In this article we will discuss about:- 1. Meaning of Interferons 2. Characteristics of Interferons 3. Production 4. Mechanism of Action 5. Applications.
Meaning of Interferons:
Interferons are natural glycoproteins produced by virus-infected eukaryotic cells which protect host cells from virus infection. They were discovered by Isaacs and Lindenmann in 1957 in course of a study of the effect of UV-inactivated influenza virus on chick chorioallantoic membrane kept in an artificial medium.
They observed that the infected membrane produced a soluble substance in the medium which could inhibit the multiplication of active influenza virus inoculated in fresh chick chorioallantoic membranes. The substance was called interferon because it interfered with intracellular multiplication of viruses.
Later observations confirmed that such host-produced antiviral substances were common to many viruses. Viral interference is a phenomenon observed when multiplication of one virus is inhibited by another virus. For instance, when influenza-A virus is inoculated into the allantoic cavity of an embryonated egg followed after 24 hr by influenza-B virus, the multiplication of influenza-B virus is partly or completely inhibited. The reason why influenza-B virus cannot multiply is that the influenza-A virus infected cells produce interferon which partly or totally inhibits multiplication of B virus. The interferon also protects cells from influenza A virus.
Characteristics of Interferons:
An outstanding feature of interferons is that they are host-cell-specific and not virus-specific. This means that interferons produced by mouse or chicken will not protect human cells against the same virus which induced interferon in the experimental animals. On the other hand, an interferon produced by a virus X in an animal will protect the animal also from other viruses.
This is because interferons do not interact directly with the viruses. But they induce the virus infected cells to synthesize antiviral proteins which inhibit viral multiplication. These proteins have a wide inhibitory spectrum. As a result, not only the interferon-inducing virus, but others are also inhibited.
The reason why interferon produced by one species does not protect another species is that the same virus produces different interferons in different species. It has been observed that interferons produced by different host species following infection by the same virus differ in molecular weight as well as in other properties, like isoelectric point etc. Not only different species produce different interferons, different tissues of the same animal produce different interferons.
All types of interferons are proteins having a comparatively low molecular weight ranging between 15,000 to 40,000 Daltons. Hence, they are non-dialyzable and destroyed by proteolytic enzymes. Interferons are fairly stable at low pH (pH2) and can withstand moderate temperature being stable at 37°C for an hour or so. They are produced in minute amounts by the infected cells as a longer precursor having 23 amino acid residues more than the mature molecule.
Human interferons are of three main types. These are called alpha interferons (α-IFN), beta-interferons (β-IFN) and gamma-interferons (γ-IFN). Alpha-interferon contains many subtypes. The total subtypes exceed 20 in number.
It is produced by the B-lymphocytes, monocytes and macrophages. β-IFN is produced by the fibroblasts in the connective tissues. γ-IFN is synthesized by the T-lymphocytes after they are activated by antigens. α-IFN has been shown to be coded by as many as 20 distinct chromosomal genes, indicating thereby that the subtypes of this interferon represent a family of closely related proteins.
β-IFN appears to be a glycoprotein. It is coded by a single human gene. All the genes of α-IFN and β-IFN are located on the short arm of human chromosome 9. α-IFN proteins are all 166 amino acid long (except one). They are non-glycosylated and the proteins are monomeric. The single β-IFN protein is also 166 amino acid long and a glycoprotein. It is dimeric.
Production of Interferons:
Interferons are produced by living animal cells, both in vivo as well as cultured cells. Interferon production and its antiviral activity require expression of cellular genes, and these functions are blocked by inhibitors of transcription and translation. Thus, virus-infected host cells fail to produce interferon in presence of actinomycin D, an inhibitor of eukaryotic RNA polymerase. When the inhibitor is added after 2 hr of infection, interferon production is not inhibited, suggesting that transcription is completed by that time.
Interferon production starts after initiation of viral maturation and continues for 20 to 50 hr after that. Then the production stops, due to formation of a repressor which presumably is formed or activated only when the interferon concentration in the producing cell exceeds a certain threshold concentration. Most of the interferon is transported from the producing cell to other neighbouring cells.
The substance in a virus that is responsible for interferon synthesis by the host cell is known as interferon inducer. The nature of this substance was identified by Merigan (1970) as double-stranded RNA. The activity seems to reside in polyribonucleotide’s with a high helical content. The double- stranded RNA viruses — like reoviruses — can act as interferon inducer without replication. Single- stranded RNA viruses can act as inducers only after replication when they form double-stranded replicative intermediates. DNA-viruses can also induce interferons, presumably due to overlapping transcription of viral DNA as observed in case of vaccinia vinus (Fig. 6.39).
Fungal viruses which have mostly double-stranded RNA genomes are also efficient inducers of interferons. Some synthetic polymers containing riboinosinic acid, ribocytidylic acid (Poly I: C) as well as those containing riboadenylic acid and ribouridylic acid (Poly A: U) are also good inducers. All interferon inducers are characterized by high molecular weight, high density of anionic groups and resistance to enzymatic degradation. DNA and DNA-RNA hybrids have been found to be ineffective as interferon inducers.
The induction of interferon synthesis concerns α- and β-interferon’s which belong to a single class, called Type I. Gamma-interferon belongs to a separate class, called Type II. The human Υ-interferon is the single representative of its type. The gene coding the y-interferon protein is located on the long arm of chromosome 12. The gene has three introns, while the genes of α- and β- interferons are without any introns. Gamma-interferon (human) has 146 amino acids and is an N-glycosylated tetrameric protein. It is induced by antigenic stimulation of T-lymphocytes.
In presence of the inducer which is viral ds-RNA, the α- and β-interferon genes of the host chromosome(s) are activated to produce interferon m-RNAs. Those are then translated intoα- and β- interferon proteins. These proteins at first accumulate in the producing cell and eventually leave the cell to reach neighbouring host cells.
As the interferon concentration in the producing cell rises above a threshold level, it activates another gene of the producing cell which codes for a repressor protein which feeds back and stops further synthesis of interferon. As a result, virus-infected cells generally produce only small quantities of interferons.
The interferon molecules that leave the producing cell reach the neighbouring uninfected host cells and interact with the cell membrane or nuclear membrane receptors of these cells. Thereby these cells are induced to synthesise antiviral proteins. These antiviral proteins are the actual agents that provides protection to these host cells against viral infection.
Mechanism of Action of Interferons:
Type I interferons:
Type I interferons include α-IFN and β-IFN. These interferons do not interact with the viruses directly causing their inhibition, but they induce the formation of antiviral proteins which are activated to inhibit viral multiplications. These interferon-regulated proteins (IRPs) act presumably by blocking synthesis of the macromolecular components necessary for viral multiplication.
A general scheme for mechanism of action of type I interferons is shown in Fig. 6.40:
Several interferon regulated host proteins (IRPs) have been identified, though all of them have not been fully characterized. Among the better known of these proteins are a protein kinase and an enzyme catalyzing the formation of a short polymer of adenylic acid, the 2′, 5′-oligoadenylate synthetase (2′-5′ A synthetase).
The protein kinase is induced by Type I interferons. It has to be activated by ds-RNA. The activated kinase catalyses phosphorylation of initiation factor (el F-2) thereby causing inhibition of protein synthesis (Fig. 6.41).
The 2′-5′-oligoadenylate synthetase is an enzyme also induced by Type I interferons which requires activation by ds-RNA like the protein kinase. The activated synthetase acts as an activator of an endonuclease, RNase L. The activated RNAse degrades viral ss-RNA (Fig. 6.42).
Another group of proteins, called Mx-proteins induced by α- and β-IFN are known to possess intrinsic antiviral activity, although the exact molecular mechanism by which they inhibit viral multiplication is not known. Mx-proteins have been reported to play a major controlling role in infections caused by influenza viruses in experimental animals as well as in humans.
Type II interferon:
Type II interferon includes g-IFN which is also known as immune IFN. Although g-IFN also possesses anti-viral activity, its major role is in the immunity through activation of cytotoxic T-lymphocytes which can destroy virus infected cells. Besides T-lymphocytes, other naturally occurring killer cells like macrophages and monocytes are also activated by g-IFN. Thus, in contrast to that of Type I interferons, the antiviral effect of g-IFN is expressed through activating the killer cells of the body which destroy the virus-infected cells.
Type II interferon induces the major histocompatibility antigens of human cells. Expression of these antigens is essential for immuno-potent cells to present foreign antigens to the T-lymphocytes during generation of specific immune responses.
IFN induced expression of these major histocompatibility antigens represents an important contribution of the antiviral activity of g-IFN through enhancement of the activity of cytotoxic T-lymphocytes. The activation of cytotoxic T-lymphocytes by y-IFN also implies its possible role in elimination of cancer cells which are recognized by the immune system of the body as foreign objects.
Applications of Interferons:
Interferons could be ideal agents for combating viral diseases. They inhibit viral multiplication at such low concentration which is non-toxic to uninfected cells. One interferon can inhibit many viruses. But there are certain draw-backs which stand in their use.
Firstly, for application in humans, interferon must be of human origin, though interferons produced in monkey kidney cell cultures are also effective in humans. Interferons are produced in very small quantities and it is difficult to get them in sufficient quantity in pure form for clinical application. Another factor is that interferons are effective only for short periods and as such can be used against only acute infections, like influenza.
The difficulty of obtaining sufficient quantity of pure interferon for clinical use has been overcome by cloning the α-IFN and β-IFN human genes in bacteria and yeast. By growing these transgenic organisms in mass culture, it has been possible to obtain clinically usable interferons in sufficiently large quantities. Alpha-interferon has been marketed in 1984 under the trade name Intron A.
In the following years, this biotechnologically produced interferon has been approved for clinical use against diseases like genital herpes caused by herpes-virus, hepatitis B and C. Beta-interferon has also been biotechnologically produced and marketed under the trade name Betaseron. It has been used in a disease called multiple sclerosis. A recombinant g-interferon has been found effective against an inherited chronic disease, called granulomatous disease.
The neutrophils of the affected individual are unable to kill the infectious bacteria. Application of y-IFN to such persons restores the ability of the neutrophils to kill bacteria. As the disease is chronic and inherited, the affected persons must take g-IFN throughout their life to remain normal.
Interferons are not only antiviral, but they have also anticancer activity. Clinical trials have shown that interferons have effect against only some types of tumours. Alpha-interferon has been approved for treating hairy-cell leukemia, and Kaposi’s sarcoma, a cancer that occurs in AIDS patients.
Gamma-interferon has been mainly used as an immuno-stimulant in cancer patients. Resistance against tumours in the body is controlled by the immune response against tumour antigens. The cytotoxic T-lymphocytes recognize these antigens as foreign and destroy them. Gamma-interferon can stimulate the cytotoxic function of T-lymphocytes and other natural killer cells of the body, thereby helping to control the tumour cells.