Interferon: An era in molecular biology begins

Clinical Microbiology Newsletter NoverrPoer,i980

Copyright © 1980 by G. K. Hall & Co.

ISSN 0196-4399

Interferon: An Era in Molecular Biology Begins Sandra Panem, Ph.D. Department of Pathology and the Comnlitte on Virology University of Chicago Chicago, lllinois 60637 Interferons are glycoproteins that protect cells from viral cytolysis. The infected cell synthesizes low levels of interferon, which is released into the extracellular milieu. Neighboring cells bind the interferon at their plasma membranes and, subsequently, become resistant to viral infection (3). The molecular events that determine the synthesis of interferon in the primary cell and induce the socalled antiviral state in the secondary, protected cell have yet to be defined. Until recently, interferon research has been concentrated mainly on these virus-associated phenomena; other studies, however, t/ave shown that interferons can be synthesized in response to nonviral stimuli. This suggests that the role of interferon may be more extensive than originally thought. Because some cancer patients who received interferon therapy have shown remission of their disease, interferon has recently received a great deal of public attention. The "antitumor" potential of interferon has spurred increased interest in interferon research. Interferons produced by different cell types may differ in their structure, function and target cell (3). Although an individual cell generally produces only one kind of interferon, it is known that cells contain the genes for at least several distinct interferons, and that they may produce more than one type. Interferon research has been hampered by the difficulty in obtaining sufficient quantities of pure material. Because

many experiments utilized imput, and heterogeneous preparations, it is not known which of the many different effects attributed to interferon are really a consequence of its action, or whether different interferons have the same or diverse effects on cells. Within the past year, studies of the molecular structure and mode of interferon action have produced exciting results. Using recombinant DNA technology, several interferon genes have been cloned (1,4) and analyzed. Small quantities of interferon have been purified and subjected to amino acid analysis (2,5). There is great interest in these experiments because, for the first time, they provide definitive proof of the complexity of interferons at the genetic level. Interferon preparations of sufficient purity and quantity are now potentially available to determine if their spectrum of activity represents functional diversity of different interferons. Molecular Biology of Human Interferons Interferons are species and tissue specific (3), that is, interferons produced by human cells protect human, but not rabbit or mouse, cells from viral infection, and different cell types synthesize different kinds of interferon. The interferons synthesized by h u m a n cells have been divided into two major types, I and II, based on their acid stability and whether the agent that induces their synthesis is a virus op a mitogen (an agent stimulating cell division). Acidstable, Type I interferons are elicited by viral infection and, depending on their cell of origin, are either fibroblast (FilF) or leukocyte (LelF) I

interferons. Both LelF and FilF are glycoproteins whose molecular weight is 16,000 to 26,000 daltons. Although LelF and FilF have similar effects on ceils, they are encoded by different genes and can be distinguished by their I) diversity at the amino end of mature interferon polypeptides, 2) target-cell specificities and 3) neutralization with type-specific anti-interferon antibodies. The genetic control of LelF and FilF is not yet understood, but different interferons are probably regulated independently because most cells produce either LelF or FilF, but some can produce both simultaneously. The nucleic acid sequences of several interferons produced by human fibroblasts and of one interferon from human Ieukocytes have been isolated and cloned by recombinant D N A techniques. As described below, several sequential steps were required. First, messenger RNA (mRNA) was isolated from cells producing interferon after viral challenge. Second, a DNA copy (complementary DNA [cDNA] of the interferon mRNA was prepared using RNA-dependent DNA-polymerase. Next, this interferon cDNA was inserted into bacterial plasmids (IF plasmids) and used to transform Escherichia coli. In the final and crucial step, colonies of E. coli containing IF plasmids were identified using several hybridization techniques. First, colonies were detected whose DNA hybridized with mRNA from interferon-producing cells but not with mRNA from cells that did not synthesize interferon. The DNA from these colonies was then used in successful' 'hybridization-arrest" experiments. In this type of experi-

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The isolation ofE. cofiwith IF plasmids has made it possible to describe interferon at the molecular level. This exciting description has shown that a) several interferon genes are unique but share similarity, b) different interferons may have evolved from a common ancestral interferon gene, c) interferons may be more numerous than previously supposed. The molecular description of interferon diagrammed in Figure 1 was obtained by determining the DNAbase sequence of interferon plasmids from FilF and LelF. The sequences of several clones were compared and the first codons (nucleotide triplets) for initiation of protein synthesis were aligned. The amino acid composition of FilF and LelF was then predicted and found to correspond with the sequences determined by direct amino acid analysis of purified interferons. The data show that interferon is probably derived by cleavage from a pre-interferon protein that

ment, the DNA from colonies thought to contain IF plasmids is hybridized to mRNA from interferon-producing cells. If the colonies really contain IF plasmid, the interferon mRNA enters into a hybrid that can be removed from the mRNA population. When placed in an in vitro protein-synthesizing system, the remaining mRNA is no longer able to direct the synthesis of biologically active interferon. Therefore, hybridization with DNA from IF plasmids "arrests" the ability of interferon mRNA to direct the synthesis of interferon. One aim of these recombinantDNA cloning experiments was to isolate pure interferon genes. With these, it was planned eventually to "engineer" bacteria that synthesize pure interferon protein. It was exciting to find, therefore, that some of the original IF plasmid-containing E. coli synthesized biologically active interferon.

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contains the mature interferon polypeptide and a signal peptide. Signal peptides are characteristic of proteins, such as insulin, that are synthesized in one cell but act in others. It is thought that the signal peptide allows the transport of the newly synthesized protein from the ribosome to its site of release from the cell. There, the signal peptide is removed and the protein is released into the extracellular milieu. A finding of particular interest is that FilF and LelF share extensive DNA-sequence homology (45070) that clusters in four regions or domains (Fig. 1). This may mean that the IF protein is organized into four functional areas, but this speculation must still be proven. Analysis of several different LelF and FilF plasmids indicates, however, that human cells contain multiple, distinct interferon genes. Type II interferons are acid-labile proteins induced in lymphoblastoid cells by mitogenic stimuli (e.g, concanavalin A, phytohemagglutinin). The molecular study of these immune interferons has not advanced as quickly as that of FilF and LelF. There is great interest in immune interferon, however, because one of the in vivo effects of interferon therapy is modulation of natural' 'killercell" activity. There is speculation that this killer-cell regulation may be the "antitumor" activity of interferon. Another technical breakthrough in interferon research has been the preparation of monoclonal antibody to LelF. Rapid purification of interferon by affinity chromatography using these pure antibodies should now be possible.

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Fig. 1. Schematic drawing o f LelF and FilF mRNAs. This diagram shows the postulated functional regions and the shared areas of LelF and FilF mRNAs. Each line drawing is related to the nucleotide sequence of interferon cDNAs shown at the bottom of the diagram in number of base pairs. The postulated signal peptide anc~mature interferon polypeptide are located within a typical mRNA (ca. 850 base pairs long) that includes untranslated sequences at both ends. This structure was deduced by comparing the base sequence of the LelF and FilF and aligning the first initiation codon. Comparison o f the sequences revealedfour domains or clusters of shared sequences (hatch marks) in LelF and FilF.

The Action of Interferon The mechanism(s) of interferon action are still under investigation. It is known that interferon acts at the cell surface and does not enter the cell. Its mode of action therefore resembles that of some hormones. Both LelF and FilF induce antiviral states that are characterized by the synthesis and/or activation of at least three enzymes: a protein kinase, 2',5'-

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oligoadenylate synthetase and an endonuclease. One effect of these enzymes is modulation of ribosomalassociated elongation factor, an important element for protein construction. Since interferon has long been suspected of altering protein synthesis, the induction of these enzymes may explain the antiviral state. Many different biochemical effects have been reported as occurring specifically as a consequence of interferon treatment. Now that it is clear that ceils contain the genetic information for many interferons, it is hoped that discrete biochemical effects can be linked to individual interferons. For examp!~ three common interferon effects are a) altered plasma membrane-associated phenomena, b) altered protein synthesis, and c) decreased cell division. The Future of Interferon Research Future molecular research will allow the number, diversity, and or-

ganization of interferon genes within the human chromosome to be determined. With monoclonal antibodies, it will be possible to isolate different interferons and immunologically compare their structures. Combined use of genetic and immunologic approaches will allow the different effects of interferon to be assigned to individual gene sequences and will also clarify the mechanisms controlling interferon expression, and species and tissue specificity. The eventual payoff of basic studies will be the production of high quality reagents for definitive clinical trials and the possible laboratory engineering of new interferon molecules that possess the desirable effects of several naturally occurring interferons. Finally, there is the tantalizing possibility that once interferon is understood, it will be found to be not only therapeutic, but also an important regulatory mechanism of normal, physiologic processes.

References 1. Derynck, R. el ai. 1980. Isolation and structure of a human fibroblast interferon gene. Nature (London) 285: 542-547. 2. Knight, E., Jr. et al. 1980. Human fibroblast interferon: Amino acid analysis and amino terminal amino acid sequence. Science 207: 525-526. 3. Stewart, W. E., II. 1979. The interferon system. Springer-Verlag, Inc., New York. 4. Taniguchi, T. et al. 1980. Human leukocyte and fibroblast interferons are structurally related. Nature (London) 285:547-549. 5. Zoon, K. C. et al. 1980. Amino terminal sequenceof the major component of human lymphoblastoid interferon. Science 207:527-528.

media would have to be opened and the liquid solidified in cement (obviously an unacceptably expensive procedure) before it would be accepted. The Beatty, Nevada, and Richland, Washington clump sites had already been shut down. On June 27, 1979, Johnston Laboratories sent out a memo urging hospitals in all states to obtain a general license that would permit them to discard used blood-culture bottles "in the normal laboratory waste." Our response to this memo was to query the State Department of Environmental Protection. We were informed that it is illegal to bury radioactive waste in any landfill in Connecticut except in a dump site approved for disposal of radioactive waste. Since there is no appro.ved dump site in Connecticut, simply obtaining a general license from the Nuclear Regulatory Commission (NRC) does not permit a Bactec user to dump the

used media with "the normal laboratory waste." Connecticut, then, is like the other 24 nonagreement states whose laws governing the dumping of radioactivity are stricter than NRC regulations. To date, there is no government agency with a compilation of state regulations governing the disposal of low-level radioactive waste, but approved methods of disposing of low-level radioactivity in individual states can be determined by contacting the Radiological Health Section of the State Department of Environmental Protection. In October 1979 we began uncapping the used bottles and pouring the contents down the drain. Radioactive waste disposal is not only a complicated but also an emotional issue. Someone, apparently concerned about radioactive contamination of the environment, called the local newspaper, which ran a story on our disposal problem. An educational

Editorial

Disposal of Radioactive Blood Culture Media James C. McLaughlin, Ph.D. Division of Microbiology Hartford Hospital Hartford, Connecticut 06115 The acceptable means of disposal of Bactec (Johnston Laboratories, Inc.) blood culture media is a question with which many clinical microbiologists have struggled during the last few years. The problems associated with the disposal of highlevel radioactive wastes have served to complicate the disposal of Bactec blood culture bottles, containing only 2#Ci of C '* each. In September 1979, Chem-Nuclear Systems, Inc., which operates the Barnwell, South Carolina radioactive dump site, announced that it would no longer accept liquid waste. This meant that bottles of radioactive blood culture