The study of the induction of type I IFN genes has been greatly facilitated by the fact that their promoter regions are appropriately regulated when transfected into cultured cell hnes (Canaani and Berg 1982, Hauser et al 1982, Mantei and W eissmann 1982, Ohno and Taniguchi 1982, Pitha et al 1982, Zinn et al 1982).
1.2.2.1. Nature of Inducing Signals
The synthesis of type I IFNs is not detectable in normally growing cells, but reaches high levels after induction. In vivo, almost all viruses can act as inducers, whether their genome consists of DNA, single-stranded RNA, or double-stranded RNA. Furthermore, many viruses can induce IFNs ex vivo in isolated tissues and cell suspensions, or in vitro in primary fibroblast cultures, and in many established fibroblastoid and lymphoblastoid cell lines. However, several viruses that are efficient IFN-inducers in vivo, are poor inducers, or not inducers at all, of cultured cells.
In addition to viral infection, IFN-6 can be induced in vitro by treatment of cells with double-stranded RNAs, such as synthetic poly(I)-poly(C). Poly(I)-poly(C), though an effective inducer in vitro, is a rather poor inducer in vivo, due to RNA-degrading enzymes in serum. The exact structural features of dsRNA molecules important for induction are not clear, presumably uninterrupted double-stranded stretches of certain length are necessary (Marcus 1983).
It has been believed that the viral induction of IFN-6 gene is also mediated by dsRNA that either forms the viral genome, or is generated from it as an intermediate at some stage of a viral replication cycle (Marcus 1984). However, the induction pathways by viruses and dsRNA are clearly not identical, and at least some viruses seem to provide an inducing factor, or elicit a cellular signal transduction pathway, that is different from, or additional to, those provided by dsRNA. For example, certain ssRNA viruses can induce IFN under conditions non-permissive for replication, and certain replication-defective mutants of reovirus do not induce IFN, even if their genome is dsRNA (Lai and Joklik 1973). Furthermore, the viral induction of the IFN-a genes does not appear to be mediated solely
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by dsRNA, since they are inducible by NDV but not by poly(I)-poly(C) in primary human fibroblasts, whereas the IFN-6 gene can be induced by both agents (Havell et al 1978). In addition, partial induction of otherwise priming-dependent cell lines, can be reached by Sendai virus [a paramyxovirus; genome (-)ssRNA] without the need to pretreat cells with IFN (King and Goodboum 1994 ^ ). A difference between Sendai virus and dsRNA has also been reported at the level of the DNA binding factors that bind to the DNA elements within the IFN-6 promoter: in differentiated mouse embryonal carcinoma cells, Sendai can induce the PRD II binding activity NF-kB, whilst dsRNA cannot (Ellis and G oodboum 1994). The nature of the non dsRNA com ponent(s) provided by the Sendai vim s is not known, although it has been suggested that the viral protein C could function as an efficient IFN-6 inducer (Taira et al 1987).
The signal pathway generated by dsRNA, and leading to specific gene activation, has not been well elucidated. It should be emphasized that the induction o f IFN-6 transcription does not require de novo protein synthesis, suggesting that the effect on the transcription factors is posttranslational, perhaps mediated by specific phosphorylation events, known to modulate the activity of many DNA binding proteins. Inhibition of IFN-6 induction can be achieved by the purine analog 2-aminopurine (Marcus and Sekellick 1988, Zinn et al 1988), a rather nonspecific kinase inhibitor, known to inhibit PKR among other kinases. It is interesting that PKR, a kinase induced by IFNs and implicated as a mediator of IFN response, is activated by dsRNA - thus it can be an effector molecule functioning at more than one level o f the IFN system. It has indeed been shown that by virtue o f its phosphorylation activity, PKR can activate in vitro one transcription factor, NF-kB, important for the regulation of the IFN-6 promoter (Kumar et al 1994). Also, the selective ablation of the PKR mRNAs in HeLa cells inhibits the dsRNA mediated activation of NF-
k B (Maran et al 1994). It remains to be investigated whether the other DNA binding regulators of the IFN-6 promoter could be targets for regulatory phosphorylations by PKR, either as direct substrates or at the end of a signal cascade where PKR would be an upstream effector.
It has also been suggested that under some circumstances the accumulation of naturally occurring cellular double stranded RNAs can induce IFN production (Belhumeur et al
1993). If this kind of endogenous induction machinery exists, it would have to be tightly regulated to prevent inappropriate IFN expression, which would inhibit cell proliferation. Cellular RNA unwindases could be such regulators.
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The induction o f IFN-6 has been shown to occur primarily at the level of transcriptional initiation (figure 1.2.; Raj and Pitha 1983, Nir et al 1984). In uninduced cells the IFN-6 mRNA is undetectable. The induction cycle begins with a lag period after the introduction of an inducer. This period does not appear to result from the delayed entry of an inducer into the cell (Hauser et al 1982), nor does it reflect a need for synthesis of poly(I)-poly(C)- induced proteins, since IFN-6 mRNA is inducible in the presence of protein synthesis inhibitors. Rather, the lag period is likely to reflect the time required to derepress the prom oter to allow efficient transcription. The lag period is followed by an IFN synthesis phase peaking 6-12 hours after the cells have encountered an inducer, during which a substantial proportion of the newly synthesized mRNA is IFN-6-specific - that is, several thousand transcripts per cell. The induction of the IFN-6 is transient, and at the final postinduction turn-off stage, the IFN production rapidly decreases back to undetectable levels. The lengths of different phases vary depending on the cell type and inducer.
The dramatic changes in the IFN-6 expression during the induction cycle reflect the biological properties of IFN-6 - the potent cytostatic effects would make expression in uninduced cells incompatible with cellular growth, while overproduction in induced cells serves to minimize the spread of viral infection.
Induction can occur without the requirement for de novo protein synthesis, indicating that all the factors necessary for these events pre-exist in the cell in some form (Cavalieri et al 1977). In fact, simultaneous treatment of dsRNA-induced cells with metabolic inhibitors of protein synthesis, such as cycloheximide which blocks the elongation of a peptide chain, causes an enhancement in the degree of IFN-6 induction, a phenomenon referred to as superinduction. As discussed in section 1.2.2.5., in many cell lines, the inhibition of protein synthesis interferes with the postinduction shutoff of the promoter.
In some cultured cell lines IFN-6 induction can be strongly increased by pretreating cells with IFN before induction, a phenomenon known as priming. Furthermore, the kinetics o f induction^accelerated by priming (Abreu et al 1989, Content et al 1980, Fujita and Kohno 1981). The basis of the priming phenomenon remains somewhat unclear, but has been shown to operate at the level of transcription (Nir et al 1985). W ithin the IFN-6 promoter, the priming effect cannot be localized to any specific sequence element, suggesting that a cellular function is induced that allows the inducer dsRNA to activate independent cellular targets (King and G oodboum 1994). On the basis of complem entation in cell fusion experiments, it seems that priming provides an IFN-inducible factor required for dsRNA- induction that is constitutively present in priming-independent cells (Enoch et al 1986), but