Corrado Baglioni

Interferon-induced enzymatic activities and their role in the antiviral state

Department of Biological Sciences State University of New York at Albany Albany, New York 12222 lnterferons are glycoproteins secreted by virus-in- fected cells which promote the establishment of an antiviral state in uninfected cells (Finter, 1973). Treat- ment of animal cells in culture with interferon reduces their ability to support replication of RNA and DNA viruses by mechanisms that are not yet understood. Early investigations of the mechanism of interferon action were carried out with virus-infected cells (re- viewed by Friedman, 1977). These studies showed an inhibition of accumulation and/or translation of viral templates, but failed to reveal the causes of this inhi- bition. Recent work has focused on the enzymatic differences between extracts of interferon-treated and control cells reported by several investigators. The precise relationship between these enzymatic activi- ties and the establishment of the antiviral state has not yet been clarified, but it seems probable that some explanation for the molecular basis of the antiviral state will be forthcoming shortly. Double-stranded RNA (dsRNA) is the most potent inducer of interferon synthesis (reviewed by Colby and Morgan, 1971). Both synthetic and viral dsRNAs induce synthesis and secretion of interferon in animal cells (see Torrence and De Clercq, 1977). These facts led to the hypothesis that double-stranded replicating viral RNA (the “replicative intermediate” formed by RNA viruses) triggers interferon synthesis by an un- known mechanism. Replicative intermediate obtained from mengovirus-infected cells is indeed an effective inducer of interferon synthesis (Falcoff and Falcoff, 1970). The enormous potency of dsRNA in its inter- action with the cell has been elegantly shown by Marcus and Sekellick (1977). A single defective inter- fering particle of vesicular stomatitis virus (VSV) con- taining dsRNA can induce synthesis of interferon. The defective interfering particle is thought to inject into a cell a single molecule of dsRNA, which is sufficient to induce interferon synthesis. Moreover, dsRNA is a potent inhibitor of protein synthesis in extracts of interferon-treated cells (Kerr, Brown and Ball, 1974). Investigations of this enhanced sensitivity to inhibition by dsRNA led to the discovery of two interferon-in- duced dsRNA-dependent enzymatic activities (Figure 1 )-an oligonucleotide polymerase, which synthe- sizes a series of oligonucleotides containing unusual 2’5’phosphodiester bonds from ATP (Kerr and Brown, 1978) and a protein kinase, which phosphorylates the small subunit of initiation factor elF-2 (Farrell et al., 1977). The product of the oligonucleotide polym- erase, pppA(2’p5’A). (designated here 2’5’oligo(A) or 2,5A), is the activator of an endoribonuclease (Cle- mens and Williams, 1978; Eppstein and Samuel, 1978; Baglioni, Minks and Maroney, 1978b; Ratner et al., 1978; Zilberstein et al., 1978). The enzymatic activity which synthesizes the oligonucleotides is des- ignated 2,5A polymerase. This review focuses on recent work on the protein kinase and 2,5A polymerase/endoribonuclease sys- tem and describes studies examining the stability of 2,5A in cell extracts. These studies may help us to understand how the endoribonuclease functions in virus-infected interferon-treated cells. Other studies on the inhibition of viral mRNA methylation (Sen et al., 1975, 1977) are also briefly described. Although mechanism of this inhibition has not yet been char- acterized at the molecular level, it is discussed here because it is an interferon-dependent activity in ceil extracts which does not require addition of dsRNA. Inhibition of viral mRNA methylation may also provide an explanation for impairment of its translation.

Evidence for role of m7G5'-phosphate group in recognition of eukaryotic mRNA by initiation factor IF-M3.

7-methylguanosine 5′-monophosphate inhibits protein synthesis in a fractionated, messenger-dependent, reticulocyte cell-free system. This compound also inhibits binding of histone mRNA to reticulocyte ribosomes as well as interaction of VSV mRNA and histone mRNA but not EMC virus RNA with purified initiation factor IF-M3. These studies provide evidence that the role of 7-methylguanosine in the mechanism for initiation of eukaryotic mRNA translation may be related to specific recognition of mRNA by initiation factor IF-M3.

The role of platelet-activating factor in inflammation

Inflammation and hypersensitivity reactions are reactions of vascularized tissue to exogenous or endogenous noxa. Inflammation suggests a protective, immunologically or nonimmunologically mediated response that tends first to localize and then to neutralize noxious material. The inflammatory response is strictly intertwined with a complex series of events that serve to heal and repair the damaged tissue. However, inflammation and repair may be potentially harmful. Hypersensitivity reactions imply an incongruous or sustained activation of the immunologic reactions involved in the inflammatory response, which results not in protection, but in destruction of tissue integrity.

Protection from tumor necrosis factor cytotoxicity by protease inhibitors.

Tumor necrosis factor (TNF) is cytocidal for human and murine cells when protein synthesis is inhibited by cycloheximide, but some protease inhibitors completely protect these cells from TNF cytotoxicity. Inhibitors of chymotrypsin-like proteases are active at lower concentrations than inhibitors of trypsin-like proteases. Both irreversible inhibitors, such as alkylating compounds, and reversible inhibitors, such as substrates of proteases, protect cells from the cytocidal activity of TNF. This protection is most effective when the cells are pretreated with these inhibitors before addition of TNF. When the protease inhibitors are removed, the cells gradually lose resistance to TNF cytotoxicity. The inhibitors do not interfere with the functioning of TNF-receptor complexes, since SK-MEL-109 melanoma cells treated with a protease inhibitor synthesize a TNF-induced protein. These findings suggest that a protease in involved in the cytocidal action of TNF.

Direct evidence that the gene product of the human chromosome 21 locus, IFRC, is the interferon-α receptor

Abstract The gene, IFRC , on human chromosome 21 has been considered, on the basis of indirect functional and antibody blocking evidence, to code for an interferon receptor. To obtain direct evidence for this conclusion, the specific binding of 125 I-HuIFN-αA to matched sets of fibroblasts diploid and aneuploid for chromosome 21 has been determined. Although the dissociation constant for IFN-α is the same for both trisomic and diploid cells, trisomic cells bind more IFN-α and monosomic cells less than do diploid cells. When the data for all determinations are combined, the monosomy 21:diploid:trisomy 21 binding ratios are 0.5:0.8:1.5, in good agreement with the values of 0.5:1.0:1.5 expected on the basis of strict gene dosage considerations for a product coded for by chromosome 21. We conclude, therefore, that the chromosome 21 gene product determined by IFRC and recognized by antibodies which block interferon action is truly a specific cell surface receptor for human interferon-α.

Synthesis of H1 histones by BHK cells in G1

The synthesis of histones and DNA was examined in BHK cells arrested in G1 by isoleucine starvation and in cells progressing into the S phase upon isoleucine refeeding. Approximately 2-3% of the cells were not arrested in G1 and synthesized DNA. The rate of synthesis of DNA and nucleosomal histones observed in cells starved for isoleucine could be accounted for by the presence of these asynchronous cells. Synthesis of H1 histones by cells in G1, however, was 3 times that of the nucleosomal histones and approximately 15% of the rate of H1 histone synthesis in mid-S. Upon entry into S, the histones were synthesized in the same molar ratio in which they are present in chromatin. The possible biological significance of H1 histone synthesis in G1 cells and its implications for the regulatory mechanisms controlling histome synthesis are discussed.

A relation between inhibition of protein synthesis and conformation of 5′-phosphorylated 7-methylguanosine derivatives

The inhibition of protein synthesis by 5′-phosphorylated derivatives of 7-methylguanosine and 7-methylinosine has been studied in a wheat germ cell-free system programmed by exogenous messenger RNA. The di- and triphosphate derivatives were found to inhibit protein synthesis at lower concentrations than 7-methylguanosine-5′-monophosphate. Inosine derivatives were consistently poor inhibitors when tested with different eucaryotic and viral RNAs. Dinucleotides derived from G5′ppp5′A by methylation of guanosine in the 7 position and/or of the adenosine in the 2′-O position were also tested as inhibitors of protein synthesis. A dinucleotide without the 7-methyl group is not inhibitory. Dinucleotides containing m7G ‡ inhibit protein synthesis at about the same concentration as m7G5′ppp. The presence of the 2′-O methyl group has no effect on the inhibition of protein synthesis. A correlation between inhibition of protein synthesis and conformation in solution of the phosphorylated derivatives of m7G has been sought by nuclear magnetic resonance analysis. In aqueous solutions m7G exhibits considerable conformational freedom and various conformers are present. In the 5′-phosphorylated derivatives of m7G a very dramatic effect on the conformational freedom of the backbone is observed. This results in an increase in conformational purity and loss of the flexibility about the C-4′−C-5′ bond and a significant increase in the population of 3E conformers of the ribofuranose ring. Little conformational difference is observed between the di- and triphosphate derivatives of m7G. It seems likely that the electrostatic interaction between the positively charged N-7 and the negatively charged phosphate groups plays the dominant role in imparting conformational “rigidity” to the backbone of the 5′-phosphorylated derivatives of m7G.

Tumor necrosis factor alters cytoskeletal organization and barrier function of endothelial cells

Treatment of human umbilical cord vein endothelial cells with tumor necrosis factor results in marked changes in cell shape and cytoskeletal organization. After 4 h of treatment, these cells loose reciprocal contacts with the formation of intercellular gaps. This retraction reaches a maximum after 6 h when most stress fibers staining for F-actin disappear and vinculin becomes diffused in the cytoplasm. Such changes spontaneously reverse after 24 h in the presence of tumor necrosis factor or after 2 h of incubation in fresh medium. After treatment with tumor necrosis factor, endothelial monolayers become permeable to albumin because of gaps that form between cells. Normal human serum, plasma alpha 1-proteinase inhibitor and an anti-inflammatory peptide that decrease synthesis of platelet-activating factor inhibit the changes induced by tumor necrosis factor. Furthermore, receptor antagonists of platelet-activating factor have the same effect. These findings suggest that platelet-activating factor is a secondary mediator responsible for the changes in cell shape and cytoskeletal organization, and for the leakiness of endothelial monolayers.

Viral messenger RNA unmethylated in the 5′-terminal guanosine in interferon-treated HeLa cells infected with vesicular stomatitis virus

The distribution of viral mRNA between polysomal and nonpolysomal fractions was investigated in interferon-treated cells infected with vesicular stomatitis virus (VSV). More than half of the viral mRNA synthesized by these cells is in the nonpolysomal fraction, whereas less than one-third of the mRNA synthesized by control cells is found in this fraction. Polysomal and nonpolysomal mRNA sediment identically on sucrose density gradients, but the nonpolysomal mRNA from interferon-treated cells in under-methylated, as shown by labeling experiments with [methyl-3H]methionine. Analysis of VSV mRNA after digestion with nucleases shows that all molecules are capped but about 60% of the nonpolysomal mRNA isolated from interferon-treated cells is unmethylated in the 5′-terminal G. When tested in an assay for initiation of protein synthesis, only 45% of this mRNA binds to ribosomes, as compared to 80% binding for polysomal mRNA from either control or interferon-treated cells.

Low interferon binding activity of two human cell lines which respond poorly to the antiviral and antiproliferative activity of interferon

Abstract Two human cell lines (HT1080 and MRC5) were found to be resistant to the antiproliferative effects of human interferon α. These cells showed also a reduced antiviral response, when virus yield was measured by a plaque assay. The binding of 125 I-labeled α interferon to HT1080 and MRC5 cells was greatly reduced, relative to another human cell line (A549) which responded well to the antiproliferative and antiviral activities of interferon. These results suggested that the poor response of HT1080 and MRC5 cells was due to the presence of fewer interferon receptors than in A459 cells. Some functional receptors, however, appeared to be present in HT1080 and MRC5 cells, since treatment with interferon resulted in a slight reduction in virus yield and in a modest increase in 2′,5′-oligo(A) polymerase activity.