Modulation of viral replication in macrophages persistently infected with the DA strain of Theiler's murine encephalomyelitis virus.

Background: Demyelinating strains of Theiler's murine encephalomyelitis virus (TMEV) such as the DA strain are the causative agents of a persistent infection that induce a multiple sclerosis-like disease in the central nervous system of susceptible mice. Viral persistence, mainly associated with macrophages, is considered to be an important disease determinant that leads to chronic inflammation, demyelination and autoimmunity. In a previous study, we described the establishment of a persistent DA infection in RAW macrophages, which were therefore named DRAW.

R75761, a lead compound for the development of antiviral drugs in late stage poliomyelitis eradication strategies and beyond.

In this study the antiviral activity of a panel of 18 out of 240 pyridazinamine analogues was evaluated against the Sabin strains of the three poliovirus types. We found one compound, R75761 which had a comparable 50% effective concentration (EC50) value against all three poliovirus Sabin strains. Virus multiplication was reduced by 10(4.

Theiler's virus strain-dependent induction of innate immune responses in RAW264.7 macrophages and its influence on viral clearance versus viral persistence.

Infection of susceptible mice with the DA strain of Theiler's murine encephalomyelitis virus (TMEV) induces a persistent central nervous system infection accompanied by demyelination that resembles multiple sclerosis. In contrast, Theiler's GDVII strain does not persist, because infected animals either clear the virus or die. Previously, the authors have shown that in vitro infection of RAW264.

Persistent infection of RAW264.7 macrophages with the DA strain of Theiler's murine encephalomyelitis virus: An in vitro model to study viral persistence.

Theiler's murine encephalomyelitis virus (TMEV) is a member of the Picornaviridae family and causes a virus strain dependent pathology in the central nervous system of mice. The GDVII strain induces an acute and mostly fatal encephalomyelitis. In the few mice that survive, the virus is cleared by the immune system.

The morphogenesis of Theiler's murine encephalomyelitis virus is strain specific.

During a single cycle infection with the neurovirulent GDVII- and demyelinating DA-strain of Theiler's murine encephalomyelitis virus (TMEV) in L-929 cells, different subviral particles were found for both strains. Early in the assembly process, the DA-strain generated 14 S pentamers composed of the viral proteins VP0, VP1 and VP3, while in GDVII-infected cells, particles with the same protein composition but with a sedimentation coefficient of 20 S were found. These newly discovered 20 S particles are probably virion assembly precursors considering their capsid protein composition and their early time of appearance in infected cells.

Uptake of poliovirus into the endosomal system of HeLa cells.

To understand the topology and mechanism of poliovirus uncoating, the question of whether intact virions can be endocytosed by the host cell was studied by a combination of various techniques. In order to prevent alteration of the virus to subviral particles, Hela cells were infected at 26 degrees C. At this temperature the majority of cell-associated virions remained at the plasma membrane, whereas a smaller amount accumulated in vesicles having the same mobility (upon free-flow electrophoresis) and migration behaviour on Nycodenz density gradients as early and late endosomes.

Dexamethasone alters messenger RNA levels but not synthesis of collagens, fibronectin, or laminin by cultured rat fat-storing cells.

Glucocorticoids have been shown to suppress collagen synthesis and gene expression by fibroblasts. However, little is known about their effects on fat-storing cells, the major matrix-producing cells in liver fibrosis. In this study we investigated the effect of dexamethasone on the extracellular matrix expression by cultured rat fat-storing cells.

Heat stabilized, infectious poliovirus.

Polioviruses type 1 (Mahoney) and type 3 (Sabin) were treated with the antiviral pyridazinamine R78206 by first binding the compound to the virus and then removing unbound compound by dialysis. As a result of this treatment, both poliovirus strains were protected against thermal inactivation at 46 degrees C. The R78206 treatment did not cause inactivation except with the Sabin 3 strain at high R78206 concentrations.

Poliovirus infection without accumulation of eclipse particles.

Poliovirus type 1 (Mahoney) was treated with the capsid-binding pyridazinamine R 78206, followed by dialysis to remove free compound. Upon infection of HeLa cells by R 78206-pretreated virus, the formation of intra- and extracellular modified particles was completely inhibited, except for a small amount of empty capsids. The synthesis of viral proteins and first cycle progeny virus was delayed by 1 h.

Intracellular proteolysis of poliovirus eclipse particles is abortive.

A series of weak bases and the ionophore monensin were tested for their effect on the intracellular processing of type 1 poliovirus in HeLa cells. At concentrations that did not inhibit plaque formation or viral protein synthesis, the compounds suppressed the proteolytic processing of 135S particles and the formation of 110S particles. In addition, some compounds strongly reduced transit of modified particles to the lysosomes.

Postabsorption neutralization of poliovirus.

Nineteen neutralizing murine monoclonal antibodies against poliovirus type 1, including representatives reacting with each of the antigenic sites on the virion, were tested for their abilities to neutralize the virus either before or after attachment to susceptible cells. All antibodies neutralized unattached virus; six had reasonable titers of postabsorption neutralization (PAN). Experiments with antibodies lacking PAN activity showed that Fc-specific rabbit anti-mouse antibodies could confer PAN activity.

Effect of a capsid-stabilizing pyridazinamine, R 78206, on the eclipse and intracellular location of poliovirus.

R 78206 (a pyridazinamine derivative) inhibits the formation of poliovirus eclipse particles. Its effect on the intracellular location of poliovirus was studied by separating subcellular fractions in iso-osmotic Nycodenz gradients. The compound did not inhibit internalization of intact virus into small lipid vesicles, but it did inhibit the release of virus from these vesicles and its entry into lysosomes.

Compartmentalization of subviral particles during poliovirus eclipse in HeLa cells.

HeLa cells were preincubated with radiolabelled poliovirus type 1 at 26 degrees C, such that the 160S virions were internalized, but not altered structurally. The temperature was then shifted to 37 degrees C to study the intracellular redistribution of the virions and the modifications they undergo at that temperature. Using subcellular fractionation in isoosmotic Nycodenz gradients, we obtained evidence for the rapid loss of virions from the plasma membrane and from a vesicular fraction, as well as for the formation of two populations of intracellular 135S particles.

Localization and cellular source of the extracellular matrix protein tenascin in normal and fibrotic rat liver.

The distribution and the cellular source of the novel extracellular matrix glycoprotein tenascin were studied in normal and fibrotic rat liver. Cryostat sections of normal rat livers, livers of rats treated with intraperitoneal injections of CCl4 and 4-day-old and 8-day-old primary fat-storing cell cultures were stained for tenascin and desmin using an immunoperoxidase procedure or a double-label immunofluorescence technique. Fat-storing cell cultures were metabolically labeled with 3H-proline.

Antigenic N to H conversion of poliovirus by a monoclonal antibody at low ionic strength.

Monoclonal antibody 35-1f4 at low ionic strength converted native virions (N antigen) to noninfectious H-antigenic, empty capsids. The reaction was stoichiometric, as the amount of N antigen that could be converted to H was limited to an average of 2 virions per molecule of antibody. The antibody remained associated with virus aggregates after antigenic conversion.

Internalization of intact poliovirus by HeLa cells as shown by subcellular fractionation in isoosmotic Nycodenz gradients.

HeLa cells were infected with radiolabelled poliovirus at different temperatures, and the intracellular distribution of input radioactivity was studied. To this end, homogenates were fractionated by rate zonal centrifugation in linear isoosmotic (2 to 30%) Nycodenz gradients. Further purification of subcellular fractions was achieved by recentrifugation to equilibrium in 10 to 30% Nycodenz.

Chloroquine induces empty capsid formation during poliovirus eclipse.

The poliovirus capsid (160S) is modified during eclipse in HeLa cells, which results in at least three types of particles having sedimentation coefficients of 135, 110, and 80S. The lysosomotropic agent chloroquine redirected the production of eclipse products from 135 and 110S particles (containing RNA) to 80S particles (without RNA). The effect started at 5 microM and was fully developed with 20 microM chloroquine.

Formation of subviral particles by in vitro translation of subgenomic poliovirus RNAs.

Rabbit reticulocyte lysates were programmed with either RNA extracted from purified poliovirus or a mixture of mRNAs encoding the capsid precursor, P1, and proteinase 3CD. In both cases, 14S subunits were formed at 30 degrees C and empty capsids at 37 degrees C. Both the 14S subunits and empty capsids had the expected polypeptide composition and neutralization epitopes.

Antibodies may act synergistically or additively with interferon to inhibit poliovirus.

We investigated the protective effects of combinations of interferon-alpha (IFN-alpha) and neutralizing monoclonal antibodies (mAbs) and serum antibodies against poliovirus type 1 (PV-1) in vitro. Our results indicate that the antiviral effects of IFN-alpha and most neutralizing mAbs to PV-1 act synergistically to inhibit PV-1. However, the antiviral effects of IFN-alpha and one type specific mAb to PV-1 were additive.

Metabolic activation of quercetin mutagenicity.

The mutagenicity of quercetin was reinvestigated using the Salmonella/microsome test. The mutagenicity of quercetin was enhanced by the cytosolic fraction of liver extract (S100), or by ascorbate, and even more by the complete liver supernatant (S9) in the presence of cofactors (NADP and glucose-6-phosphate). The formation of metabolites by the S9 enzymes was demonstrated by reverse-phase HPLC.

New evidence for the precursor role of 14 S subunits in poliovirus morphogenesis.

In poliovirus-infected HeLa cells incubated as 30 degrees, 14 S subunits are selectively labeled and no virions are assembled. When the temperature is subsequently shifted to 37 degrees, the radioactivity of the 14 S material is transferred mainly to virions, showing that 14 S subunits are virion precursors. When 14 S subunits synthesized at 30 degrees are incubated in vitro at 37 degrees, they assemble to empty capsids.

Eclipse products of poliovirus after cold-synchronized infection of HeLa cells.

The particles derived from radioactively labeled poliovirus, in cold-synchronized infection of HeLa cells, were studied. After temperatures shift-up, radioactivity was transferred rapidly from the infecting virions (160 S) to 135 S, and from there more slowly to 110 S, a previously unreported eclipse product. Both the 135 and 110 S particles contained RNA, lacked VP4, and were H-antigenic.

In vitro differentiation of fat-storing cells parallels marked increase of collagen synthesis and secretion.

Fat-storing cells were isolated and purified from livers of normal adult rats and maintained in primary culture. By light and electron microscopy it was established that they underwent phenotypic changes into cells with the ultrastructural characteristics of myofibroblasts, between the third and sixth day in culture. These morphological changes were accompanied by a 2-fold increase of L-[3H]proline incorporation into secretory proteins and an 11-fold increase into secreted collagenase-sensitive proteins.

Denaturation of poliovirus procapsids.

The denaturation of poliovirus procapsids at pH 6.5 was studied, both in the frozen and liquid condition. Denaturation involved alteration of antigenic and physical features (isoelectric pH, alkali dissociability, sedimentation coefficient).

Antiviral activity of flavones and potentiation by ascorbate.

We compared the anti-poliovirus activities of three flavones, quercetin, luteolin and 3-methylquercetin, which differ only at ring position 3. 3-Methylquercetin was the most potent compound. Quercetin exhibited antiviral activity only when protected against oxidative degradation by ascorbate.