1-infected cells is an intrinsic Us3 function and defines a new role for this alphaherpesvirus Us3 kinase in regulating MAPK activation in infected cells.     

Pyrrolidine Dithiocarbamate Inhibits Herpes Simplex Virus 1 and 2 Replication,and Its Activity May Be Mediated through Dysregulation of the Ubiquitin-Proteasome System

Pyrrolidine dithiocarbamate (PDTC) is widely used as an antioxidant or an NF-κB inhibitor. It has been reported to inhibit the replication of human rhinoviruses, poliovirus, coxsackievirus, and influenza virus. In this paper, we report that PDTC could inhibit the replication of herpes simplex virus 1 and 2 (HSV-1 and HSV-2). PDTC suppressed the expression of HSV-1 and HSV-2 viral immediate early (IE) and late (membrane protein gD) genes and the production of viral progeny. This antiviral property was mediated by the dithiocarbamate moiety of PDTC and required the presence of Zn²⁺. Although PDTC could potently block reactive oxygen species (ROS) generation, it was found that this property did not contribute to its anti-HSV activity. PDTC showed no activity in disrupting the mitogen-activated protein kinase (MAPK) pathway activation induced by viral infection that was vital for the virus''s propagation. We found that PDTC modulated cellular ubiquitination and, furthermore, influenced HSV-2-induced IκB-α degradation to inhibit NF-κB activation and enhanced PML stability in the nucleus, resulting in the inhibition of viral gene expression. These results suggested that the antiviral activity of PDTC might be mediated by its dysregulation of the cellular ubiquitin-proteasome system (UPS).     

Inhibition of Herpes Simplex Virus-1 Replication by Natural Compound Honokiol

Honokiol is a pleiotropic natural compound isolated from Magnolia and has multiple biological and clinically relevant effects, including anticancer and antimicrobial function. However, the antiviral activity of honokiol has not yet been well studied. Here we showed that honokiol had no effect on herpes simplex virus-1(HSV-1) entry, but inhibited HSV-1 viral DNA replication, gene expression and the production of new progeny viruses. The combination of honokiol and clinical drug acyclovir augmented inhibition of HSV-1 infection. Our results illustrate that honokiol could be a potential new candidate for clinical consideration in the treatment of HSV-1 infection alone or combination with other therapeutics.     

IGFBP-2 stimulates calcium/calmodulin-dependent protein kinase kinase 2 activation leading to AMP-activated protein kinase induction which is required for osteoblast differentiation

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins-2 (IGFBP-2) function coordinately to stimulate osteoblast differentiation. Induction of AMP-activated protein kinase (AMPK) is required for differentiation and is stimulated by these two factors. These studies were undertaken to determine how these two peptides lead to activation of AMPK. Enzymatic inhibitors and small interfering RNA were utilized to attenuate calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) activity in osteoblasts, and both manipulations resulted in failure to activate AMPK, thereby resulting in inhibition of osteoblast differentiation. IGFBP-2 and IGF-I stimulated an increase in CaMKK2, and inhibition of IGFBP-2 binding its receptor resulted in failure to induce CaMKK2 and AMPK activation. Injection of a peptide that contained the IGFBP-2 receptor-binding domain into IGFBP-2^−/− mice activated CaMKK2 and injection of a CaMKK2 inhibitor into normal mice inhibited both CamKK2 and AMPK activation in osteoblasts. We conclude that induction of CaMKK2 by IGFBP-2 and IGF-I in osteoblasts is an important signaling event that occurs early in differentiation and is responsible for activation of AMPK, which is required for optimal osteoblast differentiation.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

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.     

Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy

Viruses rely on the metabolic network of their cellular hosts to provide energy and building blocks for viral replication. We developed a flux measurement approach based on liquid chromatography-tandem mass spectrometry to quantify changes in metabolic activity induced by human cytomegalovirus (HCMV). This approach reliably elucidated fluxes in cultured mammalian cells by monitoring metabolome labeling kinetics after feeding cells (13)C-labeled forms of glucose and glutamine. Infection with HCMV markedly upregulated flux through much of the central carbon metabolism, including glycolysis. Particularly notable increases occurred in flux through the tricarboxylic acid cycle and its efflux to the fatty acid biosynthesis pathway. Pharmacological inhibition of fatty acid biosynthesis suppressed the replication of both HCMV and influenza A, another enveloped virus. These results show that fatty acid synthesis is essential for the replication of two divergent enveloped viruses and that systems-level metabolic flux profiling can identify metabolic targets for antiviral therapy.     

Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.     

The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host

The human herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), HHV-6B, HHV-7 and HHV-8, establish persistent infections with possible recurrence during immunosuppression. HCMV replication is inhibited by the nucleoside analogue ganciclovir (GCV), the compound of choice for the treatment of HCMV diseases and preemptive treatment of infections. The viral UL97 protein (pUL97) which shares homologies with protein kinases and bacterial phosphotransferases is able to monophosphorylate GCV. Homologues of pUL97 are found in HSV (UL13), VZV (ORF47), EBV (BGLF4), HHV-6 (U69), HHV-8 (ORF36) as well as in murine CMV (M97) or rat CMV (R97). Several indolocarbazoles have been reported to be specific inhibitors of pUL97. The protein is important for efficient replication of the virus. Autophosphorylation of pUL97 was observed using different experimental systems. Most recently, it has been shown that pUL97 interacts with the DNA polymerase processivity factor pUL44. Indolocarbazole protein kinase inhibitors are promising lead compounds for the development of more specific inhibitors of HCMV.     

A human cytomegalovirus function inhibits replication of herpes simplex virus.

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.