Inhibitory Effect of Acylphloroglucinol Derivatives on the Replication of Vesicular Stomatitis Virus

The antiviral activity of natural phloroglucinols and of synthesized mono- and diacylphloroglucinols, and 2,6-diacyl-4,4-dialkylcyclohexa-1,3,5-triones was investigated. A correlation between the acyl chain length and inhibitory activity against vesicular stomatitis virus (VSV) was observed. Potent antiviral activity was found in di-isovalerylphoroglucinol. 2,6-Diacyl-4,4-dialkylcyclohexa-1,3,5-triones inhibited replication of the virus with low cytotoxicity.     

Inhibitory effect of acylphloroglucinol derivatives on the replication of vesicular stomatitis virus.

The antiviral activity of natural phloroglucinols and of synthesized mono- and diacylphloroglucinols, and 2,6-diacyl-4,4-dialkylcyclohexa-1,3,5-triones was investigated. A correlation between the acyl chain length and inhibitory activity against vesicular stomatitis virus (VSV) was observed. Potent antiviral activity was found in di-isovalerylphoroglucinol. 2,6-Diacyl-4,4-dialkylcyclohexa-1,3,5-triones inhibited replication of the virus with low cytotoxicity.     

Stimulation of pp60c-src tyrosyl kinase activity in polyoma virus-infected mouse cells is closely associated with polyoma middle tumor antigen synthesis

We have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product, pp60c-src. The results of our studies demonstrate that infection of mouse cells with wild-type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c-src tyrosyl kinase activity. The level of pp60c-src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c-src kinase activity was not observed in mouse cells infected with transformation-defective strains of polyoma virus. In examining the kinetics of pp60c-src kinase stimulation in mouse cells at various times following wild-type polyoma virus infection, we found that the level of pp60c-src kinase activity correlated directly with the synthesis of polyoma virus-encoded tumor antigens. By comparing wild-type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c-src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.     

Effect of urea and uric acid on the hemolytic activity of Sendai virus

It was found that haemolytic activity of Fushimi strain of Sendai virus multiplied in allantoic cavity of chicken embryos is independent on its haemagglutinating titer and also on allantoic fluid urea and uric acid content. It was shown in experiments with embryonated eggs that these two compounds have no also influence on haemolytic activity induction in Sendai virus. Moreover, the results of an experiment in which allantoic fluid was replaced by Eagle's liquid suggest that most probably the other components present in allantoic fluid do not also influence the appearance of haemolytic activity of this virus.     

Metabolic Requirements for the Development of Hemadsorption Activity and Virus Formation in BHK-21 Cells Infected with Newcastle Disease Virus

Differential effect of various metabolic inhibitors on the development of hemadsorption activity and virus formation in cells infected with Newcastle disease virus (NDV) was investigated. It was found that, in BHK-21 cells infected with NDV, cycloheximide did not prevent the development of hemadsorption activity, whereas protein synthesis and virus formation by the cell were rapidly inhibited by the drug. When the drug was added to the culture at 4.5 h after infection or later, hemadsorption activity of the cell continued to develop normally for about 1 h. Similar increase in hemadsorption activity was found in cells which were treated with anti-NDV serum (to neutralize their hemadsorption activity) and then washed and incubated with cycloheximide. However, when cells were treated with the drug early in the infection (1.5 or 3.0 h), they did not show any detectable hemadsorption reaction throughout the infection. In contrast to cycloheximide, iodoacetate added to the culture together with sodium azide inhibited completely both the development of hemadsorption activity and the formation of progeny virus. These results suggest that the change of cell surface to become hemadsorptive may depend upon the energy generating system but not upon de novo synthesis of protein, whereas production of infectious virus may require continuous synthesis of protein.     

Switch in antiviral specificity of a GTPase upon translocation from the cytoplasm to the nucleus.

The Mx2 protein of rats is a cytoplasmic GTPase that protects cells against vesicular stomatitis virus but not against influenza virus. Since vesicular stomatitis virus replicates in the cytoplasm and influenza virus replicates in the nucleus, it was possible that the antiviral specificity of rat Mx2 protein was determined solely by the protein's subcellular localization. Here, we found that, indeed, rat Mx2 protein lost its anti-vesicular stomatitis virus activity and gained anti-influenza virus activity when it was directed to the nucleus by way of a foreign nuclear-transport signal appended to its amino terminus. These data show that rat Mx2 protein possesses an antiviral activity that is revealed only when the protein is shuttled to the nucleus.     

Seroepidemiological study of Herpes simplex virus type 2 in adult women of Costa Rica]

The frequency of genital Herpes simplex type 2 infections in a group of twenty adult Costa Rican women was studied by isolation of the virus and the measurement of neutralizing antibody activity in sera. The virus could not be isolated in any of the vaginal secretions. Neutralizing antibody activity to herpes virus types 1 and 2 was found in sera from sixteen subjects. An antibody II/I index equal to or larger than 87, indicative of infection with Herpes simplex type 2 was found in fifty per cent of the population studied, a second segment was composed by the subjects with indices below 87. Evaluation of antibody activity to Herpes simplex type 2 revealed that: a) only a small percentage of the women lacked detectable antibody activity to the virus; b) there is a significant difference (p < 0.005) between the mean number of years of sexual experience among the two population segments; and c) there is a positive correlation (p < 0.05) between II/I index values and age among the women of the population segment with a II/I index equal to or larger than 87.     

Synthesis and antiviral activity of hydrazides and substituted benzalhydrazides of betulinic acid and its derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N'-benzalhydrazides, were synthesized. Their antiviral activities toward of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.     

Polymerase Activity of Pichinde Virus

Pichinde virus, a member of the arenavirus group, was examined for polymerase activity. Purified virus was found to contain RNA-dependent RNA polymerase but not RNA-dependent DNA polymerase activity. Since RNase but neither DNase nor actinomycin D inhibited the endogenous polymerase reaction, RNA of the virus appeared to be used as the template. The divalent cations Mg(2+) and Mn(2+) were required for optimal reactivity. The RNA product was partially resistant to RNase and the resistant portion had a sedimentation coefficient of 22 to 26S in sucrose gradients.     

The role of target membrane sialic acid residues in the fusion activity of the influenza virus: the effect of two types of ganglioside on the kinetics of membrane merging

The influenza virus enters target cells via the action of hemagglutinin proteins (HA) inserted into the viral envelope. HA promotes membrane fusion between the viral envelope and endosomal membrane at low pH, following viral binding to sialic acid-containing receptors on target cells, and internalization by endocytosis. The effect of target membrane sialic acid residues on the fusion activity of the influenza virus towards model membranes was evaluated by both reduction, (i.e. treating somatic cells with neuraminidase- (NA-) prior to virus-cell interactions), and by supplementing liposomes with the gangliosides GD1a and GT1b. The harshness of the neuraminidase pretreatment of target cells required to affect virus-induced membrane merging was found to greatly depend on the assay conditions, i.e. whether a virus-cell prebinding step at neutral pH was included prior to acidification. Minor concentrations of neuraminidase were found to greatly reduce virus fusion, but only in the absence of a prebinding step; they had no effect if this step was included. Although membrane merging was greatly reduced following cell neuraminidase pretreatment, virus-cell association at low pH was not disturbed proportionately. This probably reflects unspecific virus-cell binding under these conditions, probably of inactivated or aggregated virus particles, which does not translate into membrane merging. This seems to suggest both that target membrane sialic acid can protect the virus from losing its activity before triggering membrane merging, and that the importance of this interaction is not merely to ensure virus-target proximity. With liposomes, we found that both types of ganglioside supported efficient fusion, with GD1a promoting a slightly faster initial rate. However, in this case, virus-target proximity closely mirrored fusion activity, thus pointing to differential specificity between targets routinely used to assay influenza virus fusion activity.     

An assay for the receptor-destroying activity of influenza C virus

We have developed a convenient method for assaying the receptor-destroying enzyme (RDE) activity of influenza C virus. This method measures the ability of the RDE to destroy the hemagglutination-inhibition activity of a potent inhibitor present in rat serum. Some physico-chemical properties of the RDE of influenza C virus were investigated by using this method. The temperature optimum for maximal activity of this enzyme was found to be 45 C to 53 C. There was little difference in thermostability between the RDE and hemagglutinating activities of influenza C virus. When influenza C virions were treated with various concentrations of trypsin, the RDE activity decreased in parallel with the decrease in the amount of residual gp88 glycoprotein, suggesting association of RDE with this glycoprotein.     

Expression of extremely low levels of thymidine kinase from an acyclovir-resistant herpes simplex virus mutant supports reactivation from latently infected mouse trigeminal ganglia

A single-cytosine-deletion in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. A laboratory strain engineered to carry this mutation did not generate sufficient TK activity for detection by plaque autoradiography, which detected 0.25% wild-type activity. However, a drug sensitivity assay suggested that extremely low levels of TK are generated by this virus. The virus was estimated to express 0.09% of wild-type TK activity via a ribosomal frameshift 24 nucleotides upstream of the mutation. Remarkably, this appeared to be sufficient active TK to support a low level of reactivation from latently infected mouse trigeminal ganglia.     

Mechanisms of disrupting DNA repair in human cells. IV. Interferon protects DNA of noninfected and chronically infected human cells from damage caused by cadmium chloride

The protective activity of interferon on the cadmium chloride-treated human cells (Hep-2), infected chronically with meals virus and uninfected, was studied. It was found that cadmium chloride induced the formation of partially non-repairable DNA lesions. Decrease in cell repair activity was observed in the cells chronically infected with virus. Pretreatment of cells with interferon protected cell DNA from formation of DNA breaks and caused more effective resynthesis of DNA breaks.     

High-frequency phenotypic reversion and pathogenicity of an acyclovir-resistant herpes simplex virus mutant

A double-guanine-insertion mutation within a run of guanines in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. This mutation was engineered into strain KOS, and stocks were generated from single plaques. Plaque autoradiography revealed that most plaques in such stocks exhibited low levels of TK activity, while approximately 3% of plaques exhibited high levels of TK activity, indicating a remarkably high frequency of phenotypic reversion. This virus was able to reactivate from latency in mouse ganglia; a fraction of the reactivating virus expressed a high level of TK activity due to an additional G insertion, suggesting that the observed genetic instability contributed to pathogenicity.     

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 μM) and La Crosse virus (EC(50)=0.27 μM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).     

Enhancement of influenza virus hemolysis by physical and serological treatments

The mode of hemolysis by influenza A virus was compared with that of Sendai virus. The WSN strain of influenza virus grown in either eggs or MDCK cells expressed hardly any hemolytic activity by itself. Treatment of the MDCK cell-grown WSN virus with sonication or freezing and thawing moderately enhanced the hemolytic activity, but the maximum level attainable was considerably lower than that of Sendai virus. A high level of hemolytic activity comparable to that of Sendai virus was obtained only after treatment of the virus with antibody and complement. An electron microscopic study revealed that non- or low-hemolytic WSN virions were not permeable to uranyl acetate stain in contrast with the hemolytic virions obtained after treatment with antibody and complement, indicating that the hemolytic virions had sustained some injury to their envelopes. These phenomena were comparable to those found with Sendai virus, showing that damage to the envelope is also responsible for the hemolysis of influenza virus. The influenza viruses, however, remained spherical after every treatment and the stain did not penetrate into the core of the virion. These observations suggest that the envelope of influenza virus is more rigid than that of Sendai virus but that the hemolytic process of influenza virus is nevertheless mediated through envelope-membrane fusion as in the case of Sendai virus.     

Thymidine kinase activity in mouse embryo fibroblast cells infected with murine cytomegalovirus

Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-³H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication.     

Synthesis and Antiviral Activity of Hydrazides and Substituted Benzalhydrazides of Betulinic Acid and Its Derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N"-benzalhydrazides, were synthesized. Their antiviral activities toward viruses of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.