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An antiviral substance from Penicillium funiculosum
Abstract
Helenine exerts a therapeutic effect against Semliki Forest virus infections of mice. Cures, that is to say the survival of treated animals, were more frequently observed in Semliki Forest virus infections than they were in SK virus infections. It is believed that this difference in end-result probably represented only a quantitative difference in the therapeutic effect of helenine against these two viruses and not a qualitative difference in its mechanism of therapeutic action. The findings reported in this paper with regard to the treatment of Semliki Forest virus infections with helenine parallel very closely those described in an accompanying paper which deals with the action of helenine on SK. virus infections.
... Calling the extracts of the fungus " Helenine " , Shope found that these could induce an interferon response in animals. [13] A next chapter in this early story was carried out by Maurice Hillemans group at Merck, who used Shopes fungus as a starting point to purify the material that was actually responsible for the viral resistance. In a paper published in 1967, [14] they showed that double-stranded RNA was present in the fungal extracts and was responsible for the induction of resistance. ...
I would like to thank the Nobel Assembly of the Karolinska Institute for the opportunity to describe some recent work on RNA-triggered gene silencing. First a few disclaimers, however. Telling the full story of gene silencing would be a mammoth enterprise that would take me many years to write and would take you well into the night to read. So we'll need to abbreviate the story more than a little. Second (and as you will see) we are only in the dawn of our knowledge; so consider the following to be primer … the best we could do as of December 8th 2006. And third, please understand that the story that I am telling represents the work of several generations of biologists, chemists, and many shades in between. I'm pleased and proud that work from my laboratory has contributed to the field, and that this has led to my being chosen as one of the messengers to relay the story in this forum. At the same time, I hope that there will be no confusion of equating our modest contributions with those of the much grander RNAi enterprise.
... His most notable success came from a fungus (Penicillium funiculosum) that he found in Guam growing on a picture of his wife Helen. Calling the extracts of the fungus " Helenine " , Shope found that these could induce an interferon response in animals [13]. A next chapter in this early story was carried out by Maurice Hilleman's group at Merck, who used Shope's fungus as a starting point to purify the material that was actually responsible for the viral resistance. ...
The December 2007 article labelled as Dr. Andrew Fire's Nobel Lecture "Gene Silencing By Double Stranded RNA" was an abridged version that was prepared and published by Cell Death and Differentiation with the consent of the Nobel Foundation but without Dr. Fire's knowledge or consent. The Nobel Foundation did not explicitly give permission for it to be edited, and the editors have withdrawn the abridged article from the Cell Death and Differentiation archive. Readers can download the original and complete version of Dr. Fire's Nobel Lecture at the Nobel Foundation website: http://nobelprize.org/nobel_prizes/medicine/laureates/
2006/fire-lecture.html
Helenine, a substance obtained from broth cultures of Penicillium funiculosum and known to exert a protective effect in vivo in experimental animals against several unrelated viruses, has been shown to elicit the formation in cell cultures and in intact mice of an inhibitor of viral plaque formation. Because the biological and chemical characteristics of the viral inhibitor induced by helenine are similar to those of interferon, it is suggested that the antiviral effect of helenine may be mediated through the formation of interferon.
1. Helenine injected intraperitoneally 24 hr prior to a regularly fatal dose of Semliki Forest virus saves most of the mice to which it is administered. 2. Mice saved by helenine develop no viral immunity and regularly succumb when rechallenged 2 wk later with the same dose of virus from which they were originally saved. 3. The time during which helenine is optimally effective in protecting mice from death by Semliki Forest virus covers a period of approximately 36 hr beginning after about 12 hr and extending to 48 hr before virus infection. When periods of less than 12 hr, or more than 48 hr, elapse between the time of helenine administration and virus inoculation, its protective effectiveness diminishes progressively. 4. Repeated injections of helenine at 2- or 3-day intervals, if continued long enough, exhaust the capacity of a host to respond favorably to helenine administered 24 hr before virus inoculation. 5. Helenine injections at intervals of 4, 3, and 2 wk before its administration 24 hr prior to infection do not decrease the effectiveness of this final dose in protecting mice from fatal infection by the virus. The experimental results here reported indicate that, as suggested by the findings of earlier work, helenine does not act directly as an antiviral substance, but instead exerts its effect through some substance that it induces the host to elaborate. The nature of this induced antiviral substance is as yet unknown though, to judge from the failure of spared mice to acquire viral immunity, it appears to act at a stage in viral replication prior to that at which antigenic viral protein is produced. The findings with helenine and those thus far reported for interferon afford no factual basis for judging the relationship of the two, if any.
1. Helenine prevents the establishment in mice of passive viral immunity by anti-Semliki serum of swine, rabbit, or guinea pig origin. 2. A period of 12 days must elapse, between the antiviral serum administration and challenge with virus, for prevention of the establishment of passive immunity to become apparent. This period is believed to correspond to that in which injected antibody persists in circulation in the injected host. 3. Helenine is effective in preventing the establishment of passive viral immunity by heterologous antiviral sera when it is administered any time during a period of 6 days, extending from 4 days before to 2 days after injection of the antiviral serum. 4. Helenine does not prevent the establishment of passive viral immunity by antiviral sera of mouse origin (homologous). 5. Evidence is presented to indicate that the phenomenon of the prevention of the establishment of passive viral immunity by heterologous antiviral sera is not effected directly, but rather is mediated through some substance that helenine induces the injected host to elaborate. 6. The capacity to prevent the establishment of passive viral immunity could not be exhausted by repeated preceding injections of helenine at 2- or 3-day intervals. 7. Evidence is presented to indicate that the helenine-induced material does not act upon antiviral antibody per se but rather on heterologous foreign protein that happens to be labeled as Semliki Forest virus antibody. This helenine-induced material, whatever its nature, appears to enhance the capacity of the injected host to recognize and dispose of foreign protein. 8. Statolon, a material that like helenine is a known inducer of interferon, is, like helenine, also capable of preventing the establishment of passive viral immunity by heterologous antiviral sera. 9. Experiments designed to determine whether the induced material responsible for the antipassive immunity effect of helenine is interferon have yielded inconclusive answers thus far.
Two new oxaphenalenone dimers, talaromycesone A (1) and talaromycesone B (2), and a new isopentenyl xanthenone, talaroxanthenone (3), together with six known diphenyl ether derivatives, e.g., Δ(1',3'),-1'-dehydroxypenicillide (4), 1',2'-dehydropenicillide (5), vermixocin A (6), vermixocin B (7), 3'-methoxy-1'2'-dehydropenicillide (8), and AS-186c (9), were isolated from the culture broth and mycelia of a marine fungus Talaromyces sp. strain LF458. Compound 2 represents the first example of 1-nor oxaphenalenone dimer carbon skeleton. All isolated compounds were subjected to bioactivity assays. Compounds 1, 2, and 9 exhibited potent antibacterial activities with IC50 3.70, 17.36, and 1.34 μM, respectively, against human pathogenic Staphylococcus strains. Compounds 1, 3, and 9 displayed potent acetylcholinesterase inhibitory activities with IC50 7.49, 1.61, and 2.60 μM, respectively. Interestingly, phosphodiesterase PDE-4B2 was inhibited by compounds 3 (IC50 7.25 μM) and 9 (IC50 2.63 μM).
Ich danke der Nobelversammlung des Karolinska-Instituts für die Gelegenheit, einige neuere Arbeiten zur RNA-aktivierten Gen-Stummschaltung vorstellen zu können. Eines muss ich aber vorausschicken: Die gesamte Geschichte der Gen-Stummschaltung zu erzählen, wäre ein gigantisches Unternehmen, das den Rahmen dieses Vortrags weit übersteigen würde. Es wird daher nicht zu vermeiden sein, dass ich die Geschichte mehr als nur ein bisschen verkürze. Des Weiteren befinden sich unsere Kenntnisse der Gen-Stummschaltung noch in den Anfängen, weshalb Sie den Vortrag nur als Einführung in das Thema betrachten sollten – so gut es eben geht an diesem 8. Dezember 2006. Als drittes sollten Sie wissen, dass die Geschichte, die ich erzählen werde, die Arbeiten mehrerer Generationen von Biologen und Chemikern (und sämtlichen Schattierungen dazwischen) zusammenfasst. Ich bin geschmeichelt, dass Arbeiten meiner Forschungsgruppe zu diesem Feld beitragen konnten und dazu geführt haben, dass ich nun hier über diese Entwicklungen berichten darf. Gleichzeitig hoffe ich, dass man unsere bescheidenen Beiträge nicht mit den sehr viel größeren Errungenschaften der RNAi-Forschungen gleichsetzt.
Picornaviruses are among the best understood animal viruses in molecular terms. A number of important human and animal pathogens are members of the Picornaviridae family. The genome organization, the different steps of picornavirus growth and numerous compounds that have been reported as inhibitors of picornavirus functions are reviewed. The picornavirus particles and several agents that interact with them have been solved at atomic resolution, leading to computer-assisted drug design. Picornavirus inhibitors are useful in aiding a better understanding of picornavirus biology. In addition, some of them are promising therapeutic agents. Clinical efficacy of agents that bind to picornavirus particles has already been demonstrated.
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2
A material capable of rendering mice resistant to Semliki Forest virus is present in the livers but not in the muscles, brains, or spleens of mice that have survived a usually fatal dose of virus.
This review article deals with the following items: (1) distribution and discovery of fungal viruses; (2) structure and biochemistry; (3) replication; (4) virulence; and (5) transmission of fungal viruses. The literature list mentions 223 references.
A single intraperitoneal injection of statolon was shown to exert a therapeutic effect on mice previously injected with the large plaque-forming variant of mengovirus. The ld(50) and survival time data demonstrated that such treatment was effective when given 2 to 48 hr after infection. No protective effect was apparent when statolon was administered 3, 4, or 5 days after the viral challenge. It was concluded that statolon, or other similar interferon inducers, may be of therapeutic value in instances of accidental or other known exposure to hazardous viral agents.
The effect of statolon-induced interferon on immune response was investigated. Young adult Swiss albino mice were immunized with chicken red blood cells or infectious mengovirus. The red blood cells were injected three times and the virus twice at 2-week intervals, with or without treatment with statolon 24 hr before the inoculations. The effect of interferon on antibody formation was measured by determinations of hemagglutinating, hemolytic, and virus-neutralizing antibodies in the sera of treated and nontreated groups. The hemagglutinin and hemolysin titers after each successive inoculation were equal in both groups, indicating that interferon had no effect on their production. A decrease in the primary response to the viral antigen was associated with interferon induction; the booster response was unaffected. The possible use of interferon inducers as attenuants for immunization with live virulent viruses is discussed.
A polyhedral virus has been found in a strain of Penicillium cyaneo-fulvum. Double-stranded RNA isolated from the virus was active in stimulating interferon production.
INTERFERON inducers seem to be the most practical way of making use of interferons in clinical practice. Helenine1 obtained from a fungus Penicillium funiculosum is a powerful interferon inducer and Lampson et al.2 found that the activity was present in a fraction consisting of double stranded RNA. Other double stranded RNA inducers have been discovered, for example, that from Escherichia coli infected with RNA bacteria virus MS2 (ref. 3) and also certain copolymers of synthetic polyribonucleotides.
1. Helenine injected intraperitoneally 24 hr prior to a regularly fatal dose of Semliki Forest virus saves most of the mice to which it is administered.
2. Mice saved by helenine develop no viral immunity and regularly succumb when rechallenged 2 wk later with the same dose of virus from which they were originally saved.
3. The time during which helenine is optimally effective in protecting mice from death by Semliki Forest virus covers a period of approximately 36 hr beginning after about 12 hr and extending to 48 hr before virus infection. When periods of less than 12 hr, or more than 48 hr, elapse between the time of helenine administration and virus inoculation, its protective effectiveness diminishes progressively.
4. Repeated injections of helenine at 2- or 3-day intervals, if continued long enough, exhaust the capacity of a host to respond favorably to helenine administered 24 hr before virus inoculation.
5. Helenine injections at intervals of 4, 3, and 2 wk before its administration 24 hr prior to infection do not decrease the effectiveness of this final dose in protecting mice from fatal infection by the virus.
The experimental results here reported indicate that, as suggested by the findings of earlier work, helenine does not act directly as an antiviral substance, but instead exerts its effect through some substance that it induces the host to elaborate. The nature of this induced antiviral substance is as yet unknown though, to judge from the failure of spared mice to acquire viral immunity, it appears to act at a stage in viral replication prior to that at which antigenic viral protein is produced.
The findings with helenine and those thus far reported for interferon afford no factual basis for judging the relationship of the two, if any.
Helenine, a substance obtained from broth cultures of Penicillium funiculosum and known to exert a protective effect in vivo in experimental animals against several unrelated viruses, has been shown to elicit the formation in cell cultures and in intact mice of an inhibitor of viral plaque formation.
Because the biological and chemical characteristics of the viral inhibitor induced by helenine are similar to those of interferon, it is suggested that the antiviral effect of helenine may be mediated through the formation of interferon.
The present findings indicate that the wide fluctuation observed in the antiviral activity of various crude helenine preparations may be attributable to the presence of varying amounts of inhibitor. Antiviral activity could be enhanced by removal of the inhibitor. A meaningful value for helenine titer in a preparation clearly should be determined in the inhibitor-free zone.
1. Helenine prevents the establishment in mice of passive viral immunity by anti-Semliki serum of swine, rabbit, or guinea pig origin.
2. A period of 12 days must elapse, between the antiviral serum administration and challenge with virus, for prevention of the establishment of passive immunity to become apparent. This period is believed to correspond to that in which injected antibody persists in circulation in the injected host.
3. Helenine is effective in preventing the establishment of passive viral immunity by heterologous antiviral sera when it is administered any time during a period of 6 days, extending from 4 days before to 2 days after injection of the antiviral serum.
4. Helenine does not prevent the establishment of passive viral immunity by antiviral sera of mouse origin (homologous).
5. Evidence is presented to indicate that the phenomenon of the prevention of the establishment of passive viral immunity by heterologous antiviral sera is not effected directly, but rather is mediated through some substance that helenine induces the injected host to elaborate.
6. The capacity to prevent the establishment of passive viral immunity could not be exhausted by repeated preceding injections of helenine at 2- or 3-day intervals.
7. Evidence is presented to indicate that the helenine-induced material does not act upon antiviral antibody per se but rather on heterologous foreign protein that happens to be labeled as Semliki Forest virus antibody. This helenine-induced material, whatever its nature, appears to enhance the capacity of the injected host to recognize and dispose of foreign protein.
8. Statolon, a material that like helenine is a known inducer of interferon, is, like helenine, also capable of preventing the establishment of passive viral immunity by heterologous antiviral sera.
9. Experiments designed to determine whether the induced material responsible for the antipassive immunity effect of helenine is interferon have yielded inconclusive answers thus far.
Helenine is moderately stable in solution at refrigerator temperature and can be kept for long periods of time without evident loss of activity if stored frozen at the temperature of solid CO2. It is filterable through a Seitz pad but not dialyzable. Crude SPS preparations of helenine do not lose activity when dried from the frozen state. Some conditions are described, however, which influence the preservation or inactivation of acetone-precipitated helenine when freeze-dried. Helenine is partially inactivated by exposure for 3 minutes to the temperature of a boiling water bath and is completely inactivated by autoclaving at 15 pounds' pressure for 15 minutes.
The data presented suggest that helenine acts, either directly or by triggering some mechanism of the host itself, to destroy virus by a process which renders the latter non-antigenic. This effect may be exerted by action upon the virus itself or by interference with some stage in the developmental cycle of the virus.
While the chemical nature of helenine is not known, the presence of a large proportion of polysaccharide in crude active preparations might suggest the possible importance of this class of substance in helenine activity. It is believed that helenine differs from the polysaccharide reported by Horsfall and McCarty and the penicillin impurity reported by Groupé and Rake to be active against certain viruses. It may be related, however, to the antiviral substance recently reported by Powell and his co-workers.
Helenine exerts a therapeutic effect against Semliki Forest virus infections of mice. Cures, that is to say the survival of treated animals, were more frequently observed in Semliki Forest virus infections than they were in SK virus infections. It is believed that this difference in end-result probably represented only a quantitative difference in the therapeutic effect of helenine against these two viruses and not a qualitative difference in its mechanism of therapeutic action.
The findings reported in this paper with regard to the treatment of Semliki Forest virus infections with helenine parallel very closely those described in an accompanying paper which deals with the action of helenine on SK. virus infections.
A culture of P. funiculosum isolated on Guam proved capable of elaborating a substance which exerted a favorable therapeutic effect against swine influenza virus infections in white mice. The culture was extremely variable and irregular in its production of the antiviral substance, and during maintenance in the laboratory for several years gradually lost this property. Efforts to restore it were unsuccessful.
Subsequently it was found that the mold elaborated a substance, now designated helenine, which is therapeutically effective against Columbia SK encephalomyelitis virus infections in mice. Helenine appears to differ from the substance earlier procured from the mold, which was active against swine influenza virus infections in mice. It is frequently present in greater or lesser amount in the fluid portions of stationary cultures of P. funiculosum but is more regularly obtained and in larger amount, from the cellular components of the pellicles. When liberated from these latter by mechanical bruising and fracturing, it goes into solution in the culture fluids. It is precipitable from aqueous solution by 50 per cent acetone.
Infected mice injected with helenine in amounts less than the amount which produces a maximal therapeutic effect exhibit a dosage response. Increasing the dose above the optimum fails to increase the therapeutic effect.
Helenine exerts its maximum effect when given within the first 10 hours after viral infection but its influence is apparent even when treatment is delayed for up to 24 hours. It is not effective against massive amounts of virus and gives the best therapeutic results when used in the treatment of animals infected with from 10 to 1000 fatal doses of virus. Treatment of infected mice with helenine delays the entrance of virus into their brains for from 24 to 48 hours.
The mechanism by which helenine exerts its therapeutic effect against SK virus is not known but the findings presented suggest either that it causes an inhibition or interruption of multiplication of the virus, slowing down the whole process of infection and spread to the central nervous system, or that in some way it interferes temporarily with the neuroinvasiveness of the virus.
This historical account covers 50 years of seminal research work on interferon done since its discovery in 1957. Topics related to molecular structure, production and action of interferons are considered from the viewpoint of how our insights have expanded and deepened within the context of evolving tools and general knowledge in cellular and molecular biology. Lines of thought that linked each discovery to the next are expounded.
Helenine is moderately stable in solution at refrigerator temperature and can be kept for long periods of time without evident loss of activity if stored frozen at the temperature of solid CO2. It is filterable through a Seitz pad but not dialyzable. Crude SPS preparations of helenine do not lose activity when dried from the frozen state. Some conditions are described, however, which influence the preservation or inactivation of acetone-precipitated helenine when freeze-dried. Helenine is partially inactivated by exposure for 3 minutes to the temperature of a boiling water bath and is completely inactivated by autoclaving at 15 pounds' pressure for 15 minutes.
The data presented suggest that helenine acts, either directly or by triggering some mechanism of the host itself, to destroy virus by a process which renders the latter non-antigenic. This effect may be exerted by action upon the virus itself or by interference with some stage in the developmental cycle of the virus.
While the chemical nature of helenine is not known, the presence of a large proportion of polysaccharide in crude active preparations might suggest the possible importance of this class of substance in helenine activity. It is believed that helenine differs from the polysaccharide reported by Horsfall and McCarty and the penicillin impurity reported by Groupé and Rake to be active against certain viruses. It may be related, however, to the antiviral substance recently reported by Powell and his co-workers.
Helenine exerts a therapeutic effect against Semliki Forest virus infections of mice. Cures, that is to say the survival of treated animals, were more frequently observed in Semliki Forest virus infections than they were in SK virus infections. It is believed that this difference in end-result probably represented only a quantitative difference in the therapeutic effect of helenine against these two viruses and not a qualitative difference in its mechanism of therapeutic action.
The findings reported in this paper with regard to the treatment of Semliki Forest virus infections with helenine parallel very closely those described in an accompanying paper which deals with the action of helenine on SK. virus infections.
A culture of P. funiculosum isolated on Guam proved capable of elaborating a substance which exerted a favorable therapeutic effect against swine influenza virus infections in white mice. The culture was extremely variable and irregular in its production of the antiviral substance, and during maintenance in the laboratory for several years gradually lost this property. Efforts to restore it were unsuccessful.
Subsequently it was found that the mold elaborated a substance, now designated helenine, which is therapeutically effective against Columbia SK encephalomyelitis virus infections in mice. Helenine appears to differ from the substance earlier procured from the mold, which was active against swine influenza virus infections in mice. It is frequently present in greater or lesser amount in the fluid portions of stationary cultures of P. funiculosum but is more regularly obtained and in larger amount, from the cellular components of the pellicles. When liberated from these latter by mechanical bruising and fracturing, it goes into solution in the culture fluids. It is precipitable from aqueous solution by 50 per cent acetone.
Infected mice injected with helenine in amounts less than the amount which produces a maximal therapeutic effect exhibit a dosage response. Increasing the dose above the optimum fails to increase the therapeutic effect.
Helenine exerts its maximum effect when given within the first 10 hours after viral infection but its influence is apparent even when treatment is delayed for up to 24 hours. It is not effective against massive amounts of virus and gives the best therapeutic results when used in the treatment of animals infected with from 10 to 1000 fatal doses of virus. Treatment of infected mice with helenine delays the entrance of virus into their brains for from 24 to 48 hours.
The mechanism by which helenine exerts its therapeutic effect against SK virus is not known but the findings presented suggest either that it causes an inhibition or interruption of multiplication of the virus, slowing down the whole process of infection and spread to the central nervous system, or that in some way it interferes temporarily with the neuroinvasiveness of the virus.
Evidence is presented which indicates that certain polysaccharide preparations derived from various bacterial species, as well as similar materials not of bacterial origin, are capable of lessening the severity of infection with pneumonia virus of mice (PVM) and inhibiting multiplication of the virus in mouse lungs infected with this agent. It seems probable that modification with respect to the virus is mediated by a substance which may be polysaccharide in nature.
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