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Figure 4.4. Outline of how the 2-5' synthetase system promotes its anti-viral effect. The 2-5' phosphodiesterase ‘off switch’ is omitted for clarity. Refer to text for details
Figure 4.5. (a) Structural detail of the 2'-5' oligonucleotides (2'-5'An) generated by 2-5' An synthetase. Compare the 2'-5' phosphodiester linkages with the 3'-5' linkages characteristic of normal cellular oligonucleotides, such as mRNA (b)
The sole biochemical function of 2'-5'An (and hence 2'-5' An synthetase) appears to be as an activator of a dormant endo-RNase, which is expressed constitutively in the cell. This RNase, known as RNase L or RNase F, cleaves all types of single-stranded RNA (ssRNA). This inhibits production of both viral and cellular proteins, thus paralizing viral replication. Presumably cellular destruction of the invading ssRNA will be accompanied by destruction of any additional viral components. Removal of dsRNA would facilitate deactivation of the endo RNase, allowing translation of cellular mRNA to resume. A 2'-5'-phosphodiesterase represents a third enzymatic component of this system. It functions as an off switch, as it rapidly degrades
THE CYTOKINES—THE INTERFERON FAMILY 207
the 2'-5' An oligonucleotides. Although this enzyme also appears to be expressed constitutively, IFN binding appears to increase its expression levels in most cells.
The eIF-2a protein kinase system
Intracellular replication of viral particles entirely depends upon successful intracellular transcription of viral genes with subsequent translation of the viral mRNA. Translation of viral or cellular mRNA is dependent upon ribosome formation. Normally several constituent molecules interact with each other on the mRNA transcript, forming the smaller ribosomal subunit. Subsequent formation/attachment of the larger subunit facilitates protein synthesis.
Exposure of cells to IFN normally results in the induction of a protein kinase termed eIF-2a protein kinase. The enzyme, which is synthesized in a catalytically inactive form, is activated by exposure to dsRNA. The activated kinase then phosplorylates its substrate, eIF-2a, which is the smallest subunit of initiation factor 2 (eIF2). This in turn blocks construction of the smaller ribosomal sub-unit, thereby preventing translation of all viral (and cellular) mRNA (Figure 4.6).
Induction of eIF-2a protein kinase is dependent upon both IFN type and cell type. IFN-a, -b and -g are all known to induce the enzyme in various animal cells. However, in human epithelial cells the kinase is induced only by type I IFNs, while none of the IFNs seems capable of inducing synthesis of the enzyme in human fibroblasts. The purified kinase is highly selective for initiation factor eIF-2, which it phosplorylates at a specific serine residue.
Interferon, in particular type I IFN, is well adapted to its anti-viral function. Upon entry into the body, viral particles are likely to quickly encounter IFN-a/b-producing cells, including macrophages and monocytes. This prompts IFN synthesis and release. These cells act like sentries, warning other cells of the viral attack. Most body cells express the type I IFN receptor, thus the released IFN-a or -b will induce an anti-viral state in such cells.
The ability of IFNs (especially type I IFNs) to induce an anti-viral state is unlikely to be solely dependent upon the enzymatic mechanisms discussed above. Furthermore, the 2'-5'An synthetase and eIF-2a kinase systems may play important roles in mediating additional IFN actions. The ability of such systems to stall protein synthesis in cells may play a role in IFN-induced alterations of cellular differentiation or cell cycle progression. They may also be involved in mediating IFN-induced anti-proliferative effects on various transformed cells.
Major histocompatibility complex (MHC) antigens and b2-macroglobulin are amongst the best known proteins whose synthesis is also induced in a variety of cell types in response to various interferons.
Additional studies focus upon identification and characterization of gene products whose cellular levels are decreased in response to IFN binding, e.g. studies using various human and animal cell lines found that IFN-a and -b can induce a significant decrease in the level of c-myc and c-fos mRNA in some cells. IFN-g has also been shown to inhibit collagen synthesis in fibroblasts and chondrocytes. Such studies, elucidating the function of gene products whose cellular levels are altered by IFNs, will eventually lead to a more complete picture of how these regulatory molecules induce their characteristic effects.
The anti-viral and anti-proliferative activity of IFNs, as well as their ability to modulate the immune and inflammatory response, renders obvious their potential medical application. This has culminated in the approval for clinical use of several interferon preparations (Table 4.8).