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Biomedical subjects

R M Friedman

Publications and source records attributed to R M Friedman.

At least 235 records · Page 13Linked to original sources
PubMed1969

Inhibition of arbovirus assembly by cycloheximide.

Addition of cycloheximide (100 mug/ml) to cultures of chick cells infected with Semliki Forest virus (SFV) halted subsequent increase in virus titers. When added after 4 hr of infection, the drug had no effect on the rate of viral ribonucleic acid (RNA) synthesis, although marked inhibition of protein synthesis was seen. All of the previously identified forms of SFV RNA were seen in the drug-treated cells at higher concentrations than were present in untreated controls. The latter observation appeared to result from a failure to form viral "cores" or nucleocapsids in the cycloheximide-treated cells, resulting in sequestration of viral RNA intracellularly. The failure to form new virus cores was correlated with the failure of type II cytopathic vacuoles to appear in thin sections. Virus budding from the cell surface and the formation of type I cytopathic vacuoles persisted in cycloheximide-treated cells. The cellular pool of the major protein present in the virus core appeared to be small. None of this protein was found in a free pool in cytoplasm. The results indicated that, in the presence of cycloheximide, virus assembly was impaired because of the small size of the cellular pool of the major protein required for virus core formation.

Journal Article↗
PubMed1969

Chromatography of arbovirus ribonucleic acid forms on columns of benzoylated-diethylaminoethyl cellulose.

Benzoylated-diethylaminoethyl cellulose (BD-cellulose) column chromatography was found to be useful in resolving most of the ribonucleic acid (RNA) forms from the replicative cycle of group A arbovirus Semliki Forect virus (SFV). The elution patterns were independent of molecular weight and appeared to be related to the degree of secondary structure in the molecule. Fractions of RNA were taken from a sucrose density gradient of cytoplasmic extracts of SFV-infected chick cells pretreated with actinomycin D. In a linear salt gradient, 16S material cochromatographed with the rapidly eluted ribonuclease resistant core of the double-stranded SFV-RNA and with the homopolymer duplex polyinosinic acid: polycytidylic acid. This fraction, therefore, probably contains an SFV-RNA form similar to the completely double stranded replicative form (RF) of several RNA viruses and bacteriophages. Faster moving (>20S) sucrose gradient fractions eluted more slowly, suggesting a decreasing proportion of secondary structure with increasing sedimentation value. The fractions, therefore, seemed to contain replicative intermediate (RI) structures. The two single stranded forms of SFV-RNA (42S and 26S) could only be eluted from BD-cellulose in the presence of urea or dimethyl sulfoxide, suggesting the presence of minimal secondary structure. Under these conditions, the single-stranded viral RNA forms could not be resolved. Molecular sieve chromatography of the single-stranded RNA forms, performed by passage through an agarose column, also failed to resolve these forms. The viral RNA forms containing a high degree of secondary structure, probably the RF and the RI, could, therefore, be rapidly separated from each other and from the single-stranded forms.

Animals↗
PubMed1968

Actinomycin D: inhibition of phospholipid synthesis in chick embryo cells.

When chick embryo fibroblasts are exposed to actinomycin D for 30 minutes to 3 hours, there is progressive inhibition of phospholipid synthesis, so that by 3 hours the inhibition is 40 percent. All phospholipids are affected. Since this inhibition is twice as great as the inhibition of protein synthesis, some effects of actinomycin D previously ascribed to decreased protein synthesis must be reevaluated.

Animals↗
PubMed1968

Interferon: production by chick erythrocytes activated by cell fusion.

In the presence of Sendai virus inactivated with ultraviolet light, nucleated chick erythrocytes can be fused with several types of human cells to form heterokaryons. Although chick erythrocytes alone cannot be stimulated by Sendai virus to produce interferon, fusion with a human cell (AH-1) which itself may produce human interferon results in heterokaryons in which the erythrocyte genome is activated and chick interferon is produced. When nucleated chick erythrocytes are fused with another type of human cell (HeLa clone S-3) which does not produce human interferon when stimulated, no chick interferon is detectable, despite morphologic changes suggestive of activation of the erythrocyte nuclei.

Animals↗
PubMed1968

Protein synthesis directed by an arbovirus.

In contrast to chick embryo fibroblast protein synthesis, the bulk of the protein synthesis directed by Semliki Forest virus is carried out on membranes. Under conditions where more than 95% of cell protein synthesis was inhibited, viral polysomes could be demonstrated. Viral protein appeared to be produced on polysomes associated with nascent ribonucleic acid strands still attached to the base-paired, double-stranded replicative form of the virus. Very rapid incorporation of virus protein into 140S virus core particles was also demonstrated.

Animals↗
PubMed1968

Structural and nonstructural proteins of an arbovirus.

Purified Semliki Forest virus (SFV) contains three structural proteins while its core (nucleocapsid) contains two of these proteins. To identify all of the proteins synthesized under virus direction, cells were infected with SFV in the presence of actinomycin D and guanidine. Cell protein synthesis was markedly and irreversibly inhibited under these conditions; virus growth was reversibly inhibited by guanidine and began when the cells were washed to remove the guanidine. When cells were treated with guanidine for 4 hr after virus infection and then were washed, five major proteins were produced early in infection. Three of these proteins corresponded to virus structural proteins. None of these five proteins was a major protein of uninfected cells or of virus-infected cells which had been incubated with partially purified interferon before infection. Late in infection, three major proteins, the virus structural proteins, were produced.

Animals↗
PubMed1968

Inhibition of arbovirus protein synthesis by interferon.

Infection of cells treated with guanidine and actinomycin D and then washed to remove the guanidine inhibition of virus growth had no effect on antiviral activity already established by interferon. Protein synthesis in interferon-treated cells infected under these conditions was decreased as compared to control cells similarly treated but not exposed to interferon. In these control cells, analysis by polyacrylamide gel electrophoresis indicated that six proteins were produced during the first hour after guanidine reversal. Five of these proteins have been previously identified as probably being viral in origin. In interferon-treated cells, only a single major protein was produced. Ribonucleic acid (RNA) synthesis by Semliki Forest virus during the first hour after guanidine reversal was markedly depressed by incubation at 42 C, but no inhibition of total virus protein synthesis was seen; this finding suggested that much of the virus protein produced in the first hour after guanidine reversal was carried out by input virus RNA. Interferon was fully active in cells incubated at 42 C. The results suggested that interferon inhibits the production of Semliki Forest virus proteins ordinarily produced under the direction of the virus genome.

Carbon Isotopes↗
PubMed1968

Cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis.

Unique cytoplasmic structures, herein designated as type I cytopathic vacuoles (CPV-I), are found in chick embryo cells early in the logarithmic phase of Semliki Forest virus replication. High resolution autoradiography demonstrated that the CPV-I are loci of (3)H-uridine incorporation. This evidence correlates well with previous biochemical data and electron microscopy of the subcellular fractions active in Semliki Forest virus ribonucleic acid synthesis. Origin of the CPV-I within host cell cytoplasm is confirmed by the distribution of electron-dense tracer particles and sequential ultrastructural observations.

Animals↗
PubMed1968

Replicative intermediate of an arbovirus.

One hour after infection of chick fibroblasts with Semliki Forest virus (SFV), a viral ribonucleic acid (RNA) structure is present which has many of the properties described for the replicative intermediate of several RNA bacteriophages. These properties include a polydisperse nature on sucrose density gradient analysis, ribonuclease resistance, a variation in sedimentation pattern associated with changes in salt concentration, and recovery of infectious viral RNA upon denaturation. Most of the replicative intermediate present in SFV infection appears to be membrane-associated.

Animals↗
PubMed1967

Interferon binding: the first step in establishment of antiviral activity.

Chick cells incubated at 1 degrees C with interferon fail to develop antiviral activity, but this activity appears subsequent to a 7-hour incubation at 37 degrees C after removal of interferon by repeated washings. Treatment with actinomycin D blocks the development of the latter activity. Cells incubated with interferon at 1 degrees C for up to 1 hour and then washed and incubated for 2 hours at 37 degrees C develop a degree of antiviral activity proportional to the concentration of interferon at initial incubation; at any concentration, the antiviral activity increased with the duration of initial incubation at 1 degrees C, but a maximal response was reached at 10 or 20 minutes. Treatment with trypsin after incubation with interferon at 1 degrees C inhibited development of antiviral activity. Interferon is rapidly bound to a superficial cell site, and this binding is necessary for development of antiviral activity in chick cells.

Animals↗
PubMed1967

Cytoplasmic fractions associated with Semliki Forest virus ribonucleic acid replication.

When actinomycin D-treated chick fibroblasts were labeled with (3)H-uridine for varying periods during the log phase of Semliki Forest virus infection, radioactivity was found associated with different cytoplasmic fractions. After a 1-min period of labeling, it appeared in a large cytoplasmic structure which was seen in electron micrographs of infected cells. Sediments of sucrose density gradients of cytoplasmic extracts of these cells also contained these structures. Three forms of viral ribonucleic acid (RNA) were associated with this cytoplasmic structure: a ribonuclease-sensitive 42S form identical to the RNA of the mature virus, a ribonuclease-sensitive 26S form, and a ribonuclease-resistant 20S form. After a 5- to 10-min labeling period, radioactivity was associated with a ribonuclease-sensitive 65S cytoplasmic fraction which contained only the 26S RNA form. Finally, after a 1-hr labeling period, a 140S ribonuclease-resistant particle was the most prominent radioactive structure in the cytoplasm. This particle contained only 42S viral RNA. Negative-contrast electron micrographs of the 140S particle and the virion demonstrated structural differences between them. The base compositions of the 42S and 26S viral RNA forms were not significantly different. The base composition of the 20S form differed significantly from that of the other two viral RNA forms, but the values obtained for the mole fractions of the bases present in the 20S form differed, and depended on the period during the virus growth cycle in which (32)P was present. These results suggested that viral RNA originated in the large cytoplasmic body. The 20S RNA appeared to be a structure engaged in viral RNA replication and the 140S particle appeared to be a virus precursor.

Animals↗
PubMed1967

Interferon action on parental Semliki forest virus ribonucleic acid.

Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.

Animals↗