Metabolomic Analysis Reveals Vitamin D-induced Decrease in Polyol Pathway and Subtle Modulation of Glycolysis in HEK293T Cells.

We combined 1H NMR metabolomics with functional and molecular biochemical assays to describe the metabolic changes elicited by vitamin D in HEK293T, an embryonic proliferative cell line adapted to high-glucose concentrations. Activation of the polyol pathway, was the most important consequence of cell exposure to high glucose concentration, resembling cells exposed to hyperglycemia. Vitamin D induced alterations in HEK293T cells metabolism, including a decrease in sorbitol, glycine, glutamate, guanine. Vitamin D modulated glycolysis by increasing phosphoglycerate mutase and decreasing enolase activities, changing carbon fate without changing glucose consumption, lactate export and Krebs cycle. The decrease in sorbitol intracellular concentration seems to be related to vitamin D regulated redox homeostasis and protection against oxidative stress, and helped maintaining the high proliferative phenotype, supported by the decrease in glycine and guanine and orotate concentration and increase in choline and phosphocholine concentration. The decrease in orotate and guanine indicated an increased biosynthesis of purine and pyrimidines. Vitamin D elicited metabolic alteration without changing cellular proliferation and mitochondrial respiration, but reclaiming reductive power. Our study may contribute to the understanding of the metabolic mechanism of vitamin D upon exposure to hyperglycemia, suggesting a role of protection against oxidative stress.

Inactivation of Dengue and Yellow Fever viruses by heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX.

AIMS: To investigate the effect of heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX (CoPPIX and SnPPIX), macrocyclic structures composed by a tetrapyrrole ring with a central metallic ion, on Dengue Virus (DENV) and Yellow Fever Virus (YFV) infection. METHODS AND RESULTS: Treatment of HepG2 cells with heme, CoPPIX and SnPPIX after DENV infection reduced infectious particles without affecting viral RNA contents in infected cells. The reduction of viral load occurs only with the direct contact of DENV with porphyrins, suggesting a direct effect on viral particles. Previously incubation of DENV and YFV with heme, CoPPIX and SnPPIX resulted in viral particles inactivation in a dose-dependent manner. Biliverdin, a noncyclical porphyrin, was unable to inactivate the viruses tested. Infection of HepG2 cells with porphyrin-pretreated DENV2 results in a reduced or abolished viral protein synthesis, RNA replication and cell death. Treatment of HepG2 or THP-1 cell lineage with heme or CoPPIX after DENV infection with a very low MOI resulted in a decreased DENV replication and protection from death. CONCLUSIONS: Heme, CoPPIX and SnPPIX possess a marked ability to inactivate DENV and YFV, impairing its ability to infect and induce cytopathic effects on target cells. SIGNIFICANCE AND IMPACT OF THE STUDY: These results open the possibility of therapeutic application of porphyrins or their use as models to design new antiviral drugs against DENV and YFV.

Dengue induces platelet activation, mitochondrial dysfunction and cell death through mechanisms that involve DC-SIGN and caspases.

BACKGROUND: Worldwide, dengue is the most prevalent human arbovirus disease. Dengue infection may cause a range of clinical manifestations from self-limiting febrile illness through to a life-threatening syndrome accompanied by both bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease; however, the mechanisms involved in DENV-induced platelet activation and thrombocytopenia are incompletely understood. PATIENTS AND METHODS: Freshly isolated platelets from patients with dengue were evaluated for markers of activation, mitochondrial alteration and activation of cell death pathways. In parallel, we examined direct DENV-induced activation and apoptosis of platelets obtained from healthy subjects. RESULTS: We found that platelets from DENV-infected patients exhibited increased activation by comparison to control subjects. Moreover, platelets from DENV-infected patients exhibited classic signs of the intrinsic pathway of apoptosis that include increased surface phosphatidylserine exposure, mitochondrial depolarization and activation of caspase-9 and -3. Indeed, thrombocytopenia was shown to strongly associate with enhanced platelet activation and cell death in DENV-infected patients. Platelet activation, mitochondrial dysfunction and caspase-dependent phosphatidylserine exposure on platelets were also observed when platelets from healthy subjects were directly exposed to DENV in vitro. DENV-induced platelet activation was shown to occur through mechanisms largely dependent on DC-SIGN. CONCLUSIONS: Together our results demonstrate that platelets from patients with dengue present signs of activation, mitochondrial dysfunction and activation of the apoptosis caspase cascade, which may contribute to the development of thrombocytopenia in patients with dengue. Our results also suggest the involvement of DC-SIGN as a critical receptor in DENV-dependent platelet activation.

Dengue virus-induced regulation of the host cell translational machinery

Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5 percent of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score ≥ ±2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.

Dengue virus-induced regulation of the host cell translational machinery.

Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5% of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score >or= +/-2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.

Viral inactivation based on inhibition of membrane fusion: understanding the role of histidine protonation to develop new viral vaccines.

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells, what makes it a potentially attractive target for viral inactivation approaches. Fusion is mediated by viral surface glycoproteins that undergo conformational changes triggered by interaction with specific cellular receptors or by the exposition to low pH of endossomal medium. Here we review how several studies on the structural rearrangements of vesicular stomatitis virus (VSV) glycoprotein G during cellular recognition and fusion led us to propose a crucial role of the protonation of His residues for G protein activity. Moreover, we demonstrated that using diethylpyrocarbonate (DEPC), a histidine-modifying compound, it was possible to abolish viral infectivity and pathogenicity in mice, and to elicit neutralizing antibodies that confer protection in these animals against challenge using lethal doses of the virus. The presence of conserved His residues in a wide range of viral fusion proteins and the use of DEPC as a more general means for vaccine development will be also discussed.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Low pH-induced conformational changes in vesicular stomatitis virus glycoprotein involve dramatic structure reorganization.

Membrane fusion is the key step in the entry of enveloped animal viruses into their host cells. Fusion of vesicular stomatitis virus with membranes occurs at acidic pH and is mediated by its envelope glycoprotein, the G protein. To study the structural transitions induced by acidic pH on G protein, we have extracted the protein from purified virus by incubation with nonionic detergent. At pH 6.0, purified G protein was able to mediate fusion of either phospholipid vesicles or Vero cells in culture. Intrinsic fluorescence studies revealed that changes in the environment of Trp residues occurred as pH decreases. In the absence of lipidic membranes, acidification led to G protein aggregation, whereas protein-protein interactions were substituted by protein-lipid interactions in the presence of liposomes. 1,1'-Bis(4-aniline-5-naphthalene sulfonate) (bis-ANS) binding was utilized to probe the degree of exposure of hydrophobic regions of G protein during acidification. Bis-ANS binding was maximal at pH 6.2, suggesting that a hydrophobic segment is exposed to the medium at this pH. At pH 6.0, a dramatic decrease in bis-ANS binding was observed, probably due to loss of tridimensional structure during the conformational rearrangement. This hypothesis was confirmed by circular dichroism analysis at different pH values, which showed a great decrease in alpha-helix content at pH values close to 6.0, suggesting that a reorganization of G protein secondary structure occurs during the fusion reaction. Our results indicate that G protein undergoes dramatic structural changes at acidic pH and acquires a conformational state able to interact with the target membrane.

Kinetics of intracellular viral disassembly and processing probed by Bodipy fluorescence dequenching.

A novel method is described for the study of viral disassembly and processing in live cells. Vesicular stomatitis virus (VSV) was labelled with the fluorescent probe Bodipy-FL and the resulting conjugate was 97.6% self-quenched due to fluorescence resonance energy transfer between neighbouring Bodipy molecules. In vitro experiments showed a four-fold increase in Bodipy fluorescence after extraction of VSV G protein from the virus envelope with Triton X-100 or beta-octylglucoside. Bodipy-labelled virus retained its capacity to mediate fusion of viral membrane with phosphatidylserine liposomes. Incubation of Bodipy-VSV with proteases in the presence of detergent promoted a total fluorescence enhancement of ca. 20 fold, showing that the conjugate fluorescence was also sensitive to proteolysis. Fluorescence microscopy and flow cytometry experiments with macrophages incubated with Bodipy-VSV revealed that intracellular relaxation of fluorescence self-quenching resulted from a combination of viral disassembly due to pH-induced membrane fusion and viral protein degradation inside the endosomes. When macrophages were incubated simultaneously with ammonium chloride and protease inhibitors, the increase in fluorescence was abolished completely due to inhibition of both endosomal acidification and proteolysis. In addition, experiments carried out in the presence of protease inhibitors alone allowed, for the first time, isolated observation of G protein-mediated fusion of viral envelope with the endosomal membrane in living cells. The results indicate that this methodology may find wide application for further studies of viral infection.

Partially folded states of the capsid protein of cowpea severe mosaic virus in the disassembly pathway.

The different partially folded states of the capsid protein that appear in the disassembly pathway of cowpea severe mosaic virus (CPSMV) were investigated by examining the effects of hydrostatic pressure, sub-zero temperatures and urea. The conformational states of the coat protein were analyzed by their intrinsic fluorescence, binding of bis(8-anilinonaphthalene-1-sulfonate) (bis-ANS) and susceptibility to trypsin digestion. CPSMV could be disassembled by pressure at 2.5 kbar. Intrinsic fluorescence and hydrodynamic measurements showed that pressure-induced dissociation was completely reversible. Virus pressurization in the presence of ribonuclease revealed that viral RNA was not exposed, since it was not digested by the enzyme, suggesting the maintenance of protein-nucleic acid interactions under pressure. When the temperature was decreased to -10 degrees C under pressure, CPSMV disassembly became an irreversible process and in this condition, viral RNA was completely digested by ribonuclease. These results suggest a relationship between protein-RNA interactions and CPSMV assembly. Bis-ANS binding and trypsin digestion of coat proteins revealed that they assume a different conformation when they are denatured by low temperatures under pressure or than when they are denatured by urea at atmospheric pressure. The results indicate that the coat proteins can exist in at least four states: (1) The native conformation in the virus capsid; (2) bound to RNA when the virus is dissociated by pressure at room temperature, assuming a conformation that retains the information for reassembly; (3) free subunits in a molten-globule conformation when the virus is dissociated by low temperature under pressure; and (4) free subunits completely unfolded by high concentrations of urea.

Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.

The use of hydrostatic pressure as a tool to study viruses and other macromolecular assemblages.

Recent studies on the effect of pressure on macromolecular assemblages have provided new information on protein-protein and protein-nucleic acid interactions. New findings have recently emerged on the use of hydrostatic pressure to assess intermediate states in the assembly pathways of viruses, multimeric proteins and protein-nucleic acid complexes, addressing many questions of macromolecular recognition.

Concentration dependence of the subunit association of oligomers and viruses and the modification of the latter by urea binding.

A theoretical model is presented that accounts for the facilitation of the pressure dissociation of R17 phage, and for the partial restoration of the concentration dependence of the dissociation, by the presence of subdenaturing concentrations of urea. As an indifferent osmolyte urea should promote the stability of the protein aggregates under pressure, and the decrease in pressure stability with urea concentration demonstrates that such indirect solvent effects are not significant for this case, and that the progressive destabilization is the result of direct protein-urea interactions. By acting as a "homogenizer" of the properties of the phage particles, urea addition converts the pressure-induced deterministic dissociation of the phage into a limited stochastic equilibrium. The model establishes the origin of the uniform progression from the stochastic equilibrium of dimers, to the temperature-dependent and partially concentration-dependent association of tetramers, to the fully deterministic equilibrium observed in many multimers and in the virus capsids.

Cold denaturation of an icosahedral virus. The role of entropy in virus assembly.

Assembly of icosahedral viruses is not completely understood at the molecular level. The main puzzle is to answer how chemically identical protein subunits take up unique positionally dependent conformations during the process of assembly. The stability of the ribonucleoprotein particles of cowpea mosaic virus (CPMV) to pressures and subzero temperatures has been studied. At room temperature, reversible pressure denaturation of CPMV is obtained only in the presence of 5.0 M urea. On the other hand, when the temperature is decreased to -15 degrees C, the ribonucleoprotein components denature, at 2.5 kbar, in the presence of 1.0 M urea. At temperatures close to -20 degrees C, denaturation is obtained even in the absence of urea. Whereas the denaturation promoted by pressure and urea at room temperature is reversible, virus particles denatured when the temperature is decreased under pressure cannot reassemble. Bis-ANS binding data suggest that this irreversibility may be related to protein release from RNA, which probably does not occur under denaturating conditions at room temperature. The contributions of enthalpy (delta H*) and entropy (delta S*) for the free energy of association of CPMV are calculated from the cold denaturation curves under pressure. The entropy change is positive and large, making the assembly of ribonucleoprotein components an entropy-driven process, suggesting that the burial of nonpolar side chains during the process of assembly is the structural foundation for CPMV assembly.

Arc repressor will not denature under pressure in the absence of water.

Hydration forces are believed to play a determining role in protein folding. We have examined the contribution of water for the stability of the native dimer state of Arc repressor, a DNA-binding protein. Hydrostatic pressure was utilized to convert Arc repressor protein from a native state to a denatured, molten-globule state at decreasing concentrations of water. The volume change associated with Arc denaturation fell linearly with the increase in concentration of glycerol, whereas the free energy of the reaction increased. The pressure that promotes 50% denaturation (p1/2) increased in direct proportion to the concentration of glycerol or the decrease of water. Extrapolated to zero concentration of water, the data indicate that pressure denaturation would not occur without water. It is concluded that water plays a crucial role in decreasing the stability of a protein to a level that is compatible with its biological properties.

Differences in pressure stability of the three components of cowpea mosaic virus: implications for virus assembly and disassembly.

A comparison of pressure stability of empty capsids and ribonucleoprotein particles of cowpea mosaic virus (CPMV) is presented. A combination of high pressure and subdenaturing concentrations of urea was utilized to promote dissociation and denaturation. We found that RNA plays an important role in stabilizing the particles as well as in conferring reversibility to the pressure-induced denaturation. Dissociation and denaturation of the top component (empty capsid) was observed at 2.5 kbar and in the presence of 2.5 M urea. The pressure-dissociated state of the capsid protein had the characteristics of a denatured conformation as suggested by fluorescence spectra, lifetime of tryptophans, and binding of bis-ANS. The properties of the dissociated capsid protein were more similar to those of a molten-globule conformation, different from the more drastically unfolded state obtained using high concentrations of urea. Whereas the fluorescence of bis-ANS increased for the pressure-dissociated protein (1.5 M urea and 2.5 kbar), it decreased for the virus denatured by 6.0 M urea. Middle and bottom components underwent less than 50% change in center of spectral mass at 2.5 kbar and 2.5 M urea. The particles containing RNA could be fully affected by pressures of 2.5 kbar--as measured by the spectral shift--only in the presence of 5.0 M urea. RNA-containing capsids denatured by pressure did not bind bis-ANS, suggesting that the capsid protein continues to be bound to the RNA after the protein-protein contacts are broken by pressure. Reassembly of the nucleoprotein particles was obtained after decompression, reinforcing the idea that proteins had not dissociated from RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversible pressure dissociation of R17 bacteriophage. The physical individuality of virus particles.

In the absence of urea, pressures up to 2.5 kbar promote only 10% dissociation of the whole particles of R17 bacteriophage. In the presence of concentrations of urea between 1.0 and 5.0 M, pressure promotes complete, reversible dissociation of the virus particles. At the lower urea concentrations reversible dissociation of R17 virus particles shows no dependence on protein concentration indicating a high degree of heterogeneity of the particles, but higher urea concentrations, 2.5 to 5.0 M, result in progressive restoration of the protein concentration dependence of the pressure dissociation. At still higher urea concentrations, 5.0 to 8.0 M, irreversible dissociation of virus takes place at atmospheric pressure. In contrast, the dissociation of the isolated dimers of the capsid protein was dependent on protein concentration to the extent predicted for a stochastic equilibrium, and dimers were much less stable than the whole virus both to dissociation by pressure or urea. In contradistinction, the reversible whole-virus dissociation observed at urea concentrations below 2.5 M appears to be a typical deterministic equilibrium, without appreciable dynamic exchange between whole particle and subunits during the lengthy experiments. The experiments demonstrate that the "thermodynamic individuality" of the virus particles arises in conformational differences in the assembled viruses, and that there is a direct relation between the stability of the particles and their heterogeneity.