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Interferon research: a brief history

Authors:

Abstract

Interferons are the antiviral early inflammatory proteins produced in the cells in response to the infectious agents. The characterization of the interferon genes, their expression, and their function was advanced with the development of novel techniques in molecular and cellular biology. Using genetically modified mice revealed the critical role of the interferons in innate and acquired immune response. The critical steps and discovery that lead to the understanding of the interferon system and its role in the antiviral immune response are summarized in this chapter.
Interferon Research 25
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From:
Methods in Molecular Medicine, Vol. 116: Interferon Methods and Protocols
Edited by: D. J. J. Carr © Humana Press Inc., Totowa, NJ
2
Interferon Research
A Brief History
Myriam S. Kunzi and Paula M. Pitha
Summary
Interferons are the antiviral early inflammatory proteins produced in the cells in response to
the infectious agents. The characterization of the interferon genes, their expression, and their
function was advanced with the development of novel techniques in molecular and cellular
biology. Using genetically modified mice revealed the critical role of the interferons in innate
and acquired immune response. The critical steps and discovery that lead to the understanding
of the interferon system and its role in the antiviral immune response are summarized in this
chapter.
Key Words: Innate immunity; interferon; genes; receptors; clinical use; Toll receptors;
viruses.
1. Introduction
Interferon (IFN) was described in 1957 by Isaacs and Lindenmann (1) as an
antiviral protein synthesized by the cell in response to viral infection. The char-
acterization of this protein, its expression, and its function has been closely
linked to the availability of new methods and advances in cellular and molecu-
lar biology. Indeed, the isolation and detection of antiviral proteins synthe-
sized by infected cells was dependent on the development of techniques
enabling the cultivation of eukaryotic cells and the ability to use them for in
vitro viral replication. Later, the availability of specific antibodies and molecu-
lar biology techniques made it possible to recognize that IFN is represented by
a family of closely related, but distinct genes, to characterized IFN genes and
to purify IFNs, as well as produce sufficient amounts for clinical studies.
From the onset, researchers held the hope that IFNs could be used as a gen-
eral antiviral agent in the fight against viral infections, much like antibiotics
are used to control bacterial infections, thanks to their ability to inhibit a variety
of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Unfortu-
26 Kunzi and Pitha
nately, the broad antiviral application has gone largely unfulfilled, mostly because
of the pleiotropic effects that IFNs exert on the cells. Nevertheless, the critical role
of IFNs in the antiviral immune response and cancer editing is just emerging
from studies using the genetically modified mice, and IFNs have been used in
the clinic for the treatment of selective viral infections and malignancies.
2. Purification and Characterization of IFNs
IFNs were initially identified as a group of proteins secreted by cells upon
viral infection and able to inhibit the growth of a wide range of unrelated
viruses. Whereas IFN did not appear to be virus-specific, it was recognized
to be species-specific. Human white blood cells were shown to produce IFN
upon infection, and they were regarded as a possible source of IFN for clinical
purposes. A number of experiments using actinomycin D at doses that inhib-
ited cell RNA synthesis, but not viral replication, demonstrated that IFN was a
product of the cell genome. The use of protein synthesis inhibitors further sug-
gested that IFN exerted its antiviral effect via the synthesis of one or more
proteins, which were the actual antiviral effectors. Quickly, it was recognized
as well that IFNs had properties able to regulate both cell growth and function.
IFN preparations available at that time, however, contained a number of impu-
rities, and the purification of small quantities of highly active IFN proved dif-
ficult. It was not until the advent of IFN-specific antibodies (2), which
permitted the isolation of IFN to near purity by column chromatography, that
the cell antigrowth effects of IFN could be confirmed.
The development of molecular biology techniques led to the detection mes-
senger RNA (mRNA) and genomic DNA in cells. The translation of interferon
mRNA in eukaryotic cell such as Xenopus oocytes and the high specific activ-
ity of IFN allowed for the detection of interferon proteins by their antiviral
activity in cell cultures (3).
3. Identification and Cloning of the IFN Genes
Once a standard assay for IFN mRNAs was established, several laboratories
nearly simultaneously cloned the IFN genes. The cloning of the IFN genes
brought two unexpected findings. First, it became clear that IFN is represented
by large number of cellular genes. These genes known as type I IFN, are repre-
sented by a family of 13 IFNA genes expressed in cells of lymphoid origin and
one IFNB gene expressed in a majority of infected cells (4–6). Although it was
believed for some time that there was at least one more IFN-β protein, IFN-β-
2, this protein was shown to be identical to IL-6. A single IFNW gene (7), with
sequence homology to IFNA, was found to be expressed in leucocytes, and
recently one IFNk gene, with sequence homology to both IFNA and IFNB,
was found to be expressed in keratinocytes and dendritic cells (8). Second, it
Interferon Research 27
was found that all type I IFN genes are nonspliced genes, and although their
expression shows cell specificity, all the genes are localized on the short arm
of chromosome 9 in human cells and on chromosome 4 in the mouse. All type
I IFNs are secreted proteins, although secretion of IFN-κ seems to be very
inefficient. IFN-β is modified by glycosylation, whereas the majority of IFN-α
are unglycosylated (9).
Finally, IFN-γ, or type II IFN, is encoded by a spliced gene localized on
chromosome 12 and has been shown to be synthesized selectively in cells of
the immune system, such as natural killer cells, CD4 Th-1 cells, and CD8 sup-
pressor cells (10,11). The ability to express IFN genes in bacterial expression
systems, coupled with affinity purification, provided sufficient amounts of IFN
proteins to study their specificity and ultimately provided sufficient amounts
for clinical studies
4. IFN Gene Regulation
The optimization of DNA transfection into eukaryotic cells has facilitated
the identification of the regulatory regions of the IFN genes. In this method,
genomic fragments localized at the 5' or 3' end of an IFN gene are cloned in
front of a reporter gene encoding an easily detectable protein, transfected into
cultured cells, and then their ability to induce expression of the reporter gene in
infected and uninfected cells is analyzed. These studies have identified a virus-
regulated element (VRE) in the promoter region of IFNA and IFNB, which
alone confer responsiveness to virus infection (12–14).
Studies of the molecular mechanism involved in the virus-mediated activa-
tion of type I IFN genes has brought about the discovery of IFN regulatory
factors (IRFs), a new group of transcriptional factors (15). The IRFs play a
critical role in the induction of type I IFN genes; chemokine genes; and genes
mediating antiviral, antibacterial, and inflammatory responses. Three of these
IRFs, IRF-3, IRF-5, and IRF-7, function as direct transducers of virus-medi-
ated signaling (16–18). In uninfected cells, these IRFs are expressed in the
cytoplasm, whereas in infected cells, they are activated by a C’ terminal serine
phosphorylation, which results in their translocation from cytoplasm to nucleus
(19). Recently, an IKK kinase, TBK-1, was shown to be responsible for the
phosphorylation and activation of IRF-3 and IRF-7 in infected cells, as well as
cells treated with double-stranded RNA (dsRNA)-polyIC (20,21). The target
of TBK-1 phosphorylation is a cluster of 4 serines in the carboxy terminus of
the IRF-3 polypeptide (22). In infected cells, the ubiquitously expressed IRF-3
mediates the induction of IFNB (23,24). Activation of this gene involves co-opera-
tive assembly of several transcription factors: nuclear factor (NF)κB, ATF-2/c-
june, IRF-3, and IRF-7 on the VRE of the IFNB promoter (25). This complex-
enhanceosome recruits two coactivators, acetyltransferase CBP/P300 and
28 Kunzi and Pitha
holoenzyme polII (26), whereas in the uninfected cells the IFNB promoter is
under a negative control (27). Most of the promoters of IFNA genes do not
contain an NFκB site, and their activation depends not only on IRF-3 but also
on IRF-5 or IRF-7, both of which were shown to be components of the IFNA
enhanceosome assembled on the VRE of IFNA genes (19,28). The chromatin
precipitation assay has permitted the detection of these enhanceosomes in liv-
ing cells. IRF-5 or IRF-7 expression in infected cells, unable to express IFNA
genes, restored the expression of a number of IFNA genes and IFN-α synthesis
(18,29). In most of the cells, expression of IRF-7 can be induced by interferon
induced transcriptional factor ISGF3 (30). Type I IFN genes can be therefore
generally divided into two groups: immediate-response genes, represented by
IFNB, which requires only IRF-3 for its induction and is therefore rapidly
induced in most infected cells, and late IFNA genes, which require IFN-acti-
vated IRF-5 or IRF-7. The fact that IFNB-null mice are unable to synthesize
IFN-α supports the dependence of IFNA expression on IFNB and the hypoth-
esis of a positive feedback operation in interferon mediated antiviral response
(31,32). However, the recently developed quantitative RT-PCR analysis of
RNA transcripts, as well as the sensitive detection of proteins by intracellular
immune staining, have shown that the high IFN-α-producing pDC2 cells, con-
sidered to be natural IFN-producing cells, express high levels of IRF-7 consti-
tutively in the absence of IFN synthesis (33). Thus the requirement for IFN-β
synthesis may not apply to these cells.
The discovery of toll receptors (TLRs) and their role in the innate immune
response has brought further unexpected findings. Three of these TLRs, TLR-
3, TLR-7, and TLR-9, are intracellular and double stranded RNA (dsRNA),
single-stranded RNA (ssRNA), and CpGDNA, respectively, are their ligands.
Furthermore, binding of the dsRNA to TLR-3 activates TBK-1 and results in
phosphorylation of IRF-3 and IRF-7 and the induction of type I IFNs. In con-
trast, TLR7 and TLR9 activate IRF-5 and IRF-7 but not IRF-3 (60,61).
It is noteworthy that synthesis of IFN-β also can be induced by the binding
of lipopolysaccharide to TLR-4 and that the induction proceeds through acti-
vation of TBK-1, and activation of IRF-3 and IRF-7 (34). These results indi-
cate that although the initial recognition of the infectious entity may be distinct,
the cellular response to bacterial or viral infection shows profound similarities.
However, none of these mechanisms could have been unambiguously estab-
lished, without the availability of genetically modified null mice with various
components of the TLR-mediated signaling pathway deleted.
Experiments with genetically modified mice have also indicated a role for
the members of the IRF family in the antiviral immune response. Thus, tar-
geted disruption of IRF-1 results in an increased sensitivity to viral infection, a
defect in the development of TH-1 responses and a resistance to apoptosis.
Interferon Research 29
IRF-4-null mice have a defect in both T- and B-cell maturation and, conse-
quently, defective immune functions (35). IRF-8-null mice show an increased
sensitivity to viral infection and a defect in the development of myeloid cells
and pDC2 subtype of dendritic cells that are high IFN-producing cells (36,37).
IRF-5-null mice show a profound defect in CPG DNA mediated responses (62).
Furthermore, because that the IRF-5 is a component of the p53-mediated
growth inhibitory and pro-apoptotic pathway (38) and, thus, a recently observed
antiviral activity of p53 may be mediated by IRF-5 (39).
5. IFN Receptors
Cellular receptors for type I IFNs and IFN-γ belong to the class 2 cytokine
receptor subfamily. In recent years, these receptors and the signaling pathways
they induce have been elucidated (40–42).
With varying degrees of avidity, all type I IFNs bind to the same receptor
made of two subunits, IFNAR1 and IFNAR2, of which there are a short and a
long variant, the result of differential mRNA splicing. IFN-α or INF-β induces
the association of IFNAR1 with the long variant of IFNAR2 and initiate a sig-
naling pathway involving the tyrosine kinases Tyk2, Jak1, and the ultimate
migration of activated transcription activators signal transducer and activator
of transcription (STAT)-1 and STAT-2 to the nucleus, where they bind together
with IRF-9 to a specific sequences (i.e., IFN-stimulated response elements)
within the promoters of IFN-stimulated genes (ISGs) and initiate their tran-
scriptional activation (43,44). The IFN-γ receptor also comprises two subunits
and the signaling pathway with which it is associated, involves Jak1, Jak2, and
STAT-1. Activated STAT-1 homodimers translocate to the nucleus and bind to
the γ-IFN activation sequence, culminating in the transcriptional activation of
specific genes (11).
Infection of genetically modified mice in which type I IFNR or IFNgR recep-
tor or critical component of the IFN signaling pathways had been deleted has
shown a central but not redundant role for type I and II IFNs in the host
response to infection. Thus, elimination of type I IFNR increases sensitivity
to infection by number of RNA viruses, whereas these mice are still resistant to
some bacterial infections (45,46). However, IFNGR-null mice show increased
sensitivity to microbial infections, as well as infection with some DNA viruses
such as HSV-1 and vaccinia (47).
6. IFN-Stimulated Genes
Although IFNs were initially identified by their antiviral properties, it was
recognized early on that the actual effector was not IFN itself, but one or sev-
eral proteins induced by IFN. Recently, microarray analysis of the cellular tran-
scripts induced in cells treated with IFN has estimated that IFN stimulates more
30 Kunzi and Pitha
than 300 ISGs with homology to genes involved in signaling, host defense,
immune modulation, transcription, translation, apoptosis, cell adhesion, anti-
viral and inflammatory responses, ubiquitination, and antigen processing
(48,49).
Not surprisingly, the most studied ISGs have been those with antiviral prop-
erties. The enzymes of the 2,5-oligosynthetase family (OAS-1 and OAS-2)
catalyze the synthesis of short oligoadenylates, which binds and activate
RNAseL, an enzyme that cleaves viral and cellular RNAs, thus inhibiting pro-
tein synthesis (50). DsRNA-activated protein kinase (PKR) phosphorylates the
translation initiation factor eIF2a, also resulting in the inhibition of viral and
cellular protein syntheses (51). More recently, PKR also was found to be
required for the activation of the transcription factor NFκB, a central actor in
inflammatory cytokine induction, immune modulation, and apoptosis (52). Mx
proteins are GTPases and this intrinsic activity is required for antiviral effect
(53). Mx proteins inhibit the replication of RNA viruses by either preventing
transport of viral particles within the cell, or transcription of viral RNA (54).
Another very interesting ISG is the RNA-editing adenosine deaminase that
converts adenosine to inosine, thus causing hypermutation of viral RNA genomes,
such as those of VSV and measles virus (55,56).
A number of ISGs encode chemokines such as interleukin-8 and monokine
induced by IFN-γ (Mig), which are involved in lymphocyte recruitment to the
site of infection and inflammation and the expression of genes encoding adhe-
sion molecules, such as ICAM-1 and CD-47, which are crucial for the ability
of leukocytes to adhere to, infected cells. Other ISGs encode transcription fac-
tors, most of them activators of transcription. ISG-15 is an ubiquitin-like pro-
tein, conjugated to cellular proteins and has been shown to target Jak-1, STAT-1,
and extracellular signal-regulated kinase-1 (57,58).
7. Clinical Uses of IFN
Recombinant IFN-α (rIFN-α; Roferon A; Intron A) and recently its
pegylated form (Pegasys), either alone or in combination with an antiviral
agent, are used in the treatment of chronic hepatitis C virus infection. Because
a number of ISGs are shown to have pro-apoptotic characteristics, there is also
a renewed interest in using IFN in the clinic to control malignancies. In the
past, Roeferon A–rIFNα has been used in the treatment of malignant melano-
mas, Kaposi’s sarcoma, genital warts, and hairy cell leukemia (59). Avonex
(IFN-β), produced in hamster cells, remains an essential element in the treatment
of multiple sclerosis (MS). Peripheral blood mononuclear cells isolated from
patients with active MS show decreased sensitivity to type I IFNs, decreased
ISG expression, and hypophosphorylation of STAT-1. In vitro treatment of
these cells with IFN-β overcomes these defects, thus suggesting that IFN-β
therapy may serve to restore normal levels of ISG expression in active MS.
Interferon Research 31
One should be mindful to remember however, that IFN therapy is accompa-
nied with burdensome side effects, presumably because of the large scope of
biological processes influenced by IFN and that, at best, it has been able so far
to only forestall but not halt the progression of the diseases mentioned here.
8. Conclusion
IFNs were the first early inflammatory proteins recognized to be produce in
cells in the response to viral infection. The characterization of IFN genes, their
regulation and functions, facilitated by the newly emerging techniques of molecu-
lar biology, opened a new insight into our understanding of the basic mecha-
nisms involved in the virus cells interaction and in the innate antiviral response.
The availability of genetically modified mice allowed them to study in vivo the
role of the IFN system in the antiviral response. These studies have revealed
the importance of the IFN system not only for the innate, but also for the ac-
quired immunity and pointed out to the existence of the cross talk between inter-
feron system and other cytokines. Furthermore, it has become obvious that the
role of IFN is not limited to the antiviral response, but that the IFN system
plays an important role in the regulation of cell growth, apoptosis, and matura-
tion of lymphoid cells. Understanding the mechanisms of the cellular effects of
IFNs and their interaction with other cytokines may also provide more realistic
approach to the clinical use of IFNs.
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... Interferon (IFN) jest cytokiną zaangażowaną w immunologiczną odpowiedź organizmu na miejscowym i ogólnoustrojowym poziomie oraz uczestniczącą w ograniczaniu zakażenia wirusowego (3,6,9,11,25). IFN został opisany w 1957 przez Issacsa i Lindemanna jako czynnik, który interferuje z replikacją wirusów, jednakże prawdopodobny protekcyjny wpływ interferonu na zakażenie wirusem opryszczki opisano już w latach 30 XX wieku jako tzw. ...
... IFN został opisany w 1957 przez Issacsa i Lindemanna jako czynnik, który interferuje z replikacją wirusów, jednakże prawdopodobny protekcyjny wpływ interferonu na zakażenie wirusem opryszczki opisano już w latach 30 XX wieku jako tzw. fenomen Margassiego (2,9,11). Otrzymany metodami inżynierii genetycznej rekombinowany IFN-α wykorzystywany jest w terapii adiuwantowej czerniaka złośliwego oraz innych chorób rozrostowych, a także w zakażeniu wirusem brodawczaka ludzkiego, w leukoplakii włochatej i w niektórych chorobach autoimmunizacyjnych. ...
... Otrzymany metodami inżynierii genetycznej rekombinowany IFN-α wykorzystywany jest w terapii adiuwantowej czerniaka złośliwego oraz innych chorób rozrostowych, a także w zakażeniu wirusem brodawczaka ludzkiego, w leukoplakii włochatej i w niektórych chorobach autoimmunizacyjnych. Jego forma pegylowana stosowana jest w skojarzeniu z innymi lekami w leczeniu wirusowego przewlekłego zapalenia wątroby (1,7,9,11). Wytwarzany przez komórki układu odpornościowego IFN-α wykazuje plejotropowe działanie przeciwwirusowe. ...
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... The approval paved the way for the commercial production of the drugs. In fact, the worldwide annual sales of the prescribed IFN-cytokine products recently valued well over US$1 billion (Kunzi & Pitha, 2005). Considering the important role of IFNs for therapeutics and their values, it is therefore highly significant to discover the functions of the IFN-stimulatory gene products in the hope of categorizing extra pathways that will facilitate our understanding of the important biological events influenced by IFNs or IFN-like molecules in various organisms. ...
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The wide exploration of interferon (IFN)s in vertebrates for medical purposes has attracted researchers to investigate the existence of a similar role of interferon in other organisms such as invertebrates, including insects, and crustacea. A review of the literature indicates that there is no evidence of interferon existing either in insects such as D. melanogaster and A. gambiae which have had their genomes fully sequenced or in crustacea. However, a nonspecific antiviral state in crustacean, such as P. monodon can be efficiently triggered by both dsRNA and siRNA. The evidence suggests that anonymous cytokines, similar to interferon and not identical to any vertebrate IFNs, related to antiviral protection, do exist in crustacea. However, how widely spread of interferon immune response inducer or interferon-like molecules in this group is an important issue that remains to be explored.
... It is also not available specific prevention. Therefore, when confirmed in vivo, demonstrated in an antiviral study of co-administered interferon and isoprinosine, it is possible to include such a combination in controlling infections viral 33 . ...
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Isoprinosine is a combination of inosine used as antiviral drug without effect on viral particle itself, but instead only and acts as on immunostimulant and also acts indirectly by activation of immune cells. Aim of this study was to determine level of interferon-alpha (INF-α) with parainfluenza viruses HPIV-2, and adenoviruses HAdV-2 replication. In the present study, cytotoxic effect of isoprinosine was assessed using A549 cell line exposed to different concentrations of compound (isoprinosine: 50-800μg/mL) for 48 hours. Cytotoxic effect was examined visually using light, inverted microscopy Olympus CK2 under 400x magnification and by the MTT colorimetric assay. The yield re­duction assay (YRA), which evaluates the ability of the isoprinosine (50-800 μg/mL) to inhibit virus multiplication in cell cultures, was applied. The cytopathic effect of the virus was evaluated 48 h after infection of A549 cell cultures with viruses by means of light, inverted microsco­py. The YRA method was used to determine the 50% end point (IC50) in the presence of Isoprinosine with the controlled one. MTT cytotoxicity assay confirmed microscopic observations. There were no morphological changes, as assessed visually, in cell cultures treated with isoprinosine. After conducting the experi­ments and analyzing the results we noticed that higher concentrations of isoprinosine strongly inhibited multiplication of all viruses. HPIV-2 and HAdV-2 showed the highest sensitivity to the antiviral activity of isoprinosine as compared with the control, however, increasing concentrations of isoprinosine up to 800 μg /ml slightly enhanced the antiviral activity of 400 μg/ml isoprinosine. Our study was conducted that HAdV-2 and HPIV-2 have the highest sensitivity to the antiviral activity of isoprinosine from all tested viral strains.
... However, no natural virus infection has been documented in C. elegans 69 . By contrast, mammals may not use the RNAi pathway to respond to viral infection, having evolved an elaborate, protein-based immune system [70][71][72] . ...
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Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNAs have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate. Historically, siRNAs were believed to arise only from exogenous double-stranded RNA triggers in organisms lacking RNA-dependent RNA polymerases. However, the discovery of endogenous siRNAs in flies expanded the biological significance of siRNAs beyond viral defense. By high throughput sequencing we identified Drosophila endosiRNAs as 21 nt small RNAs, bearing a 2´-O-methyl group at their 3´ ends, and depleted in dicer-2 mutants. Methylation of small RNAs at the 3´ end in the soma, is a consequence of assembly into a mature Argonaute2-RNA induced silencing complex. In addition to endo-siRNAs, we observed certain miRNAs or their miRNA* partners loading into Argonaute2. We discovered, that irrespective of its biogenesis, a miRNA duplex can load into either Argonaute (Ago1 or Ago2), contingent on its structural and sequence features, followed by assignment of one of the strands in the duplex as the functional or guide strand. Usually the miRNA strand is selected as the guide in complex with Ago1 and miRNA* strand with Ago2. In our efforts towards finding 3´ modified small RNAs in the fly soma, we also discovered 24-28nt small RNAs in certain fly genotypes, particularly ago2 and dcr-2 mutants. 24-28nt small RNAs share many features with piRNAs present in the germline, and a significant fraction of the 24-28nt small RNAs originate from similar transposon clusters as somatic endo-siRNAs. Therefore the same RNA can potentially act as a precursor for both endo-siRNA and piRNA-like small RNA biogenesis. We are analyzing the genomic regions that spawn somatic small RNAs in order to understand the triggers for their production. Ultimately, we want to attain insight into the underlying complexity that interconnects these small RNA pathways. Dysregulation of small RNAs leads to defects in germline development, organogenesis, cell growth and differentiation. This thesis research provides vital insight into the network of interactions that fine-tune the small RNA pathways. Understanding the flow of information between the small RNA pathways, a great deal of which has been revealed only in the recent years, will help us comprehend how the pathways compete and collaborate with each other, enabling each other’s optimum function.
... IFNs (interferons) are a family of proteins which mediate antiviral, antiproliferative and immunomodulatory activities in response to viral infections or other biological inducers [1][2][3][4]. The three major classes of interferons are constituted by IFN-α, IFN-β and IFN-γ (Swiss-Prot accession numbers P01562, P01574 and P01579 respectively). ...
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Human beta-interferon is used extensively as a therapeutic agent in a wide variety of diseases, ranging from multiple sclerosis to viral infections. At present, the most common source of interferon-beta is derived from CHO (Chinese-hamster ovary) cells. Interestingly, however, the IFNB gene is characterized by a lack of intronic sequences and therefore does not undergo splicing during its expression pathway. As nuclear processing of pre-mRNA molecules has often been demonstrated to improve production yields of recombinant molecules, we have inserted a heterologous intronic sequence at different positions within the IFNB gene and analysed its effects on protein production. The results obtained in the present study show that the position of intron insertion has profound effects on the expression levels of the IFNB gene and on the nuclear/cytoplasm distribution levels of its mRNA as determined by FISH (fluorescent in situ hybridization) analysis of stably transfected clones. In conclusion, our results provide additional evidence that insertion of intronic sequences may be used to improve protein expression efficiency also in molecules that do not normally undergo any splicing process.
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Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats.
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Swift elimination of undesirable cells is an important feature in tumour suppression and immunity. The tumour suppressor p53 and interferon- and - (IFN-/) are essential for the induction of apoptosis in cancerous cells and in antiviral immune responses, respectively, but little is known about their interrelationship. Here we show that transcription of the p53 gene is induced by IFN-/, accompanied by an increase in p53 protein level. IFN-/ signalling itself does not activate p53; rather, it contributes to boosting p53 responses to stress signals. We show examples in which p53 gene induction by IFN-/ contributes to tumour suppression. Furthermore, we show that p53 is activated in virally infected cells to evoke an apoptotic response and that p53 is critical for antiviral defence of the host. Our study reveals a hitherto unrecognized link between p53 and IFN-/ in tumour suppression and antiviral immunity, which may have therapeutic implications.
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Translation of messenger RNA isolated from poly(rI)-poly(rC)-induced human fibroblasts in cell-free ribosomal systems and in Xenopus oocytes resulted in the production of biologically active proteins that had the properties of human fibroblast interferon. The translation in the oocytes was much more efficient, giving approximately 500 times higher titers of interferon activity than the cell-free systems. A control messenger RNA isolated from noninduced human fibroblasts, did not code for interferon synthesis in these systems. Both messenger RNA preparations stimulated [3H]amino-acid incorporation into trichloroacetic acid-insoluble material. The radioactive products and their immunoprecipitates were electrophoresed on polyacrylamide gels under denaturing conditions. The products resulting from the translation of the control (uninduced) messenger RNA in oocytes contained a major protein of approximately 45,000 molecular weight. The messenger RNA isolated from poly(rI)-poly(rC)-induced cells stimulated the synthesis of an additional 25,000 molecular weight protein that electrophoresed in the same position as human fibroblast interferon. These results suggest that human fibroblast interferon was synthesized by the translation of its messenger RNA in Xenopus oocytes and in cell-free ribosomal systems.
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Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor.
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During a study of the interference produced by heat-inactivated influenza virus with the growth of live virus in fragments of chick chorio-allantoic membrane it was found that following incubation of heated virus with membrane a new factor was released. This factor, recognized by its ability to induce interference in fresh pieces of chorio-allantoic membrane, was called interferon, Following a lag phase interferon was first detected in the membranes after 3 h incubation and thereafter it was released into the surrounding fluid.
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Considerable progress has been made in the understanding of the molecular biology of the human interferon system. The genes encoding the interferons, their receptors, and the proteins that mediate many of their biological effects have been molecularly cloned and characterized. The availability of complete cDNA clones of components of the interferon systems has contributed significantly to our understanding of both the biology and the biochemistry of the antiviral actions of interferons. At the biological level, the antiviral effects of interferon may be viewed to be virus-type nonspecific. That is, treatment of cells with one type or even subspecies of interferon often leads to the generation of an antiviral state effective against a wide array of different RNA and DNA animal viruses. However, at the biochemical level, the antiviral action of interferon is often virus-type selective. That is, the apparent molecular mechanism which is primarily responsible for the inhibition of virus replication may differ considerably between virus types, and even host cells. For example, the IFN-regulated Mx protein selectively inhibits influenza virus but not other viruses when constitutively expressed in mouse cells. The IFN-regulated 2',5'-oligoadenylate synthetase selectively inhibits EMC and mengo viruses, two picornaviruses, but not viruses of other families when constitutively expressed in transfected cells. Some viruses are typically insensitive to the antiviral effects of interferon, both in cell culture and in intact animals. This lack of sensitivity to IFN may result from a virus-mediated direct antagonism of the interferon system. For example, in the case of adenovirus, the activation of the IFN-regulated RNA-dependent P1/elF-2 protein kinase is blocked by the virus-associated VA RNA. The relative sensitivity to interferon of different animal viruses varies appreciably. All three of the basic components required to measure an antiviral response may play a role in determining the relative effectiveness of the antiviral response: the species of interferon administered; the kind of cell treated; and, the type of virus used to challenge the interferon-treated host cell. Thus, the relative sensitivity to interferon observed for a particular interferon-cell-virus combination is likely the result of the equilibrium between the many agonists and antagonists which contribute to the overall response. That is, the relative sensitivity of a virus to the inhibitory action of IFN is governed by the qualitative nature and quantitative amount of the individual IFN-regulated cell proteins that may collectively contribute to the inhibition of virus replication.(ABSTRACT TRUNCATED AT 400 WORDS)