An antiviral mechanism investigated with ribavirin as an RNA virus mutagen for foot-and-mouth disease virus

To prove whether error catastrophe/lethal mutagenesis is the primary antiviral mechanism of action of ribavirin against foot-and-mouth disease virus (FMDV). Ribavirin passage experiments were performed and supernatants of Rp1 to Rp5 were harvested. Morphological alterations as well as the levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected using the supernatants of Rp1 to Rp5 and control were measured by microscope, real-time RT-PCR, western-blotting and plaque assays, respectively. The mutation frequency was measured by sequencing the complete P1- and 3D-encoding region of FMDV after a single round of virus infection from ribavirin-treated or untreated FMDV-infected cells. Ribavirin treatment for FMDV caused dramatically inhibition of multiplication in cell cultures. The levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected were more greatly reduced along with the passage from Rp1 to Rp5, moreover, nucleocapsid protein could not be detected and no recovery of infectious virus in the supernatant or detection of intracellular viral RNA was observed at the Rp5-infected cells. A high mutation rate, giving rise to an 8-and 11-fold increase in mutagenesis and resulting in some amino acid substitutions, was found in viral RNA synthesized at a single round of virus infection in the presence of ribavirin of 1000 microM and caused a 99.7% loss in viral infectivity in contrast with parallel untreated control virus. These results suggest that the antiviral molecular mechanism of ribavirin is based on the lethal mutagenesis/error catastrophe, that is, the ribavirin is not merely an antiviral reagent but also an effective mutagen.     

Nitric oxide as an antioxidant.

Benzoate monohydroxy compounds, and in particular salicylate, were produced during interaction of ferrous complexes with hydrogen peroxide (Fenton reaction) in a N2 environment. These reactions were inhibited when Fe complexes were flushed, prior to the addition in the model system, by nitric oxide. Methionine oxidation to ethylene by Fenton reagents was also inhibited by nitric oxide. Myoglobin in several forms such as metmyoglobin, oxymyoglobin, and nitric oxide-myoglobin were interacted with an equimolar concentration of hydrogen peroxide. Spectra changes in the visible region and the changes in membrane (microsomes) lipid peroxidation by the accumulation of thiobarbituric acid-reactive substances (TBA-RS) were determined. The results showed that metmyoglobin and oxymyoglobin were activated by H2O2 to ferryl myoglobin, which initiates membrane lipid peroxidation; but not nitric oxide-myoglobin, which, during interaction with H2O2, did not form ferryl but metmyoglobin which only poorly affected lipid peroxidation. It is assumed that nitric oxide, liganded to ferrous complexes, acts to prevent the prooxidative reaction of these complexes with H2O2.     

Nitric oxide synthase activity and endogenous inhibitors in rats recovered from allergic encephalomyelitis

We have previously reported that in comparison with normal rats, the presence of experimental allergic encephalomyelitis (EAE) leads to decreased endogenous inhibitory activity (EIA) of Ca2+-dependent nitric oxide synthase (NOS) in both brain and serum, and increased expression of protein 3-nitrotyrosine (NT) in brain. In this work we show that animals recovered from the clinical signs of EAE are not different from controls in terms of either brain NOS activity, EIA of NOS, or NT expression. These results suggest that parallel to the reversal of the disease symptoms, a normalization of the production of nitric oxide and related species occurs.     

Efficient virus extinction by combinations of a mutagen and antiviral inhibitors

The effect of combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of foot-and-mouth disease virus (FMDV) in cell culture has been investigated. Related FMDV clones differing up to 10(6)-fold in relative fitness in BHK-21 cells have been compared. Systematic extinction of intermediate fitness virus was attained with a combination of FU and G but not with the mutagen or the inhibitor alone. Systematic extinction of high-fitness FMDV required the combination of FU, G, and H. FMDV showing high relative fitness in BHK-21 cells but decreased replicative ability in CHO cells behaved as a low-fitness virus with regard to extinction mutagenesis in CHO cells. This confirms that relative fitness, rather than a specific genomic sequence, determines the FMDV response to enhanced mutagenesis. Mutant spectrum analysis of several genomic regions from a preextinction population showed a statistically significant increase in the number of mutations compared with virus passaged in parallel in the absence of FU and inhibitors. Also, in a preextinction population the types of mutations that can be attributed to the mutagenic action of FU were significantly more frequent than other mutation types. The results suggest that combinations of mutagenic agents and antiviral inhibitors can effectively drive high-fitness virus into extinction.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5′-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.     

Target-sensitive immunoliposomes as an efficient drug carrier for antiviral activity

We have shown previously that target-sensitive immunoliposomes composed of palmitoyl antibody stabilized phosphatidylethanolamine bilayers could be destabilized by binding to the target cells (Ho, R. J. Y., Rouse, B. T., and Huang, L., Biochemistry (1986) 25, 5500-5506). Target-sensitive immunoliposome-encapsulated and free cytotoxic drugs of nucleoside analogs cytosine-beta-D-arabinoside (AraC) or acycloguanosine (acyclovir, ACV) were compared for their antiviral efficacy and cell cytotoxicity. Target-insensitive immunoliposomes and nontargeted liposomes were also investigated. When the mouse fibroblast L929 cells were infected at low multiplicity with herpes simplex virus, AraC encapsulated in target-sensitive immunoliposomes composed of transphosphatidylated egg phosphatidylethanolamine effectively inhibited virus replication and had far less cell cytotoxicity than free drug. As a measure of cytotoxicity, the drug concentration required to inhibit 50% of [3H]thymidine incorporation from 6 to 42 h (CD50) was determined. For free AraC, this value was 0.3 ng/ml, whereas for target-sensitive immunoliposome-encapsulated AraC, the CD50 exceeded 1 microgram/ml. However, target-sensitive immunoliposome-encapsulated AraC was virus inhibitory (50% effective dose = ED50) at 1.8 ng/ml. A free drug concentration of at least 1000-fold greater was required for comparable antiviral activity. A similar phenomenon was observed when ACV was administered via target-sensitive immunoliposomes. The CD50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 12.5 ng/ml and 1.4 micrograms/ml, respectively, whereas the ED50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 1.1 and 125 ng/ml, respectively. Consequently, our results indicated the superiority of target-sensitive immunoliposomes at drug delivery, especially when drugs were cytotoxic to cells. The use of liposomes of the target-insensitive variety provided some enhancement of activity, but this was several-fold less than that observed with target-sensitive immunoliposomes. In addition, the nucleoside transport inhibitors, p-nitrothiobenzylinosine and dipyridamole, were shown to inhibit the liposome-mediated antiviral activity of AraC. This finding indicated that site-specific cytosolic delivery of nucleoside analogs by target-sensitive immunoliposomes involved a cellular nucleoside transport system. A mechanism of action is proposed.     

Nitric oxide as an adaptive protection factor in hypoxia

Recent data concerning intracellular aspects of nitric oxide at hypoxia conditions and correction means pathologic states by it. On the basis of data obtained publications and own investigations about nitric oxide influence on mitochondrial respiration and oxidative phosphorylation possible mechanism its action is discussed. We conclude that breach of macroergs output at hypoxia connect with active oxygen forms, antioxidant enzymes activity and individual peculiarities physiologic reactivity of organism.     

Sendai virus targets inflammatory responses,as well as the interferon-induced antiviral state,in a multifaceted manner

We have used cDNA arrays to compare the activation of various cellular genes in response to infection with Sendai viruses (SeV) that contain specific mutations. Three groups of cellular genes activated by mutant SeV infection, but not by wild-type SeV, were identified in this way. While some of these genes are well known interferon (IFN)-stimulated genes, others, such as those for interleukin-6 (IL-6) and IL-8, are not directly induced by IFN. The gene for beta IFN (IFN-beta), which is critical for initiating an antiviral response, was also specifically activated in mutant SeV infections. The SeV-induced activation of IFN-beta was found to depend on IFN regulatory factor 3, and the activation of all three cellular genes was independent of IFN signaling. Mutations that disrupt four distinct elements in the SeV genome (the leader RNA, two regions of the C protein, and the V protein) all lead to enhanced levels of IFN-beta mRNA, and at least three of these viral genes also appear to be involved in preventing activation of IL-8. Our results suggest that SeV targets the inflammatory and adaptive immune responses as well as the IFN-induced intracellular antiviral state by using a multifaceted approach.     

Sendai virus vectors as an emerging negative-strand RNA viral vector system

The power to manipulate the genome of negative-strand RNA viruses, including the insertion of additional non-viral genes, has led to the development of a new class of viral vectors for gene transfer approaches. The murine parainfluenza virus type I, or Sendai virus (SeV), has emerged as a prototype virus of this vector group, being employed in numerous in vitro as well as animal studies over the last few years. Extraordinary features of SeV are the remarkably brief contact time that is necessary for cellular uptake, a strong but adjustable expression of foreign genes, efficient infection in the respiratory tract despite a mucus layer, transduction of target cells being independent of the cell cycle, and an exclusively cytoplasmic replication cycle without any risk of chromosomal integration. In this review we describe the current knowledge of Sendai virus vector (SeVV) development as well as the results of first-generation vector applications under both in vitro and in vivo conditions. So far, Sendai virus vectors have been identified to be a highly efficient transduction tool for a broad range of different tissues and applications. Future directions in vector design and development are discussed.     

Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

Biological activity of monomeric Sendai virus HN glycoprotein

Abstract We have developed a transformation system for Streptomyces wadayamensis , a cephamycin C producer. 1−5 × 10⁹ protoplasts can be obtained when late logarithmic phase cultures of this microorganisms are incubated with 10 mg/ml of lysozyme. Polyethylene glycol-Ca²⁺-mediated transformation of these protoplasts yielded 10⁶ transformants per μg of pIJ702 or pIJ365 DNA.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.     

Lead optimization of an acylhydrazone scaffold possessing antiviral activity against Lassa virus

Previously we reported the optimization of antiviral scaffolds containing benzimidazole and related heterocycles possessing activity against a variety of arenaviruses. These series of compounds were discovered through an HTS campaign of a 400,000 small molecule library using lentivirus-based pseudotypes incorporated with the Lassa virus envelope glycoprotein (LASV GP). This screening also uncovered an alternate series of very potent arenavirus inhibitors based upon an acylhydrazone scaffold. Subsequent SAR analysis of this chemical series involved various substitutions throughout the chemical framework along with assessment of the preferred stereochemistry. These studies led to an optimized analog (ST-161) possessing subnanomolar activity against LASV and submicromolar activity against a number of other viruses in the Arenaviridae family.     

Studies on antiviral glycosides. II. Mode of action for virucidal effects on Sendai virus.

Treatment of Sendai virus with p-(sec-butyl)-phenyl-6-chloro-6-deoxy-beta-D-glucopyranoside, followed by freezing and thawing resulted in a loss of hemolytic and cell fusion activities as well as infectivity without affecting hemagglutinating and neuraminidase activities. The anti-hemolytic activity of this compound was reversed by the addition of phosphatidyl choline to the virus samples. p-Azidophenyl-6-chloro-6-deoxy-beta-D[3H]glucopyranoside was successfully used for photoaffinity labeling of a specific virion site, and we confirmed the affected site of the glucoside to be the lipid components in the viral envelopes.     

Nitric oxide as a bioregulator of apoptosis

Nitric oxide (NO), synthesized from l-arginine by NO synthases, is a small, diffusible, highly reactive molecule with dichotomous regulatory roles under physiological and pathological conditions. NO can promote apoptosis (proapoptosis) in some cells, whereas it inhibits apoptosis (antiapoptosis) in other cells. This complexity is a consequence of the rate of NO production and the interaction with biological molecules such as iron, thiols, proteins, and reactive oxygen species. Long-lasting production of NO acts as a proapoptotic modulator by activating caspase family proteases through the release of mitochondrial cytochrome c into the cytosol, upregulation of p53 expression, activation of JNK/SAPK, and altering the expression of apoptosis-associated proteins including Bcl-2 family proteins. However, low or physiological concentrations of NO prevent cells from apoptosis induced by trophic factor withdrawal, Fas, TNFalpha, and lipopolysaccharide. The antiapoptotic mechanism can be understood via expression of protective genes such as heat shock proteins, Bcl-2 as well as direct inhibition of the apoptotic caspase family proteases by S-nitrosylation of the cysteine thiol. Our current understanding of the mechanisms by which NO exerts both pro- and antiapoptotic actions is discussed in this review article.