Effects of dolichol monophosphate on galactose incorporation into glycoconjugates of cell cultures.

Cultured-cell homogenates catalysed the incorporation of galactose from UDP-galactose into protein and sphingolipid acceptors. Dolichol monophosphate stimulated the incorporation of galactose into glycoproteins, but it did not affect the rate of glycosylation of either exogenous or endogenous glycosphingolipids. It is proposed that, under certain conditions, galactose may be incorporated into glycoproteins via polyisoprenol intermediates, as is the case with N-acetylglucosamine and mannose.     

Dolichyl monophosphate and its sugar derivatives in plants.

A glucose acceptor was isolated from soya beans by extraction with chloroform/methanol (2:1, v/v), followed by DEAE-cellulose column chromatography of the extract. This acceptor could not be distinguished from liver dolichyl monophosphate by t.l.c. It could replace dolichyl monophosphate as a mannose acceptor with a liver enzyme and its glucosylated derivative could replace dolichyl monophosphate glucose as a glucose donor in the same system. These results, together with those already reported [Pont Lezica, Brett, Romero Martinez & Dankert (1975) Biochem, Biophys. Res. Commun. 66, 980-987], indicate that the acceptor from soya bean is a dolichyl monophosphate. Gel filtration of its glucosylated derivative on Sephadex G-75 in the presence of sodium deoxycholate indicated that the acceptor contained 17 or 18 isoprene units. An enzyme preparation from pea seedlings was shown to use endogenous acceptors to form lipid phosphate sugars containing mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine. Chromatographic and degradative techniques indicated that the compounds formed were lipid monophosphate mannose, lipid pyrophosphate N-acetylglucosamine, lipid pyrophosphate chitobiose and a series of lipid pyrophosphate oligosaccharides containing both mannose and N-acetylglucosamine. None of these compounds was degraded by catalytic hydrogenation, and so the lipid moiety in each case was probably an alpha-saturated polyprenol. The endogenous acceptors for mannose and N-acetylglucosamine in peas may therefore be dolichyl monophosphate, as has been found in mammalian systems.     

Experimental group A rotaviral infection in cynomolgus monkeys raised on formula diet

Rotaviral infections in cynomolgus monkeys (Macaca fasicularis) were studied to ascertain its suitability as a model of infection and diarrhea caused by group A human rotaviruses. Formula-fed monkeys were used as they could be observed closely. Experimental rotaviral infection of cynomolgus monkeys was age-dependent; only young monkeys were readily infected. Formula-fed newborns were readily infected with cell-culture-adapted human (WA) and simian (SA11) viruses and with a rotavirus from a human fecal specimen. However, diarrhea was detected only in very young animals. A number of rotaviral shedding patterns as a function of time were observed. Although there was no typical viral shedding pattern which represented exclusive association of viral infection with diarrhea, the initial level of viral excretion and the maximum level of viral shedding attained were much higher in animals with diarrhea. Seroconversion occurred in less than half of the inoculated animals. The presence of maternal rotaviral antibodies did not prevent infection or diarrhea.     

Effect of phospholipids on the regulation of a soluble galactosyltransferase in aortic wall

A galactosyltransferase activity is located in the cell-sap of aortic intima-media cells. This enzymatic system calatyzes [14C]galactose transfer from UDP-[14C]galactose into endogenous and exogenous proteinic acceptors. Labelled products are isolated from the proteinic fraction obtained in 20% trichloroacetic acid pellet or from organic solvent extractions. Maximal [14C]galactose incorporation occurs at pH 7.8 in Tris-HCl buffer in the presence of 0.1 mM MnCl2 at 30 degrees C. The enzymatic activity is modified by phospholipids, particularly by phosphatidic acid and lysophosphatidylcholine, which behave as mixed inhibitors, while L-alpha-phosphatidylserine interacts as a competitive inhibitor. The effect of phospholipids is not stereospecific but appeared to be closely related to their polar headgroups, especially the acidic headgroups of phosphatidylcholine and phosphatidic acid. The chain length and the unsaturation degree of fatty acids involved in phospholipid structures are not a main factor of regulation. The lysophosphatidylcholine effect could be explained by its solubilization properties, as non-ionic detergents interact in the same way with galactosyltransferase activity. Exogenous phospholipids probably interact with the enzymatic environment by their own molecular arrangement and so could exert a control on galactosyltransferase activity or lead to a conformation change of this enzyme.     

Galactosyl transferase of a Golgi fraction from cultured neoplastic mast cells

Suspension cultures of neoplastic mouse mast cells were used to obtain large quantities of a homogeneous cell population as starting material for cell fractionation. A Golgi fraction was prepared by slight modification of established techniques and identified by electron microscopy. Assay of galactosyl transferase activity using ovalbumin, desialylated degalactosylated orosomucoid, and N-acetylglucosamine as galactose acceptors showed that the Golgi fraction was enriched in specific activity over the homogenate. The Golgi galactosyl transferase was examined in detail. Acceptor concentrations for optimal galactose incorporation were determined, and substrate inhibition effects were shown with higher concentrations of all three acceptors. Manganese was shown to be necessary for galactose incorporation. A higher concentration of manganese afforded some protection from substrate inhibition by acceptors, but at the same time was itself inhibitory. All three acceptors competed with one another for galactose incorporation, indicating that a single enzyme catalyzed the transfer of galactose for all acceptors.     

Viral RNA Synthesis and Levels of DNA-Dependent RNA Polymerases During Replication of Adenovirus 2

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin α-amanitin was used to determine the relative and absolute levels of RNA synthesis by RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was monitored by hybridization to viral DNA, and of viral 5.5S RNA, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides.

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.     

Viral neuraminidase and cellular ectosialyltransferase in human lymphoblastoid cells infected with influenza virus

In human lymphoblastoid cells, infected with an influenza virus, Fowl Plague Virus (FPV), glycoproteins (such as secreted IgM) are hyposialylated, through the action of viral neuraminidase. In this study, the modulation of the cellular ectosialyltransferase activity during viral infection was investigated. This activity was detectable in FPV-infected cells, was shown to be 2.5-fold higher than that of uninfected cells, and to be able to restore, at least partially, the level of sialylation of the cell surface acceptors.     

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Role of Mitochondria in Parvovirus Pathology

Proper functioning of the mitochondria is crucial for the survival of the cell. Viruses are able to interfere with mitochondrial functions as they infect the host cell. Parvoviruses are known to induce apoptosis in infected cells, but the role of the mitochondria in parvovirus induced cytopathy is only partially known. Here we demonstrate with confocal and electron microscopy that canine parvovirus (CPV) associated with the mitochondrial outer membrane from the onset of infection. During viral entry a transient depolarization of the mitochondrial transmembrane potential and increase in ROS level was detected. Subsequently, mitochondrial homeostasis was normalized shortly, as detected by repolarization of the mitochondrial membrane and decrease of ROS. Indeed, activation of cell survival signalling through ERK1/2 cascade was observed early in CPV infected cells. At 12 hours post infection, concurrent with the expression of viral non-structural protein 1, damage to the mitochondrial structure and depolarization of its membrane were apparent. Results of this study provide additional insight of parvovirus pathology and also more general information of virus-mitochondria association.     

Enzymatic therapeutic index of acyclovir. Viral versus human polymerase gamma specificity

We have examined the kinetics of incorporation of acyclovir triphosphate by the herpes simplex virus-1 DNA polymerase holoenzyme (Pol-UL42) and the human mitochondrial DNA polymerase using transient kinetic methods. For each enzyme, we compared the kinetic parameters for acyclovir to those governing incorporation of dGTP. The favorable ground state dissociation constant (6 microM) and rate of polymerization (10 s(-1)) afford efficient incorporation of acyclovir triphosphate by the Pol-UL42 enzyme. A discrimination factor of approximately 50 favors dGTP over acyclovir triphosphate, mostly due to a faster maximum rate of dGTP incorporation. Once incorporated, acyclovir is removed with a half-life of approximately 1 h in the presence of a normal concentration of deoxynucleoside triphosphates, leading to a high toxicity index (16,000) toward viral replication. To assess the potential for toxicity toward the host we examined the incorporation and removal of acyclovir triphosphate by the human mitochondrial DNA polymerase. These results suggest moderate inhibition of mitochondrial DNA replication defining a toxicity index of 380. This value is much higher than the value of 1.5 determined for tenofovir, another acyclic nucleoside analog. The enzymatic therapeutic index is only 42 in favoring inhibition of the viral polymerase over polymerase gamma, whereas that for tenofovir is greater than 1,200. Mitochondrial toxicity is relatively low because acyclovir is activated only in infected cells by the promiscuous viral thymidine kinase and otherwise, mitochondrial toxicity would accumulate during long term treatment.     

Translational control by influenza virus. Selective and cap-dependent translation of viral mRNAs in infected cells.

In cells infected by influenza virus type A, host protein synthesis undergoes a rapid and dramatic shutoff. To define the molecular mechanisms underlying this selective translation, a transfection/infection protocol was developed utilizing viral and cellular cDNA clones. When COS-1 cells were transfected with cDNAs encoding nonviral genes and subsequently infected with influenza virus, protein expression from the exogenous genes was diminished, similar to the endogenous cellular genes. However, when cells were transfected with a truncated influenza viral nucleocapsid protein (NP-S) gene, the NP-S protein was made as efficiently in influenza virus infected cells as in uninfected cells, showing that the NP-S mRNA, although expressed independently of the influenza virus replication machinery, was still recognized as a viral and not a cellular mRNA. Northern blot analysis demonstrated that the selective blocks to nonviral protein synthesis were at the level of translation. Moreover, polysome experiments revealed that the translational blocks occurred at both the initiation and elongation stages of cellular protein synthesis. Finally, we utilized this transfection/infection system as well as double infection experiments to demonstrate that the translation of influenza viral mRNAs probably occurred in a cap-dependent manner as poliovirus infection inhibited influenza viral mRNA translation.     

Induction of long-term protective antiviral endogenous immune response by short neutralizing monoclonal antibody treatment

Long-term immune control of viral replication still remains a major challenge in retroviral diseases. Several monoclonal antibodies (MAbs) have already shown antiviral activities in vivo, including in the clinic but their effects on the immune system of treated individuals are essentially unknown. Using the lethal neurodegeneration induced in mice upon infection of neonates by the FrCas(E) retrovirus as a model, we report here that transient treatment by a neutralizing MAb shortly after infection can, after an immediate antiviral effect, favor the development of a strong protective host immune response containing viral propagation long after the MAb has disappeared. In vitro virus neutralization- and complement-mediated cell lysis assays, as well as in vivo viral challenges and serum transfer experiments, indicate a clear and essential contribution of the humoral response to antiviral protection. Our observation may have important therapeutic consequences as it suggests that short antibody-based therapies early after infection should be considered, at least in the case of maternally infected infants, as adjunctive treatment strategies against human immunodeficiency virus, not only for a direct effect on the viral load but also for favoring the emergence of an endogenous antiviral immune response.     

Infection of APC by human cytomegalovirus controlled through recognition of endogenous nuclear immediate early protein 1 by specific CD4(+) T lymphocytes

Infections by human CMV are controlled by cellular immune responses. Professional APC such as monocytes and macrophages can be infected in vivo and are considered as a reservoir of virus. However, CMV-specific CD4(+) responses against infected APC have not been reported. To develop a model of CD4-infected APC interaction, we have transfected the U373MG astrocytoma cell line with the class II transactivator (CIITA). Confocal microscopy experiments showed that U373MG-CIITA cells expressed markers characteristic of APC. Functional assays demonstrated that infected U373MG-CIITA APC processed and presented both exogenous and endogenously neosynthesized nuclear immediate early (IE) protein 1 through the MHC class II pathway. More importantly, endogenous presentation of IE1 by infected APC lead to efficient control of CMV infection as revealed by decreased viral titer. Thus, these results describe the endogenous presentation of a nuclear viral protein by the MHC class II pathway and suggest that IE1-specific CD4(+) T cells may play an important role in CMV infection by directly acting against infected APC.     

Replication of endogenous avian retrovirus in permissive and nonpermissive chicken embryo fibroblasts.

Clones of chicken embryo fibroblasts exogenously infected with the endogenous avian retrovirus were analyzed to examine the replication of this virus in permissive (Gr+) and nonpermissive (Gr-) cells. The results demonstrate that the endogenous virus was capable of infecting both Gr+ and Gr- cells with equal efficiency. Infected clones of Gr+ and Gr- cells differed, however, in two significant ways. At the time of their initial characterization, the Gr+ clones produced 100- to 1,000-fold more virus than the Gr- clones. Further, the amount of virus produced by Gr+ clones did not change significantly during serial passage of the cells. In contrast, continued passage of the infected Gr- clones resulted in a gradual increase in the amount of virus produced. Individual clones of infected Gr- cells produced infectious virus at rates that, initially, differed by a factor of more than 10(4). The large differences in the production of virus by these clones could not be explained by equally large differences in the number of infected cells within the clonal populations. Greater than 80% of the clonal populations examined ultimately produced virus at rates that were not significantly different from the rates observed in infected Gr+ cells. Virus produced by these infected Gr- cells exhibited the same restricted replication upon establishing a new infection in nonpermissive cells. Analysis of the appearance of free and integrated viral DNA sequences during endogenous virus infection of Gr+ and Gr- cells demonstrated that, after an initial delay in the synthesis of free viral DNA in Gr- cells, the nonpermissive cells ultimately acquired as many integrated viral DNA sequences as were found in infected Gr+ cells. These results indicate that a majority of the infectious particles of the endogenous virus are capable of establishing infection in a Gr- cell and, ultimately, of producing virus at a rate that is not significantly different from that produced by infected Gr+ cells. The virus produced from the Gr- cells is not a stable genetic variant of the original endogenous virus that is capable of unrestricted replication in nonpermissive cells. The reduced efficiency with which the endogenous virus initially replicates in nonpermissive cells and the increased length of time required for infected Gr- cells to produce maximal virus titers suggest that the endogenous virus may utilize a different mechanism of replication in Gr+ and Gr- fibroblasts.     

Transfer of N-acetylglucosamine to nuclear endogenous acceptors of rat hepatocytes.

1. Nuclei were prepared from rat hepatocytes. A biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. 2. The transfer of [14C]N-acetylglucosamine to endogenous acceptors were studied comparatively in the nuclei and in the other subcellular fractions of rat hepatocytes. 3. In this report we describe the presence of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. We found 21% of this transfer in the nucleus fraction with an enrichment of 26 in comparison to homogenate.