In vitro antiviral activity of fullerene amino acid derivatives in cytomegalovirus infection]

The in vitro effect of 5 water soluble fullerene C60 amino acid derivatives (FAD) on the development of cytomegalovirus infection was studied in the schemes of the therapeutic, prophylactic and virucidal action. The following compounds as FAD were used: fullerene conjugated with Na salt of gamma-aminobutyric acid (C60-ABA-Na), 2 derivatives based on Na salts of fullerene-gamma-aminobutyric acid and fullerene-omega-caproic acid (C60-ABA-OH-Na and C60-ACA-OH-Na respectively) and 2 derivatives based on methyl ethers of the above mentioned fullerene amino acids (C60-ABA-OH-CH3 and C60-ACA-OH-CH3). All the FAD were able to inhibit the development of the virus cytopathogenic action in the cell culture. However, the compounds had different antiviral properties. C60-ABA-OH-Na, C60-ABA-CH3 and C60-ACA-CH3 showed marked antiviral activity in the prophylactic scheme. 50-Percent inhibition of the virus cytopathogenic action (ID50) was observed when concentrations of the compounds were 0.31, 5 and 25 mcg/ml respectively. C60-ACA-OH-Na inhibited the development of cytomegalovirus infection in the cell culture only in the scheme of the therapeutic action (ID50 4 mcg/ml). C60-ABA-Na had the highest antiviral effect. In a concentration of 0.22 mcg/ml it inhibited the cytomegalovirus plague-forming capacity by 50% in both the prophylactic and the virucidal schemes. The chemotherapeutic index of the compound was within the limits of 2500 to 5450.     

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.     

Modulation of N-ethylmaleimide-sensitive factor activity upon amino acid deprivation

Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition of N-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply.     

Proteolysis and amino acid recycling during nitrogen deprivation inSchizophyllum commune

Transfer of exponentially growing colonies ofSchizophyllum commune to nitrogen-deficient media for 6–60 h resulted in increased general proteolytic activity and decreased extractable protein when compared with controls. A concomitant increase in free radiolabeled leucine was detected in nitrogen-deprived colonies. Radiolabel was found in new surface hyphae following transfer of previously labeled colonies to media containing no label. The concentration of label present in the new growth indicates that this label is likely to have originated from proteolytic release of label from proteins in the older, central portions of the colony. The release of substantial amounts of label into the growth medium suggests that an extra-mycelial pathway may account, at least in part, for translocation of amino acids released by proteolysis.     

Human GAP-43: its deduced amino acid sequence and chromosomal localization in mouse and human

The growth-associated protein (GAP-43) is considered a crucial component of an effective regenerative response in the nervous system. Its phosphorylation by protein kinase C correlates with long-term potentiation. Sequence analysis of human cDNAs coding for this protein shows that the human GAP-43 gene is highly homologous to the rat gene; this homology extends into the 3'-untranslated region. However, the human protein contains a 10 amino acid insert. Somatic cell hybrids demonstrate localization of the GAP-43 gene to human chromosome 3 and to mouse chromosome 16.     

Human cytomegalovirus expresses novel microRNAs during productive viral infection

MicroRNAs (miRNAs) are a large class of approximately 22-nucleotide non-coding RNAs that facilitate mRNA cleavage and translation repression through the RNA interference pathway. Until recently, miRNAs have been exclusively found in eukaryotic organisms. A non-immunogenic molecule requiring minimal genomic investment, these RNAs may offer an efficient means for viruses to modulate both their own and the host's gene expression during a productive viral infection. In this study we report that human cytomegalovirus (HCMV) expresses miRNAs during its productive lytic infection of four clinically relevant human cell types: fibroblast, endothelial, epithelial and astrocyte cells. The sequences of the miRNAs, expressed from the UL23 and US24 loci of the viral genome, were conserved among all HCMV strains examined and in chimpanzee cytomegalovirus. Furthermore, their expression was detected from both a laboratory-adapted strain and a clinical isolate of HCMV. The conservation of these miRNAs and their expression in different cell types suggests that they represent an evolutionarily primitive feature in the viral genome, and that virus-encoded miRNAs may be more common than previously believed.     

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.     

Requirement for lysosomal localization of mTOR for its activation differs between leucine and other amino acids

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.     

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.     

Astrocytic amino acid metabolism under control conditions and during oxygen and/or glucose deprivation

Amino acid contents were measured in 1- and 3-week-old primary cultures of astrocytes and in their incubation media, an amino acid-free salt solution with or without glucose, during 3-h incubation under normoxic or anoxic conditions. Most essential amino acids were rapidly released to the medium during the beginning of the incubation. A subsequent slow medium increase reflected proteolysis. Glutamate and aspartate were absent from the media during all conditions, indicating fueling of their uptake by either glycolytically or oxidatively derived energy. The total content of glutamine increased, except during incubation in glucose-deprived media, when it declined or remained constant. Changes in aspartate were negligible, suggesting oxidative degradation of aspartate-derived oxaloacetate during normoxia and its reduction to succinate during anoxia, driving regeneration of NAD⁺ from NADH. An increase of alanine was reduced in glucose-free media, whereas serine showed especially large increase during isolated glucose deprivation, suggesting its production from glutamine via 3-phosphoglycerate.     

Human cytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in human fibroblasts

The effects ofhuman cytomegalovirus (HCMV) infection onCl/HCO₃exchanger activity in human lung fibroblasts (MRC-5 cells) were studiedusing fluorescent, ion-sensitive dyes. The intracellular pH(pH_i) of mock- and HCMV-infectedcells bathed in a solution containing 5%CO₂-25 mMHCO₃ were nearly the same. However,replacement of external Clwith gluconate caused anH₂DIDS-inhibitable (100 µM)increase in the pH_i ofHCMV-infected cells but not in mock-infected cells. Continuous exposureto hyperosmotic external media containing CO₂/HCO₃caused the pH_i of both cell typesto increase. The pH_i remainedelevated in mock-infected cells. However, in HCMV-infected cells, thepH_i peaked and then recoveredtoward control values. This pH_irecovery phase was completely blocked by 100 µMH₂DIDS. In the presence ofCO₂/HCO₃, there was an H₂DIDS-sensitivecomponent of net Cl efflux(external Cl wassubstituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl (in the nominal absenceofCO₂/HCO₃),the H₂DIDS-sensitive netCl efflux was much greaterfrom HCMV- than from mock-infected cells. In mock-infected cells,H₂DIDS-sensitive, netCl efflux decreased aspH_i increased, whereas forHCMV-infected cells, efflux increased aspH_i increased. All these resultsare consistent with an HCMV-induced enhancement ofCl/HCO₃exchanger activity.

     

The amino acid sequence of satyr tragopan lysozyme and its activity

The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck's tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck's tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).