Yongjun Yu

The TORrid affairs of viruses: effects of mammalian DNA viruses on the PI3K–Akt–mTOR signalling pathway

The successful replication of mammalian DNA viruses requires that they gain control of key cellular signalling pathways that affect broad aspects of cellular macromolecular synthesis, metabolism, growth and survival. The phosphatidylinositol 3′-kinase–Akt–mammalian target of rapamycin (PI3K–Akt–mTOR) pathway is one such pathway. Mammalian DNA viruses have evolved various mechanisms to activate this pathway to obtain the benefits of Akt activation, including the maintenance of translation through the activation of mTOR. In addition, viruses must overcome the inhibition of this pathway that results from the activation of cellular stress responses during viral infection. This Review will discuss the range of mechanisms that mammalian DNA viruses use to activate this pathway, as well as the multiple mechanisms these viruses have evolved to circumvent inhibitory stress signalling.

Human Cytomegalovirus Infection Induces Rapamycin-Insensitive Phosphorylation of Downstream Effectors of mTOR Kinase

ABSTRACT 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.

Human cytomegalovirus major immediate-early proteins and simian virus 40 large T antigen can inhibit apoptosis through activation of the phosphatidylinositide 3'-OH kinase pathway and the cellular kinase Akt.

ABSTRACT The temperature-sensitive cell line ts13 is mutated in CCG1, the gene encoding TAFII250, the largest of the TATA-binding protein-associated factors (TAFs) in TFIID. At the nonpermissive temperature, the temperature-sensitive phenotypes are (i) transcription defects, (ii) cell cycle arrest in G1, and (iii) apoptosis. We previously demonstrated that the human cytomegalovirus (HCMV) major immediate-early proteins (MIEPs) can rescue the transcription defects and inhibit apoptosis at the nonpermissive temperature. In the work presented, we show that activation of the cellular kinase Akt alone can inhibit apoptosis in ts13 cells grown at the nonpermissive temperature. More significantly, we show that the HCMV MIEPs can activate Akt, resulting in the inhibition of apoptosis. In parallel experiments, we found that simian virus 40 (SV40) large T antigen can mediate the same function. These experiments were done by transfecting the HCMV major immediate-early gene or a cDNA encoding T antigen into ts13 cells, and thus neither viral attachment to receptors, viral tegument proteins, nor any other viral protein is required for Akt activation. Akt is activated by the phosphatidylinositide 3′-OH (PI3) kinase pathway. Using a specific inhibitor of PI3 kinase, we show that the ability of the MIEPs and T antigen to activate Akt and inhibit apoptosis is eliminated, suggesting that the viral proteins utilize the PI3 kinase pathway for Akt activation. Transfection of plasmids which express the individual 86-kDa (IEP86; IE2579aa) and 72-kDa (IEP72; IE1491aa) MIEPs indicate that each MIEP could inhibit apoptosis via activation of the PI3 kinase pathway.

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 rapamycins inhibitory effects is incomplete.

Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection

Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead, it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to α-ketoglutarate to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle cofactor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy.

Human Cytomegalovirus Activates Glucose Transporter 4 Expression To Increase Glucose Uptake during Infection

ABSTRACT 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.

Human Cytomegalovirus Specifically Controls the Levels of the Endoplasmic Reticulum Chaperone BiP/GRP78, Which Is Required for Virion Assembly

ABSTRACT 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.

Effects of Simian Virus 40 Large and Small Tumor Antigens on Mammalian Target of Rapamycin Signaling: Small Tumor Antigen Mediates Hypophosphorylation of eIF4E-Binding Protein 1 Late in Infection

ABSTRACT We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen.

Interaction between Simian Virus 40 Large T Antigen and Insulin Receptor Substrate 1 Is Disrupted by the K1 Mutation, Resulting in the Loss of Large T Antigen-Mediated Phosphorylation of Akt

ABSTRACT The cellular kinase Akt is a key controller of cellular metabolism, growth, and proliferation. Many viruses activate Akt due to its beneficial effects on viral replication. We previously showed that wild-type (WT) simian virus 40 (SV40) large T antigen (TAg) inhibits apoptosis via the activation of PI3K/Akt signaling. Here we show that WT TAg expressed from recombinant adenoviruses in U2OS cells induced the phosphorylation of Akt at both T308 and S473. In contrast, Akt phosphorylation was eliminated by the K1 mutation (E107K) within the retinoblastoma protein (Rb) binding motif of TAg. This suggested that Akt phosphorylation may depend on TAg binding to Rb or one of its family members. However, in Rb-negative SAOS2 cells depleted of p107 and p130 by using small hairpin RNAs (shRNAs), WT TAg still mediated Akt phosphorylation. These results suggested that the K1 mutation affects another TAg function. WT-TAg-mediated phosphorylation of Akt was inhibited by a PI3K inhibitor, suggesting that the effects of TAg originated upstream of PI3K; thus, we examined the requirement for insulin receptor substrate 1 (IRS1), which binds and activates PI3K. Depletion of IRS1 by shRNAs abolished the WT-TAg-mediated phosphorylation of Akt. Immunoprecipitation studies showed that the known interaction between TAg and IRS1 is significantly weakened by the K1 mutation. These data indicate that the K1 mutation disrupts not only Rb binding but also IRS1 binding, contributing to the loss of activation of PI3K/Akt signaling.

Human Cytomegalovirus Infection Induces Adipocyte-Like Lipogenesis through Activation of Sterol Regulatory Element Binding Protein 1

ABSTRACT 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.