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Biomedical subjects

M Esteban

Publications and source records attributed to M Esteban.

At least 181 records · Page 10Linked to original sources
PubMed1978

Studies on the mechanisms of vaccina virus cytopathic effects. I. Inhibition of protein synthesis in infected cells is associated with virus-induced RNA synthesis.

The mechanism of vaccinia virus-induced inhibition of protein synthesis was studied in LLC-MK2, HeLa and L cells. Removal of cycloheximide (300 microgram/ml) from cells infected at a multiplicity of infection (m.o.i.) of 300 particles/cell at 4 h after infection resulted in the resumption of both host and virus protein synthesis in LLC-MK2 cells, but not in HeLa and L cells. In order to determine whether virus-induced RNA synthesis, which occurs in infected cells in the presence of cycloheximide, is related to the inhibition of protein synthesis, (cut-off), the rate of virus-induced RNA synthesis in the presence of cycloheximide was measured in all three cell types. In L cells and HeLa cells the rate of virus-induced RNA synthesis increased with time, whereas in LLC-MK2 cells it remained constant for at least 4 h. However, when higher multiplicities (900 and 2700 particles/cell) were used to infect LLC-MK2 cells, the rate of RNA synthesis in the presence of cycloheximide did increase with time and was greater at the higher multiplicity. Under these conditions there was a direct relationship between the extent of virus RNA synthesis and the degree of cut-off after the removal of cycloheximide. In HeLa and L cells infected at 300 particles/cell, the longer the exposure to cycloheximide, the greater was the cut-off observed upon removal of the drug. As was the case the LLC-MK2 cells, there was a direct relationship between the rate of RNA synthesis and the degree of inhibition of protein synthesis. Since virus-induced RNA synthesis occurs in the presence of cycloheximide, the effects of actinomycin D and cordycepin on host polypeptide synthesis were tested. Inhibition of host cell protein synthesis was virtually abolished when HeLA cells were infected in the presence of cordycepin (50 and 25 microgram/ml) or actinomycin D (20 microgram/ml). These results indicate that, as the rate of virus-induced RNA synthesis increased, regardless of the type of cell used, protein synthesis was inhibited at earlier times and to a greater extent. These observations are consistent with the hypothesis that the cut-off phenomenon is related to the synthesis of an early virus-induced RNA(s).

Cell Line↗
PubMed1978

Studies on the mechanisms of vaccinia virus cytopathic effects. II. Early cell rounding is associated with virus polypeptide synthesis.

Vaccinia virus-induced morphological lesions were studied in LLC-MK2, HeLa and L cells. In LLC-MK2 cells, cell rounding occurs within 30 to 60 min after infection with 300, 900 or 2700 particles/cell and the time of appearance of these changes is dependent on the multiplicity of infection (m.o.i.). When cycloheximide (300 microgram/ml) is added to cultures at the time of infection, early cell rounding is prevented regardless of the m.o.i. However, cell rounding does occur when cycloheximide is removed, and its time of appearance and extent depends upon the time of removal of the compound and the m.o.i. Upon removal of cycloheximide at I or 2 h after infection early cell rounding occurs, and virus polypeptide synthesis is evident in cells infected at all three multiplicities. However, when the drug is removed at 4 hr after infection, cell rounding and virus polypeptide synthesis occur only in cultures infected at 300 particles/cell. Early morphological changes are also prevented in HeLa and L cells infected at 300 particles/cell in the presence of cycloheximide. These changes occur only if the compound is removed up to 2 h after infection in HeLa cells and up to 40 min after infection in L cells. Early morphological lesions are not seen if the compound is removed at later times. The occurrence of early morphological changes in HeLa and L cells is also correlated with the synthesis of virus polypeptides. All cell types, when infected at 2700 particles/cell in the presence of cycloheximide, or inhibitors of RNA synthesis, display cell fusion. Thus, whereas early morphological changes require virus protein synthesis to become manifest, cell fusion occurs in the absence of virus RNA or protein synthesis and may be mediated by a component of the virion.

Cell Fusion↗
PubMed1978

DNA-binding proteins in the cytoplasm of vaccinia virus-infected mouse L-cells.

Mouse L cell fibroblasts were infected with vaccinia virus and labeled 2 to 3 h postinfection with [35S]methionine. Labeled proteins were fractionated on native and denatured DNA-cellulose columns and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Twenty-four 90,000 to 12,500, were detected. VDP-12A (molecular weight, 29,750) had affinity for denatured but not native DNA, and its synthesis was dependent on viral DNA replication. VDP-20 (molecular weight, 41,000) bound very tightly to native and denatured DNA and was displaced only after boiling the protein-DNA-cellulose matrix in 1% sodium dodecyl sulfate. VDP-8,-11,-12,-13, -and-14 behaved electrophoretically like the polypeptide species previously shown to be present in DNA-protein complexes prepared from infected cells. The molecular weights of VDP-10 (50,000), VDP-11 (36,000), and VDP-8 (67,000) were similar to the polypeptide subunits of polyadenylate polymerase and phosphohydrolase I, enzymes purified from virions which have also been shown to have affinity for DNA.

Cytoplasm↗
PubMed1978

Procedure for purification of intact DNA from vaccinia virus.

A procedure for the isolation of intact vaccinia DNA molecules by chromatography on hydroxyapatite in the presence of 6 M urea is described. When lysates of virions containing 0.5 to 10 microgram of DNA were employed, over 95% of the viral DNA could be recovered free of poteins. Vaccinia DNA molecules isolated in this manner sedimented at 68S in neutral sucrose gradients and had an average contour length of 62.3 micrometer when examined in an electron microscope, and the DNA could be cleaved with the restriction endonuclease EcoRI and BamHI. The results of these analyses showed that intact vaccinia DNA molecules of 120 X 10(6) to 130 X 10(6) molecular weight could be obtained by the procedures described.

Chromatography↗
PubMed1977

Rifampin and vaccinia DNA.

The effect of rifampin on the replication of vaccinia DNA was studied in mouse L cells by a cytochemical techinque and by alkaline sucrose sedimentation analysis of newly synthesized viral DNA molecules. By the use of a fluorescent DNA-binding compound (Hoechst 33258), the sequential appearance, size, and location of the viral "factories" in rifampin-treated, virus-infected cells were found to be indistinguishable from those observed in untreated, infected cells. Sedimentation analysis in alkaline scurose gradients of the viral DNA molecules labeled in pulse-chase experiments showed that formation of small fragments, elongation into "intermediate"-sized molecules, and maturation into full-length viral DNA and, finally, into cross-linked viral DNA molecules occurred in the absence or presence of rifampin. The results support the view that the primary effect of the drug is related to assembly or morphogenesis.

DNA Replication↗
PubMed1975

The formation of virus polyribosomes in L cells infected with vaccinia virus.

The fate of early virus messenger RNA in the cytoplasm of vaccinia-infected L cells has been studied during the first hour after infection. The RNA is made in the virus core structure from which it is rapidly released. It accumulates in the polyribsome fraction, where at least 75% is bound to ribosomes through an EDTA-sensitive link. Three distinct structures have been identified as possible intermediates in virus polyribosome formation. The first is a ribonucleoprotein complex (RNP) in which virus RNA is associated with cellular proteins. A complex having apparently similar properties, is formed when virus RNA is added to a cytoplasmic extract in vitro. The other two structures may consist of an RNP moiety associated with the small ribosomal subunit, or with a single ribosome. At least part of the RNA isolated as RNP appears to be a precursor of the virus messenger found in polyribosomes.

Animals↗
PubMed1975

The effect of interferon on the formation of virus polyribosomes in L cells infected with vaccinia virus.

The effect of interferon treatment of mouse L cells on the fate of virus messenger RNA following infection with vaccinia virus has been studied. The polyribosomes of interferon-treated, infected cells are found to be disaggregated and it is proposed that htis results from inhibition of the initiation of virus polypeptide snythesis. Evidence is presented that inhibition of polypeptide chain elongation also occurs. The block in initiation appears to be due to the failure of the small ribosome subunit to attach to the virus messenger ribonucleoprotein complex. The translation of the different vaccinia messenger species is inhibited to a comparable extent.

Animals↗