Semliki Forest virus envelope proteins function as proton channels

It has been shown that isolated nucleocapsids of Semliki Forest virus (SFV) contract upon low pH exposure (Soederlundet al., 1972). This contraction of the nucleocapsids has been used as an indicator to demonstrate that the spike proteins of SFV can translocate protons into the interior of the virus particle upon low pH (5.8) exposure. Spikeless virus particles obtained after bromelain digestion, which were used as a control, did not translocate protons. This implies that the ectodomain of the spike plays a crucial role for the proton translocation.     

Semliki Forest virus induced cell-cell fusion at neutral extracellular pH

Semliki Forest virus-induced cell-cell fusion from within was considered to exclusively occur at mildly acidic pH (<6.2). Data of this study show that such cell fusion can also be triggered by transient acidification of the cytoplasm of infected cells at an extracellular, neutral pH. Results were obtained by utilizing NH₄Cl pulses combined with covalent modification of cell surface proteins. The observation implies a revision of the current consensus regarding the mechanism of Semliki Forest virus induced cell-cell fusion. We propose a model in which at least two peptide segments of the viral spike protein E₁ may be involved in triggering the fusion event.     

Generation by lipopolysaccharide of a late-acting soluble suppressor of antibody synthesis.

Polyclonal stimulation of normal mouse spleen cells by lipopolysaccharide (LPS) from Salmonella typhimurium resulted in the generation of a factor which was capable of suppressing the humoral immune response in vitro. LPS effectively induced the release of the inhibitory material into the supernatants of these cultures within 24 hr. The suppressive mediator, which was similar in properties to the antigen-generated, transiently-acting soluble suppressor (TASS) reported earlier, partially abrogated (by 30–80%) the anti-sheep erythrocyte plaque-forming cell response when added to test cultures ~20 hr prior to assay for direct hemolytic plaques. Although LPS, in submitogenic doses, also was effective in depressing the in vitro hemolysin response, the inhibitory activity of residual mitogen present in the test supernatants, and that of the LPS-induced factor, were shown to be different. By use of antisera and complement treatment to selectively deplete spleen cell populations of T or B lymphocytes, it was demonstrated that B cells were essential for production of the suppressive mediator.     

Discovery of pyrido[2,3-d]pyrimidine-based inhibitors of HCV NS5A

Efforts to improve the genotype 1a potency and pharmacokinetics of earlier naphthyridine-based HCV NS5A inhibitors resulted in the discovery of a novel series of pyrido[2,3-d]pyrimidine compounds, which displayed potent inhibition of HCV genotypes 1a and 1b in the replicon assay. SAR in this system revealed that the introduction of amides bearing an additional ‘E’ ring provided compounds with improved potency and pharmacokinetics. Introduction of a chiral center on the amide portion resulted in the observation of a stereochemical dependence for replicon potency and provided a site for the attachment of functional groups useful for improving the solubility of the series. Compound 21 was selected for administration in an HCV-infected chimpanzee. Observation of a robust viral load decline provided positive proof of concept for inhibition of HCV replication in vivo for the compound series.     

Neurotrophins Differentially Regulate the Survival and Morphological Complexity of Human CNS Model Neurons

To determine the effect of neurotrophins on the survival and morphological differentiation of CNS neurons, we examined NT2-N cells, which provide a unique culture model for terminally differentiated and polar human neurons. Here we report the development of conditions for the long-term culture of NT2-N cells in low density and in chemically defined medium. We show that NT2-N cells express rRNAs for TrkA, TrkB, and TrkC tyrosine kinase receptors and the low-affinity nerve growth factor receptor (p75NTR). All members of the nerve growth factor-related family of neurotrophic factors promote neuronal survival in long-term cultures with approximately 1 ng/ml for half-maximal survival. At high concentrations (>20 ng/ml), the neurotrophins reversed the survival-promoting effect as judged by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] conversion. In contrast to the uniform effect of all neurotrophins on neuronal survival, brain-derived neurotrophic factor selectively induced an increased dendritic complexity. These results demonstrate that NT2-N cells provide a useful model to analyze the effect of neurotrophins on the survival and morphological differentiation of CNS neurons in vitro. In addition, the data indicate that neuronal survival and the development of morphological complexity are differentially regulated in a multireceptor context.     

Furin-mediated cleavage of the feline foamy virus Env leader protein

The molecular biology of spuma or foamy retroviruses is different from that of the other members of the Retroviridae. Among the distinguishing features, the N-terminal domain of the foamy virus Env glycoprotein, the 16-kDa Env leader protein Elp, is a component of released, infectious virions and is required for particle budding. The transmembrane protein Elp specifically interacts with N-terminal Gag sequences during morphogenesis. In this study, we investigate the mechanism of Elp release from the Env precursor protein. By a combination of genetic, biochemical, and biophysical methods, we show that the feline foamy virus (FFV) Elp is released by a cellular furin-like protease, most likely furin itself, generating an Elp protein consisting of 127 amino acid residues. The cleavage site fully conforms to the rules for an optimal furin site. Proteolytic processing at the furin cleavage site is required for full infectivity of FFV. However, utilization of other furin proteases and/or cleavage at a suboptimal signal peptidase cleavage site can partially rescue virus viability. In addition, we show that FFV Elp carries an N-linked oligosaccharide that is not conserved among the known foamy viruses.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of N-1-heteroalkyl-4-hydroxyquinolon-3-yl-benzothiadiazines

N-1-Alkylamino and N-1-alkyloxy-4-hydroxyquinolon-3-yl benzothiadiazines were synthesized and evaluated as inhibitors of genotype 1 HCV polymerase. The N-1-alkyloxy derivatives were not potent inhibitors, however N-1-alkylamino derivatives displayed comparable potency to carbon analogs. Analogs with aliphatic substituents were significantly more potent than those with benzylic substituents against genotype 1a polymerase. The most potent inhibitors contained small alkyl or carbocyclic substituents and exhibited IC50's of 50-100 and 200-400 nM against genotype 1b and 1a HCV polymerase, respectively.     

Alcohol withdrawal-induced changes in brain biogenic amines in mice: Influence of the genotype

Alterations in striatal and hippocampal dopamine (DA) and serotonin (5HT) activities were investigated in two inbred strains of mice (C57B1 and Balb/c) after 3 withdrawal periods following 5 months chronic ethanol administration. Two groups of animals with different levels of ethanol administration (15% and 30%, v/v) were examined. A striking strain dependency has been noted. Striatal dopaminergic mechanisms of the Balb/c strain are profoundly disturbed in both groups. In contrast no changes were noted for either transmitter activities in C57B1 mice at any withdrawal time studied. Strain dependency has also been noted for hippocampal serotonin neurotransmission, since only Balb/c mice showed a progressive decrease in 5HT levels. These impairments observed in striatum and hippocampus could be involved in motor incoordinations and convulsions often associated with the withdrawal syndrome. The differences in withdrawal effects we noted between the two strains may be linked to the specific chemical neuroanatomy of the strains. Such specificities could be implied in the well known variability of withdrawal induced behavior in man.