Sohan L. Gupta

Interferon action against reovirus: activation of interferon-induced protein kinase in mouse L929 cells upon reovirus infection

Abstract Experiments are presented which indicate that reovirus infection results in an activation of the interferon (IFN)-induced protein kinase in mouse L929 cells. This is indicated by (i) the phosphorylation in vivo of a 67,000 Mr (67K) polypeptide, which is characteristic of the IFN-induced protein kinase, within a few hours after reovirus infection of IFN-treated mouse L929 cells, and (ii) the phosphorylation in vitro of the 67K polypeptide as well as the α-subunit of exogenously added initiation factor eIF-2 in extracts of IFN-treated reovirus-infected cells without the addition of double-stranded RNA which is required in similar extracts from uninfected cells. In NIH 3T3 cells, which are deficient in (2′,5′)oligoadenylate-activated endonuclease, the replication of reovirus is inhibited by IFN treatment, though EMC virus replication is insensitive. It is suggested that this kinase activation observed upon reovirus infection may play a role in the antiviral action of IFN against reovirus.

Regulation of interferon action in human fibroblasts: Transient induction of specific proteins and amplification of the antiviral response by actinomycin D

Abstract We reported earlier that interferon treatment of human fibroblasts induces a number of proteins, and that this induction seems to correlate with the development of the antiviral state. The accumulation of interferon-induced proteins reaches a maximum within a few hr after interferon treatment and is then followed by a marked decline despite the continued presence of interferon in the medium indicating that this induction may be subject to regulation. Inhibitors of RNA synthesis (actinomycin D, 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole) were found to have a dual effect on the antiviral action of interferon. When added together with interferon, they blocked the development of the antiviral state. In contrast, when added at later intervals (4–8 hr after interferon), the antiviral effect was greatly amplified. This amplification was associated with increased levels of various interferon-induced proteins and 2′,5′-oligoadenylate synthetase enzyme activity. The results indicate that the cellular response to interferon is subject to regulation and can be manipulated. Evidence is also presented which indicates that the interferon-induced genetic transcription required for the development of the antiviral state may extend for several hours.

Accessibility of plasma membrane antigens

Serological techniques applied to intact cells register only those antigens of the plasma membrane that are exposed at the cell surface and are therefore accessible to antibody. Solubilization of the plasma membrane by detergent, used in the conventional surface-iodination immunoprecipitation technique, renders other plasma membrane antigens accessible. We have shown this by using a modified version of the technique in which lysis with detergent is postponed until after the cells have been reacted with antibody. Comparison of the conventional and modified methods confirms that the plasma membrane glycoprotein gp70 has antigen that is not exposed on the intact cells as well as accessible antigen, for example, GIX. The modified surface-iodination immunoprecipitation method is useful for distinguishing cell-surface antigens from plasma membrane antigens that normally are not accessible. This is exemplified by the fact that standard anti-TL and anti-X.1 sera identify gp70 antigen in the plasma membrane that is registered by the conventional, but not by the modified method.

On the inhibition of interferon action by inhibitors of fatty acid cyclooxygenase

Abstract Experiments were carried out to investigate the proposed involvement of the prostaglandin (PG) biosynthesis pathway in interferon (IFN) action. Several known inhibitors of fatty acid cyclooxygenase (indomethacin, ibuprofen, oxyphenylbutazone) were found to inhibit the antiviral action of IFN in mouse L929 and human FS-4 cells. However, the concentrations of these inhibitors required for this effect were much higher than those used to inhibit PG biosynthesis. Furthermore, we found that the inhibitors tested also blocked RNA and protein synthesis, and that the concentration dependence for these effects of the inhibitors and for the inhibition of IFN action were similar. This could account for the inhibitory effect of these compounds on IFN action without invoking an involvement of the cyclooxygenase pathway.

Interferon action in human fibroblasts: Induction of 2′, 5′-oligoadenylate synthetase in the absence of detectable protein kinase activity

SummaryIn several strains of human fibroblasts, treatment with human α- or β-interferon (IFN) resulted in an induction of 2′, 5′-oligoadenylate synthetase but no protein kinase activity. The antiviral and anticellular effects of IFN are elicited in these cells in the absence of detectable IFN-induced protein kinase activity.

INTERFERON‐INDUCED SYNTHESIS OF SPECIFIC PROTEINS IN HUMAN FIBROBLASTS AND THE DEVELOPMENT OF THE ANTIVIRAL STATE

The development of the antiviral effect of interferon (IF) is blocked by inhibitors of RNA and protein synthesis and by enucleation of cells, thus indicating that the expression of certain cellular gene(s) is required to be induced. We showed recently’ that IF treatment induced the synthesis of at least three proteins in mouse Ehrlich ascites tumor cells and five proteins in human fibroblasts. Studies carried out with human cells are summarized here. Human fibroblasts (FS-4, GM258, etc) were labeled with [%]methionine for 16-18 hr in the absence or presence of human leukocyte IF (400 reference unitdml), their extracts (200.000 x g supernatants) prepared and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in slab gels. These experiments revealed four bands of proteins whose synthesis was markedly induced by IF treatment. These proteins had molecular weights of 88,000, 80.000. 67,000, and 56,000 (referred to as P88H, P80H, P67H and P56H, respectively).’ Extracts of IF-treated cells contain two enzymes, a protein kinase(s1 and a 2’-5’-oligoadenylate synthetase, which are strongly activated by double-stranded RNA. Therefore, we fractionated these [35S]methionine-labeled extracts on poly I . poly C-agarose columns, and after extensive washing, the material retained on the columns was extracted with SDS and analyzed on slab gels. These experiments revealed two protein bands (apparent molecular weights 120,000 and 80,000) which were detected strongly in samples derived from IF-treated cells but poorly, if at all. in similar samples obtained from untreated cells. Thus at least five proteins were found to be induced in human fibroblasts by IF treatment. Knight and Korant’ have also detected four new proteins that were synthesized in human fibroblasts after treatment with interferon. The characteristics of the induction of P88H, P80H, P67H and P56H by IF have been studied as they can be easily quantitated. The experiments indicate that the induction of these proteins by IF treatment has features in common with the development of the antiviral state thus suggesting a correlation between the two. For example: (1) it was blocked by actinomycin D (2 &ml] and cycloheximide (50 ccg/ml) when added together with IF, thus indicating a requirement for de novo transcription and translation; (2) the induction of these proteins as well as the establishment of the antiviral state became resistant to inhibition by actinomycin D within 2 hr after the addition of IF, thus indicating that under the conditions employed, the transcription event induced by IF is essentially completed within 2 hr; (3) in agreement with the known species specificity, these proteins were induced by human leukocyte and human fibroblast IFs but not by mouse interferon’; and (4) the induction of these proteins and the establishment of the antiviral state show a similar dependence on the concentration of interferon. Labeling for 2-hr periods at various times intervals after the addition of IF