Virus-infected Baby Hamster Kidney Cells Research Articles

Screening of Phellinus linteus, a Medicinal Mushroom, for Anti-viral Activity

Harmful viruses, including dengue virus, human immunodeficiency virus, and hepatitis B virus are known to be extremely sensitive to glucosidase inhibition. Phellinus linteus, an edible medicinal mushroom, was screened as an α-glucosidase inhibitor. The methanol extract of P. Linteus inhibited the trafficking process of Newcastle disease virus-hemagglutinin-neuramidase, a viral glycoprotein, in virus-infected baby hamster kidney cells. These results suggest that P. Linteus extract inhibits viral glycoprotein expression on cell surface through inhibition of trafficking process rather than glycoprotein synthesis.

Anti-bacterial and Anti-viral Activities of Extracts from Terminalia chebula Barks

Effects of methanol extract of Terminalia chebula on antibacterial activity and the generation of superoxide radical in Bacillus subtilis, as well as on syncytium formation and cytopathic activity in virus-infected baby hamster kidney cells were examined. The extract effectively inhibited syncytium formation in a concentration-dependent manner, and infectious virus production was markedly reduced. However, glycoprotein synthesis was not affected. These results collectively indicate that methanol extract of T. chebula potentially inhibit glycosylation by acting as a suppressor of intracellular glycosylation trafficking.

Nonactin hinders intracellular glycosylation in virus-infected baby hamster kidney cells

Potent antiviral agents hinder virus-infected cell machinery, leading to rescue from viral damage. In this study, we aimed to identify selective intracellular glycosylation inhibitor(s) that do not suppress glycoprotein synthesis. Our results showed that nonactin is a potent inhibitor of intracellular glycosylation. First, we examined the effects of nonactin on syncytium formation and cytopathic activity in virus-infected baby hamster kidney cells. Nonactin effectively inhibited syncytium formation in a concentration-dependent manner, and infectious virus production was markedly reduced. However, glycoprotein synthesis was not affected. In the presence of 5 µg/ml nonactin, we observed the intracellular accumulation of vesicular stomatitis virus-G protein as well as syncytium formation, but no significant effects on Newcastle disease virus-hemagglutinin-neuramidase glycoprotein synthesis. Our results collectively indicate that nonactin potentially inhibits glycosylation by acting as a suppressor of intracellular glycosylation trafficking.

Two RNA polymerase complexes from vesicular stomatitis virus-infected cells that carry out transcription and replication of genome RNA.

By immunoaffinity column chromatography, we have purified two RNA polymerase complexes, the transcriptase and replicase, from vesicular stomatitis virus-infected baby hamster kidney cells. The transcriptase is a multiprotein complex, containing the virus-encoded RNA polymerase L and P proteins, and two cellular proteins, translation elongation factor-1alpha and heat-shock protein 60. In addition, the complex contains a submolar amount of cellular mRNA cap guanylyltransferase. The replicase, on the other hand, is a complex containing the viral proteins, L, P, and the nucleocapsid (N), but lacking elongation factor-1alpha, heat-shock protein 60, and guanylyltransferase. The transcriptase complex synthesizes capped mRNAs and initiates transcription at the first gene (N) start site, whereas the replicase complex initiates RNA synthesis at the precise 3' end of the genome RNA and synthesizes encapsidated replication products in the presence of the N-P complex. We propose that two RNA polymerase complexes that differ in their content of virally and host-encoded proteins are separately responsible for transcription and replication of vesicular stomatitis virus genome RNA.

Stable Protein–RNA Interaction Involves the Terminal Domains of Bluetongue Virus mRNA, but Not the Terminally Conserved Sequences

The interaction of bluetongue virus (BTV) proteins with viral RNA was investigatedin vitroby means of a biochemical approach. By subjecting cytoplasmic extracts from virus-infected baby hamster kidney cells andin vitrosynthesized radiolabeled RNA to ultraviolet cross-linking assays, we demonstrated that, of all the BTV proteins, NS2 becomes most intimately associated with the labeled viral RNA. Competition binding studies indicated that NS2 has the greatest affinity for the 3′ region of the viral transcripts. By analyzing the binding efficiency of NS2 to mutant RNA transcripts which lacked the fully conserved 5′- and/or 3′-terminal hexanucleotides, we have established that these sequences are not necessary for optimal binding. The specificity of the NS2–RNA interaction was investigated by competition experiments with unlabeled BTV-specific homologous and heterologous competitor RNAs as well as with viral double-stranded RNA (dsRNA). Although apparent differences in the ability of NS2 to bind to the different RNA transcripts were observed, it did not bind to the dsRNA.

Exposure of Semliki Forest virus-infected baby hamster kidney cells to low pH leads to a proton influx and a rapid depletion of intracellular ATP which in turn prevents cell-cell fusion. Brief report.

The conditions needed to trigger Semliki Forest virus (SFV) induced cell-cell fusion from within (FFWI) of baby hamster kidney (BHK) cells differs from some other cells (e.g., Aedes albopictus cells). In contrast to such cells it is mandatory for BHK cells to be brought back to a neutral pH after a short exposure to mildly acidic pH for fusion to occur. This phenomenon can be explained by a sudden drop of the intracellular pH observed after clamping the extracellular pH below 6.2--the pH required to trigger fusion--and by the thereby implied increased expenditure of ATP. Since the fusion from within of SFV-infected cells is energy dependent [Kempf et al. (1987) Arch Virol 95: 111-122] the fusion process is blocked due to the ATP depletion.

Immunocytochemical study of the partition and distribution of Sindbis virus glycoproteins in freeze-fractured membranes of infected baby hamster kidney cells.

Sindbis virus-infected baby hamster kidney cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies or with conventional lectin label (wheat germ agglutinin) were used in conjunction with colloidal gold-conjugated protein A or ovomucoid, respectively. In addition, intact infected cells were analyzed with both labeling procedures. Experiments with Sindbis infected-chick embryo fibroblast cells were carried out as controls. Viral transmembrane glycoproteins appeared present in freeze-fractured inner and outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes; a clear preferential partition with the exoplasmic faces of all intracellular membranes was observed. By contrast, at the plasma membrane level, Sindbis glycoproteins were found to partition preferentially with the protoplasmic face. It seems likely that this protoplasmic partition is related to the binding with the nucleocapsid that takes place during the budding of the virus. At the cell surface, viral glycoproteins always appeared clustered and were predominantly associated with budding figures: moreover, large portions of the plasma membrane were devoid of both glycoproteins and budding viruses.

Antibody-dependent cell-mediated cytotoxicity against influenza virus-infected cells.

The three major immunocompetent cells in human peripheral blood (lymphocytes, neutrophils, and monocytes) were shown to be effector cells for antibody-dependent cell-mediated cytotoxicity (ADCC) against influenza virus-infected baby hamster kidney cells in vitro. Lymphocyte cytotoxicity was mediated by FcIgG receptor-bearing null cells and T gamma cells. These effector populations were best defined by HNK-1, a monoclonal antibody to human natural killer and ADCC-mediator cells. Antibody responsible for ADCC against influenza virus-infected cells was detectable in sera of young children after natural infection and after vaccination with inactivated and live attenuated viruses. ADCC antibody appeared before hemagglutination-inhibiting antibody and persisted for at least one year after vaccination with live attenuated vaccine. ADCC antibody was subtype-specific but quite broadly reactive within a subtype. Both hemagglutinin and neuraminidase were antigenic determinants for ADCC antibody. An anamnestic response to the original strain was observed after challenge with influenza virus of a heterologous subtype.

Antibody-induced modulation of proteins in vesicular stomatitis virus-infected fibroblasts.

When vesicular stomatitis virus-infected baby hamster kidney cells were treated with rabbit anti-vesicular stomatitis virus serum, there was a loss of the viral glycoprotein G into acid-soluble products. This degradation occurred within minutes at 37 degrees C and required the presence of G protein at the cell surface. The degree of degradation depended on antiserum concentration. The antiserum, also, prevented maturation of extracellular virions and induced partial degradation of the intracellular viral proteins, without affecting host proteins. The degradation could not be prevented by the presence of lysosomotropic agents, protease inhibitors, colchicine, or cytochalasin B. Similar kinetics and specificity of degradation was obtained with cells infected with vesicular stomatitis virus mutants that were less cytopathic. These results characterize a model system for studying the parameters and consequences of antigenic modulation as well as for studying the fate of viral antigens during persistent infections.

Altered G-protein glycosylation in vesicular stomatitis virus-infected glucose-deprived baby hamster kidney cells.

The glycosylation of the G-protein was analyzed in vesicular stomatitis virus-infected baby hamster kidney cells incubated in the absence of glucose. The results indicate that the G-protein in glucose-starved cells is initially glycosylated from a lipid donor with a glucosylated oligosaccharide which is resistant to endo-beta-N-acetylglucosaminidase H and partially susceptible to alpha-mannosidase. With longer times, the protein-bound carbohydrate chain becomes much more sensitive to alpha-mannosidase while remaining endo-beta-N-acetylglucosaminidase H-resistant. Purified virions from glucose-starved baby hamster kidney cells, labeled with [35S]methionine and isolated on a sucrose gradient, contain altered forms of the G-protein, whereas the other viral proteins remain unchanged. These altered forms could also be radiolabeled with [3H]mannose, and upon analysis of labeled glycopeptides by chromatography on concanavalin A-Sepharose and Bio-Gel P-6, it was apparent that modification of the oligosaccharide portion of the G-protein occurs in baby hamster kidney cells, leading to aberrant mature carbohydrate chains.

Direct effects of 1,3-diaminopropane on reticulocyte lysate protein synthesis

The precise functions of polyamines in mammalian cells have not yet been established, but the strong association between the rate of cell growth and proliferation and the rates of putrescine and spermidine synthesis suggests that they have an important regulatory role [ 11. The short half-lives of some enzymes required for polyamine synthesis, in particular the first enzyme of the pathway ornithine decarboxylase, and the consequent very rapid and extensive variation in its activity, reinforce this suggestion. Specific inhibitors of the production of polyamines would be of great value in further elucidating their functions, and one drug which has been widely used in this respect is the putrescine analogue 1,3-diaminopropane [2-171. Diaminopropane does not directly affect the activity of isolated ornithine decarboxylase, but greatly reduces the activity of the enzyme in the intact animal or cell [2,8,10,11]. As the enzyme synthesis can also be inhibited, apparently by a feedback repression mechanism, by low concentrations of its natural product putrescine [4,18,19], it seemed likely that diaminopropane was acting by a similar mechanism, although substantially higher concentrations of the analogue were required. However, it has been inferred from studies with virus-infected baby hamster kidney cells that the effects of diaminopropane may be due at least in part of a direct effect on cellular protein synthesis [20]. The dramatic effects on ornithine decarboxylase may thus be a function of its unusually rapid turnover rate rather than, or in addition to, any specific inhibition of its synthesis by a feedback repression mechanism. Here we report that diaminopropane, at concentrations within the range normally used, does indeed have a direct inhibitory effect on the rate of protein synthesis by a cell-free system from rabbit reticulocytes. The effect of diaminopropane is not due to any antagonistic effect against naturally occurring polyamines, but is simply a cation effect consequent upon the high levels used. In the absence of normal divalent cations, diaminopropane will itself support protein synthesis in this system. These results lend strong support to the doubts about the specificity of diaminopropane as an inhibitor of polyamine synthesis raised in [20].

Translation products of foot-and-mouth disease virus-infected baby hamster kidney cells.

Electrophoretic analysis of the polypeptides present in foot-and-mouth disease virus (FMDV) infected cells indicates that the viral ribonucleic acid (RNA), as with other picornaviruses is translated to produce large polypeptide products which are subsequently cleaved into the smaller stable virus proteins (VP) and non-capsid virus proteins (NCVP). The stable genome products and their estimated molecular weights are: NCVP5 (57,000 daltons), NCVP9 (37,500 daltons), NCVP11. (15,500 daltons), VP0 (40,000 daltons), and VP1–3 (each, 28,300 daltons). The similarities of these products compared to poliovirus and encephalomyocarditis virus genome products are discussed.

Effect of actinomycin D on virus-induced ribonucleic acid polymerase formation in foot-and-mouth disease virus-infected baby hamster kidney cells.

Actinomycin D, at a concentration that inhibits cellular ribonucleic acid (RNA) synthesis, inhibited the production of foot-and-mouth disease virus-induced RNA polymerase in baby hamster kidney cells. Inhibition was proportional to exposure time and reached 85% when actinomycin D was added 90 min before infection. Polymerase production was inhibited to the same extent in growth and minimal media, and the kinetics of its appearance were slightly different than in untreated cells. Enzyme preparations from actinomycin-treated cells having one-third to one-tenth the activity of untreated samples gave products with RNA profiles similar to those of controls. The 37S viral peak, 20S ribonuclease-resistant peak, and 26 to 28S peaks were present in all cases. Actinomycin D did not consistently inhibit virus production in either medium. Insulin did not prevent the actinomycin induced inhibition of polymerase and virus production from occurring.