scholarly journals Synthesis of Genomic and Subgenomic RNA in Mosquito Cells Infected with Two Sindbis Virus nsP4 Mutants: Influence of Intracellular Nucleoside Triphosphate Concentrations

2008 ◽  
Vol 82 (14) ◽  
pp. 6880-6888 ◽  
Author(s):  
Mei-Ling Li ◽  
Tzu-Yu Kwan ◽  
H. Anne Simmonds ◽  
Victor Stollar
Keyword(s):  
Sindbis Virus ◽  
Viral Rna ◽  
Nucleoside Triphosphate ◽  
Free System ◽  
Mosquito Cells ◽  
Low Concentrations ◽  
A Cell ◽  
Infected Cells ◽  
High Concentrations ◽  
In Cells

ABSTRACT Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known. SVpzf and SVcpc are nsP4 mutants of SV which, in mosquito cells, make more G RNA than SG RNA. When low concentrations of pyrazofurin (inhibits the synthesis of UTP and CTP) were added to SVpzf-infected cells, the yield of virus was increased, and the ratio of SG/G RNA was changed from <1 to >1. These effects were reversed by uridine. In SVcpc-infected cells, but not in SVstd-infected cells, synthesis of viral RNA was inhibited by the addition of either uridine or cytidine, and viral yields were lowered. Our findings suggest that the activities of the viral RNA-synthesizing complexes in cells infected with SVpzf or SVcpc, in contrast to those in SVstd-infected cells, are sensitive to high concentrations of UTP or CTP. Using a cell-free system that synthesizes both SG and G RNA, we measured viral RNA synthesis as a function of the UTP/CTP concentrations. The results indicated that the presence of the SVpzf mutations in nsP4 and the SG promoter produced a pattern quite different from that seen with the SVstd nsP4 and SG promoter. As the UTP/CTP concentrations were increased, the SVpzf system, in contrast to the SVstd system, made more G RNA than SG RNA, reflecting the situation in cells infected with SVpzf.

scholarly journals Distinct Sites on the Sindbis Virus RNA-Dependent RNA Polymerase for Binding to the Promoters for the Synthesis of Genomic and Subgenomic RNA

2007 ◽  
Vol 81 (8) ◽  
pp. 4371-4373 ◽  
Author(s):  
Mei-Ling Li ◽  
Victor Stollar
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Sindbis Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Subgenomic Rna ◽  
Free System ◽  
Virus Rna ◽  
A Cell ◽  
Infected Cells

ABSTRACT Sindbis virus-infected cells make two positive-strand RNAs, a genomic (G) RNA and a subgenomic (SG) RNA. Here we report the amino acid sequence in nonstructural protein 4 (nsP4), the viral RNA-dependent RNA polymerase, that binds to the promoter for the synthesis of G RNA. In addition, using a cell-free system that makes both G and SG RNA, we show that specific amino acid changes in nsP4 that abolish the synthesis of SG RNA have no effect on the synthesis of G RNA. Our findings indicate that nsP4 has distinct sites for the recognition of the G and SG promoters.

scholarly journals In Vitro Synthesis of Sindbis Virus Genomic and Subgenomic RNAs: Influence of nsP4 Mutations and Nucleoside Triphosphate Concentrations

2010 ◽  
Vol 84 (6) ◽  
pp. 2732-2739 ◽  
Author(s):  
Mei-Ling Li ◽  
Hongtao Wang ◽  
Victor Stollar
Keyword(s):  
Sindbis Virus ◽  
Infected Mosquito ◽  
Nucleoside Triphosphate ◽  
Reaction Conditions ◽  
In Vitro Synthesis ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Standard Reaction ◽  
Made In

ABSTRACT Two positive-strand mRNAs are made in Sindbis virus-infected cells, the genomic (G) RNA and the subgenomic (SG) RNA. In mosquito cells infected with wild-type (wt) Sindbis virus, the latter is made in excess over the former; however, in cells infected with SVpzf or SVcpc more G RNA is made than SG RNA. Use was made of in vitro systems to investigate the effects of the SVpzf and SVcpc mutations on the synthesis of SG and G RNAs. Our findings indicate that under standard reaction conditions, the SG/G RNA ratio in vitro reflects the ratio of SG to G RNA made in infected mosquito cells. We observed further that the RNA patterns seen in vitro are affected not only by the SVpzf and SVcpc mutations but also by the nucleoside triphosphate concentrations in the reaction mixtures and that introduction of these mutations into nsP4 and the promoter/template change the relative amounts of SG and G RNAs that are made, likely through the choice of promoter. We conclude that with respect to the SVpzf and SVcpc mutations, it is mainly the nucleotide changes in the SG promoter, not the amino acid changes in nsP4, that determine the SG/G RNA ratio that results. Further, it was observed that the SVpzf mutations enhance the in vitro synthesis of SG RNA at the lowest concentrations of UTP/CTP and that the single SVcpc mutation enhances the synthesis of G RNA at the lowest concentrations of CTP tested. We also identified three Arg residues in nsP4, R545, R546, and R547, that are needed for the synthesis of G RNA but not SG RNA.

Bioenergetics Studies of the Cyanobacterium Anabaena variabilis

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1280-1284 ◽  
Author(s):  
Siegfried Scherer ◽  
Heike Sadowski ◽  
Peter Böger
Keyword(s):  
Electron Transport ◽  
Photosystem Ii ◽  
Cytochrome B ◽  
Anabaena Variabilis ◽  
Electron Donors ◽  
Free System ◽  
Low Concentrations ◽  
Cyanobacterium Anabaena ◽  
A Cell ◽  
C Complex

A cell-free system exhibiting both photophosphorylation (P/2e= 1) and oxidative phosphoryltion (P/O up to 0.8) is described for the cyanobacterium Anabaena variabilis. NADH ant NADPH were found to be equally effective as electron donors for oxidative phosphorylation. Low concentrations of UHDBT, an inhibitor of the cytochrome b/c complex of mitochondria ant loroplasts, were found to inhibit photosystem-II electron transport reactions, but did not affet the cytochrome b6/f-complex of Anabaena. The inhibition by myxothiazol, antimycin and heptyihydroxyquinoline corroborates the hypothesis that both respiration and photosynthesis share the cytochrome b6/f-complex.

scholarly journals Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells

2008 ◽  
Vol 89 (11) ◽  
pp. 2651-2661 ◽  
Author(s):  
Hua Wang ◽  
Carol D. Blair ◽  
Ken E. Olson ◽  
Rollie J. Clem
Keyword(s):  
Virus Replication ◽  
Persistent Infection ◽  
Sindbis Virus ◽  
Actinomycin D ◽  
Virus Production ◽  
Cell Types ◽  
Lytic Infection ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Apoptotic Genes

Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either pro-apoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

scholarly journals CB1 Receptor-Dependent and Independent Induction of Lipolysis in Primary Rat Adipocytes by the Inverse Agonist Rimonabant (SR141716A)

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 896 ◽  
Author(s):  
Günter A. Müller ◽  
Andreas W. Herling ◽  
Susanne Wied ◽  
Timo D. Müller
Keyword(s):  
Cannabinoid Receptor ◽  
Inverse Agonist ◽  
Hormone Sensitive Lipase ◽  
Acute Administration ◽  
Free System ◽  
Peripheral Tissues ◽  
Rat Adipocytes ◽  
A Cell ◽  
High Concentrations ◽  
Stimulation Of

(1) Background: Acute administration of the cannabinoid receptor 1 (CB1R) inverse agonist Rimonabant (SR141716A) to fed Wistar rats was shown to elicit a rapid and short-lasting elevation of serum free fatty acids. (2) Methods: The effect of Rimonabant on lipolysis in isolated primary rat adipocytes was studied to raise the possibility for direct mechanisms not involving the (hypothalamic) CB1R. (3) Results: Incubation of these cells with Rimonabant-stimulated lipolysis to up to 25% of the maximal isoproterenol effect, which was based on both CB1R-dependent and independent mechanisms. The CB1R-dependent one was already effective at Rimonabant concentrations of less than 1 µM and after short-term incubation, partially additive to β-adrenergic agonists and blocked by insulin and, in part, by adenosine deaminase, but not by propranolol. It was accompanied by protein kinase A (PKA)-mediated association of hormone-sensitive lipase (HSL) with lipid droplets (LD) and dissociation of perilipin-1 from LD. The CB1R-independent stimulation of lipolysis was observed only at Rimonabant concentrations above 1 µM and after long-term incubation and was not affected by insulin. It was recapitulated by a cell-free system reconstituted with rat adipocyte LD and HSL. Rimonabant-induced cell-free lipolysis was not affected by PKA-mediated phosphorylation of LD and HSL, but abrogated by phospholipase digestion or emulsification of the LD. Furthermore, LD isolated from adipocytes and then treated with Rimonabant (>1 µM) were more efficient substrates for exogenously added HSL compared to control LD. The CB1R-independent lipolysis was also demonstrated in primary adipocytes from fed rats which had been treated with a single dose of Rimonabant (30 mg/kg). (4) Conclusions: These data argue for interaction of Rimonabant (at high concentrations) with both the LD surface and the CB1R of primary rat adipocytes, each leading to increased access of HSL to LD in phosphorylation-independent and dependent fashion, respectively. Both mechanisms may lead to direct and acute stimulation of lipolysis at peripheral tissues upon Rimonabant administration and represent targets for future obesity therapy which do not encompass the hypothalamic CB1R.

scholarly journals POSSIBILITIES FOR INTER- AND INTRACELLULAR TRANSLOCATION OF SOME ICOSAHEDRAL PLANT VIRUSES

1969 ◽  
Vol 40 (3) ◽  
pp. 814-823 ◽  
Author(s):  
G. A. de Zoeten ◽  
G. Gaard
Keyword(s):  
Leaf Tissue ◽  
Plant Viruses ◽  
Infected Leaf ◽  
Mesophyll Cells ◽  
Low Concentrations ◽  
Infected Cells ◽  
Virus Research ◽  
High Concentrations ◽  
Pore Complexes ◽  
Intracellular Translocation

Southern bean mosaic virus (SBMV) and Tomato ringspot virus (TomRV) were compared with regard to possible ways of inter- and intracellular translocation. The pore complexes in the nuclear membranes of nuclei in leaf palisade and mesophyll cells of several plant species commonly used in plant virus research were studied. The pore structure resembled that earlier described. The diameter of the pores was great enough to allow icosahedral plant viruses between 25 and 30 mµ wide to move through. SBMV occurred in noncrystalline form in nuclei of infected cells. Although this virus forms paracrystalline structures when partially purified, no virus crystals were seen in the cytoplasm of cells containing high concentrations of SBMV. It was established that this virus could move through nuclear pores. TomRV was found in infected leaf tissue in low concentrations. This virus showed a tendency to crystallize even when present in low concentrations. TomRV was observed only in the cytoplasm, not in nuclei. This virus was present in plasmodesmata, indicating the possibility of cell to cell translocation of whole particles through these structures.

scholarly journals Glycolipid and glycoprotein transport through the Golgi complex are similar biochemically and kinetically. Reconstitution of glycolipid transport in a cell free system.

1990 ◽  
Vol 111 (2) ◽  
pp. 421-428 ◽  
Author(s):  
B W Wattenberg
Keyword(s):  
Sialic Acid ◽  
Golgi Apparatus ◽  
Chinese Hamster ◽  
Free System ◽  
Cell Free System ◽  
Glycoprotein G ◽  
A Cell ◽  
Infected Cells ◽  
Glycoprotein Transport ◽  
Kinetics Of

Glycolipid transport between compartments of the Golgi apparatus has been reconstituted in a cell free system. Transport of lactosylceramide (galactose beta 1-4-glucose-ceramide) was followed from a donor to an acceptor Golgi population. The major glycolipid in CHO cells is GM3 (sialic acid alpha 2-3 galactose beta 1-4-glucose-ceramide). Donor membranes were derived from a Chinese hamster ovary (CHO) cell mutant (Lec2) deficient in the Golgi CMP-sialic acid transporter, and therefore contained lactosylceramide as the predominant glycolipid. Acceptor Golgi apparatus was prepared from another mutant, Lec8, which is defective in UDP-Gal transport. Thus, glucosylceramide is the major glycolipid in Lec8 cells. Transport was measured by the incorporation of labeled sialic acid into lactosylceramide (present originally in the donor) by transport to acceptor membranes, forming GM3. This incorporation was dependent on ATP, cytosolic components, intact membranes, and elevated temperature. Donor membranes were prepared from Lec2 cells infected with vesicular stomatitus virus (VSV). These membranes therefore contain the VSV membrane glycoprotein, G protein. Donor membranes derived from VSV-infected cells could then be used to monitor both glycolipid and glycoprotein transport. Transport of these two types of molecules between Golgi compartments was compared biochemically and kinetically. Glycolipid transport required the N-ethylmaleimide sensitive factor previously shown to act in glycoprotein transport (Glick, B. S., and J. E. Rothman. 1987. Nature [Lond.]. 326:309-312; Rothman, J. E. 1987. J. Biol. Chem. 262:12502-12510). GTP gamma S inhibited glycolipid and glycoprotein transport similarly. The kinetics of transport of glycolipid and glycoprotein were also compared. The kinetics of transport to the end of the pathway were similar, as were the kinetics of movement into a defined transport intermediate. It is concluded that glycolipid and glycoprotein transport through the Golgi occur by similar if not identical mechanisms.

scholarly journals Murine Coronavirus Delays Expression of a Subset of Interferon-Stimulated Genes

2010 ◽  
Vol 84 (11) ◽  
pp. 5656-5669 ◽  
Author(s):  
Kristine M. Rose ◽  
Ruth Elliott ◽  
Luis Martínez-Sobrido ◽  
Adolfo García-Sastre ◽  
Susan R. Weiss
Keyword(s):  
Sindbis Virus ◽  
Sendai Virus ◽  
Hepatitis Virus ◽  
Type I ◽  
Interferon Stimulated Genes ◽  
A Cell ◽  
Infected Cells ◽  
Ifn Treatment ◽  
Β Receptor ◽  
Murine Coronavirus

ABSTRACT The importance of the type I interferon (IFN-I) system in limiting coronavirus replication and dissemination has been unequivocally demonstrated by rapid lethality following infection of mice lacking the alpha/beta IFN (IFN-α/β) receptor with mouse hepatitis virus (MHV), a murine coronavirus. Interestingly, MHV has a cell-type-dependent ability to resist the antiviral effects of IFN-α/β. In primary bone-marrow-derived macrophages and mouse embryonic fibroblasts, MHV replication was significantly reduced by the IFN-α/β-induced antiviral state, whereas IFN treatment of cell lines (L2 and 293T) has only minor effects on replication (K. M. Rose and S. R. Weiss, Viruses 1:689-712, 2009). Replication of other RNA viruses, including Theiler's murine encephalitis virus (TMEV), vesicular stomatitis virus (VSV), Sindbis virus, Newcastle disease virus (NDV), and Sendai virus (SeV), was significantly inhibited in L2 cells treated with IFN-α/β, and MHV had the ability to rescue only SeV replication. We present evidence that MHV infection can delay interferon-stimulated gene (ISG) induction mediated by both SeV and IFN-β but only when MHV infection precedes SeV or IFN-β exposure. Curiously, we observed no block in the well-defined IFN-β signaling pathway that leads to STAT1-STAT2 phosphorylation and translocation to the nucleus in cultures infected with MHV. This observation suggests that MHV must inhibit an alternative IFN-induced pathway that is essential for early induction of ISGs. The ability of MHV to delay SeV-mediated ISG production may partially involve limiting the ability of IFN regulatory factor 3 (IRF-3) to function as a transcription factor. Transcription from an IRF-3-responsive promoter was partially inhibited by MHV; however, IRF-3 was transported to the nucleus and bound DNA in MHV-infected cells superinfected with SeV.

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