Interaction with vesicular stomatitis virus-infected BALB/c3T3 cells inhibits the synthesis of nitric oxide in activated murine bone marrow culture-derived macrophages.
(17/2088)
Bone marrow-culture-derived macrophages activated with interferon-gamma and lipopolysaccharide produced less nitric oxide (NO) when cultured with vesicular stomatitis virus (VSV)-infected BALB/c3T3 (3T3-VSV) than macrophages activated in an identical manner and cultured alone, with uninfected BALB/c3T3 (3T3), or with P815. However, all four groups of macrophages produced nearly the same amount of interleukin-6 (IL-6). Addition of VSV to activated macrophages did not change the amount of NO produced. The amount of NO generated by two non-macrophage sources of NO was not affected by the presence of either P815 or 3T3-VSV. Reverse transcriptase-polymerase chain reaction showed a decrease in the amount of inducible nitric oxide synthase (iNOS) but not IL-6 mRNA from macrophages cocultured with 3T3-VSV compared with macrophages cocultured with P815. The reduction in iNOS mRNA was confirmed by ribonuclease protection assay. When RAW 264.7 transfected with an iNOS regulatory construct were activated and incubated with 3T3-VSV there was a decrease in the expression of the reporter luciferase gene and NO production but not IL-6 production compared with cells incubated with either medium alone or with P815. (+info)
Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and gqalpha /G11alpha induced by agonist stimulation.
(18/2088)
The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqalpha/G11alpha, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqalpha/G11alpha signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqalpha/G11alpha was also internalized, although much of the Gqalpha/G11alpha immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqalpha/G11alpha immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqalpha/G11alpha immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade. (+info)
Vesicular stomatitis virus G pseudotyped retrovector mediates effective in vivo suicide gene delivery in experimental brain cancer.
(19/2088)
Direct in vivo tumor-targeting with "suicide" viral vectors is limited by either inefficient gene transfer (i.e., retroviral vectors) or indiscriminate transfer of a conditionally toxic gene to surrounding nonmalignant tissue (i.e., adenoviral vectors). Retrovectors pseudotyped with the vesicular stomatitis virus G protein (VSVG) may serve as a remedy to this conundrum. These retroviral particles differ from standard murine retroviruses by their very broad tropism and the capacity to be concentrated by ultracentrifugation without loss of activity. We propose that a VSVG-typed retrovector can be used for efficient and tumor-specific herpes simplex virus thymidine kinase (TK) gene delivery in vivo. To test this hypothesis, we developed a bicistronic retroviral vector that expresses TK and green fluorescence protein (pTKiGFP). The 293GPG packaging cell line was used to generate vTKiGFP retroparticles. In cytotoxicity assays, vTKiGFP-transduced human glioma cell lines were sensitized to the cytotoxic effects of gangciclovir (GCV) 10,000-fold. Subsequently, virus was concentrated by ultracentrifugation to a titer of 2.3 x 10(10) cfu/ml. We tested the antitumor activity of vTKiGFP retroparticles in a rat C6 glioma model of brain cancer. Concentrated retrovector stock (9 microl volume) was injected stereotactically in preestablished intracerebral tumor. Subsequently, rats were treated with GCV for 10 days. Control rats (no GCV) had a mean survival of 38 days (range, 20-52 days). Sections performed on postmortem brain tissue revealed large tumors with evidence of high efficiency retrovector transfer and expression (as assessed by GFP fluorescence). Fluorescence was restricted to malignant tissue. In the experimental group (GCV treated), 8 of 12 remain alive and well >120 days after glioma implantation. In conclusion, vTKiGFP is very efficient at transducing human glioma cell lines in vitro and leads to significant GCV sensitization. Recombinant retroviral particles can be concentrated to titers that allow in vivo intratumoral delivery of large viral doses. The therapeutic efficiency of this reagent has been demonstrated in a preclinical model of brain cancer. (+info)
Secondary rearrangements and hypermutation generate sufficient B cell diversity to mount protective antiviral immunoglobulin responses.
(20/2088)
Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of "quasimonoclonal" (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, approximately 20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice. (+info)
Membrane tubule-mediated reassembly and maintenance of the Golgi complex is disrupted by phospholipase A2 antagonists.
(21/2088)
Although membrane tubules can be found extending from, and associated with, the Golgi complex of eukaryotic cells, their physiological function has remained unclear. To gain insight into the biological significance of membrane tubules, we have developed methods for selectively preventing their formation. We show here that a broad range of phospholipase A2 (PLA2) antagonists not only arrest membrane tubule-mediated events that occur late in the assembly of the Golgi complex but also perturb its normal steady-state tubulovesicular architecture by inducing a reversible fragmentation into separate "mini-stacks." In addition, we show that these same compounds prevent the formation of membrane tubules from Golgi stacks in an in vitro reconstitution system. This in vitro assay was further used to demonstrate that the relevant PLA2 activity originates from the cytoplasm. Taken together, these results demonstrate that Golgi membrane tubules, sensitive to potent and selective PLA2 antagonists, mediate both late events in the reassembly of the Golgi complex and the dynamic maintenance of its steady-state architecture. In addition, they implicate a role for cytoplasmic PLA2 enzymes in mediating these membrane trafficking events. (+info)
Optimal replication activity of vesicular stomatitis virus RNA polymerase requires phosphorylation of a residue(s) at carboxy-terminal domain II of its accessory subunit, phosphoprotein P.
(22/2088)
The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P226/227) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-type (wt) protein. Substitution of alanine for Ser-233 did not have any adverse effect on replication activity of the protein. In contrast, all the mutant proteins showed activities similar to that of the wt protein in transcription. These results indicate that phosphorylation of the carboxy-terminal domain II residues of P protein are required for optimal replication activity but not for transcription activity. Furthermore, substitution of glutamic acid residues for Ser-226 and Ser-227 resulted in a protein that was only 14% active in replication but almost fully active in transcription. Taken together, these results, along with our earlier studies, suggest that phosphorylation of residues at two different domains in the P protein regulates its activity in transcription and replication of the VSV genome. (+info)
Carboxy-terminal five amino acids of the nucleocapsid protein of vesicular stomatitis virus are required for encapsidation and replication of genome RNA.
(23/2088)
The encapsidation of vesicular stomatitis virus (VSV) genome RNA, a prerequisite step to the replication process by the nucleocapsid protein (N) was studied by its ability to package VSV leader RNA in vitro in a RNase-resistant form. The VSV leader RNA was derived from the SP6 transcription vector while the N protein was made in rabbit reticulocyte lysate. The in vitro encapsidation was carried out by translating N mRNA in the presence of 32P-labeled presynthesized leader RNA. The RNA encapsidation property of the N protein was completely abrogated when the C-terminal five amino acids (VEFDK-COOH) were deleted. Systematic mutational analyses within the C-terminal five amino acid regions reveal that the RNA encapsidation activity was lost in all mutants except K --> A and K --> R, indicating that C-terminal five amino acids, in particular the lysine residue play critical role in genome RNA encapsidation. To correlate the in vitro encapsidation abilities of these mutant N proteins with genome RNA replication, we have used a full-length cDNA clone of VSV genome RNA to rescue infectious virions from cells expressing L, P, and wt or mutant N proteins and measured the recovery of plaque forming units. The results indicate that the N mutants that are defective in in vitro encapsidation of leader RNA do not support replication, establishing the requirement of C-terminal five amino acids of the N protein in viral replication. (+info)
Neuronal expression of NOS-1 is required for host recovery from viral encephalitis.
(24/2088)
The role of nitric oxide synthase (NOS) in host defense and clearance of vesicular stomatitis virus (VSV) from the central nervous system (CNS) was examined. NOS-1, NOS-2, and NOS-3 knockout mice were infected with VSV and were treated with either IL-12 or medium. IL-12 treatment resulted in substantially decreased VSV titers in wildtype and NOS-3 knockout mice, but had a marginal effect in the NOS-1 and NOS-2 knockout mice. NOS-1 expression in neurons was associated with survival from VSV infection. The data indicate that the enzyme activity is local, since NOS-2 expression in microglia and inflammatory macrophages and NOS-3 expression in astrocytes, endothelial cells, and ependymal cells did not compensate. (+info)