Influenza virus uncoating in infected cells and effect of rimantadine.

Uncoating of influenza virus (strain WSN) in MDCK cells was studied by following the fate of the virus labelled with radioactive precursors. The accumulation of subviral components of input virus was observed in nuclear-associated cytoplasm (NAC) obtained by treatment of the nuclei with citric acid. Two types of subviral components were found there, ribonucleoproteins (RNPs) and larger subviral particles (SVP) containing RNPs in association with M protein. SVP, with different relative amounts of M protein, were revealed in NAC, suggesting that M protein was gradually released from RNPs. The released RNPs entered the nuclei while M protein accumulated within perinuclear membranes. Thus, SVP could be regarded as probable intermediates in virus uncoating. Rimantadine prevented the release of M protein from RNPs and their penetration into the nuclei provoking the accumulation of subviral components in NAC.

[Remantadine binding with influenza virus nucleoids in vitro and infected cells]. / Sviazyvanie remantadina s nukleoidami virusa grippa in vitro i v zarazhennykh kletkakh.

Interactions of the tritium-labeled antiviral preparation remantadine with nucleoids and ribonucleoprotein (RNP) of influenza virus were studied. The studies were carried out both in vivo, in infected cells, and in vitro upon direct contact of the preparation with subviral structures isolated from the infected cell and from virions. Autoradiography of the cells treated with 3H-remantadine showed its association with nuclei (possibly with nuclear membranes). The analysis of the results of gradient centrifugation showed remantadine to bind to virus nucleoids in vivo and in vitro without interacting directly with RNP. Polyacrylamide gel electrophoresis of viral proteins revealed association of remantadine with matrix M protein. Nucleoids isolated from a remantadine-resistant virus variant bound much less remantadine than those of a sensitive variant. Some suggestions on the kind of interaction leading to the formation of such stable associations are made. It is also suggested that remantadine prevents M-protein interaction with cell membranes (probably, with the nuclear membrane) thereby blocking further "uncoating". Because RNP is not released from the M-protein layer and does not penetrate into the nucleus, the nuclear stage of virus reproduction is shut down.

[Effect of remantadine on the deproteinization of influenza viruses in infected cells]. / Deistvie remantadina na deproteinizatsiiu virusa grippa v zarazhennykh kletkakh.

The effect of remantadine on penetration of influenza virus (the WSN strain) structures into MDCK cells was studied. Autoradiography of remantadine-treated cells infected with 3H-uridine-labeled virus showed remantadine to block the penetration of the parental viral structures from the perinuclear cytoplasm into the nuclei resulting in the structures aggregating in the perinuclear cytoplasm. Biophysical analysis of the viral structures recovered from the cells and analysis of their proteins by polyacrylamide gel electrophoresis showed that in remantadine-treated cells the amount of virus nucleoids (ribonucleoproteins, RNP, surrounded by a layer of M protein) increased in the perinuclear bytoplasm while the amount of intranuclear viral plasm while the amount of intranuclear viral RNPs decreased. These data suggest that the effect of remantadine is due to blocking of the second stage of deproteinization, that is, deproteinization of viral nucleoids to viral RNPs occuring in the perinuclear cytoplasm (possibly, on nuclear membrans); remantadine did not affect penetration of viral structures into the cells or the first stage of deproteinization (formation of nucleoids from virus particles). In order to inhibit deproteinization of viral structures remantadine should be present in the process of infection and be added soon after innoculation. When added 30 min after inoculation, remantadine has no effect on the intracellular distribution of viral structures.

[Localization and transport of influenza virus parental ribonucleoproteins in infected cells]. / Lokalisatsiia i transport roditel'skikh ribonukleoproteidov virusa grippa v zarazhennykh kletkakh.

MDCK cells (a continuous line of dog kidney) were infected with 3H-uridine-labeled influenza virus, the WSN strain, and localization of parental structure in the cells was determined at various intervals after inoculation using the cell autoradiography method. At 15 and 30 min postinfection radioactive granules were found in nuclei and some of them concentrated around nucleoli. One hour after infection the granules were found both in nuclei and in cytoplasm. Two hours after infection the bulk of the granules was found in the cytoplasm localized in a limited poorly strained area of the cytoplasm contiguous to the nucleus. The parental structures recovered from the cytoplasm and the nucleus were identified by centrifugation in sucrose density and cesium chloride density gradients as viral ribonucleoproteins. The amount of the granules in the nuclei at early stages of infection increased proportionately to hemagglutinin precursor cleavage in the original virus. The cleavage was achieved by treating the virus or virus-producing cells with trypsin or allantoic fluid from noninfected chick embryos. The experimental results indicate that parental ribonucleo-proteins immediately after infection penetrate into the nucleus and then are transported into the cytoplasm.

[2 stages of influenza virus deproteinization in infected cells]. / Dve stadii deproteinizatsii virusa grippa v zarazhennykh kletkakh.

MDCK cells (a continuous cell line of dog kidney) were infected with 3H-uridine- or 14C-aminoacide-labeled influenza virus, the WSN strain. The cells were fractionated 30 min and 2 hours postinfection, and viral structures in subcellular structures were identified by sedimentation and density centrifugation and protein analysis in polyacrylamide gel. At 30 min after inoculation the perinuclear cytoplasm was shown to contain structures with the properties of viral nucleoids (ribonucleoproteins--RNP--surrounded with a layer of M protein). Their sedementation rate in glycerol gradients was 70--90 S, and the buoyant density in cesium chloride 1.30 g/ml. The nuclei were found to contain viral RNP with a buoyant density of 1.39--1.41 g/ml. Two hours after inoculation the amount of RNP in the nuclei decreased and in the cytoplasm increased indicating RNP transport from the nucleus into the cytoplasm. Treatment of the cells with cytochalasine B (a substance breaking nucleus-cytoplasm bonds) resulted in redistribution of radioactivity in subcellular fractions: an increase of radioactivity in the nuclear extract and a decrease in the nuclear sediment. Autoradiography of cytochalasine-treated cells revealed accumulations of viral structures around nucleoli. These data suggest participation of the nucleoli in transportation of viral structures into the cytoplasm. Based on the foregoing data, a hypothetical scheme of the processes of deproteinization and transport of viral structures in an infected cell has been proposed.

Rimantadine hydrochloride blocks the second step of influenza virus uncoating.

Two steps of influenza virus A/WSN uncoating in MDCK cells are described. The products of the first step are cores which accumulate in the nuclear-associated cytoplasm. The products of the second step are ribonucleoproteins which penetrate the nuclei. Rimantadine blocks the second step without interfering with the first one.