Enhancement of the antiviral and interferon-inducing activities of poly r(A-U) by carminic acid

Experiments have been designed to systematically examine the effects of carminic acid (CAR) on the antiviral/interferon-inducing activity of poly r(A-U), using the human foreskin fibroblast-vesicular stomatitis virus bioassay system. Modulation of the antiviral/interferon-inducing activity of poly r(A-U) by carminic acid was examined at fixed poly r(A-U) concentrations of 0.05 mM or 0.2 mM while varying the carminic acid concentrations to produce variable CAR/ribonucleotide ratios ranging from 1:16 to 2:1. Carminic acid and poly r(A-U) were tested individually at the concentrations employed in the CAR/poly r(A-U) combinations. Neither the carminic acid alone nor poly r(A-U) alone were effective antiviral agents/interferon inducers. The antiviral/interferon-inducing activity of poly r(A-U) was potentiated twelve-fold at CAR/ribonucleotide ratios in the region of 1/6 to 1/4. These results suggest a synergism between the poly r(A-U) and the carminic acid at the concentrations employed in this study.     

Polyribonucleotide-anthraquinone interactions: in vitro antiviral activity studies.

Twelve anthraquinones (AQ) were evaluated for their ability to potentiate the antiviral activity of poly r(A-U) using a human foreskin fibroblast-vesicular stomatitis virus bioassay in which the AQ was combined with 0.2 mM poly r(A-U) to produce an AQ/ribonucleotide ratio of 1/4. Poly r(A-U) and the AQ alone were not effective antiviral agents. Five of the twelve AQs tested, mitoxantrone, adriamycin, ametantrone, carminic acid and daunomycin, enhanced the antiviral activity of poly r(A-U) 9- to 13-fold. The interferon-inducing activity of the five active AQ/poly r(A-U) combinations was equal to the sum of the interferon-inducing activities of their constituents. These five AQs appear to potentiate the antiviral activity of poly r(A-U) without superinduction of interferon.     

Potentiation of the antiviral activity of poly r(A-U) by riboflavin, FAD and FMN

The role of riboflavin (RFN), FAD or FMN in modulating the antiviral activity of poly r(A-U) was examined by the human foreskin fibroblast-vesicular stomatitis virus bioassay in which the concentrations of poly r(A-U) was fixed at 0.1 mM or 0.2 mM while the riboflavin, FAD or FMN concentration was varied to produce variable RFN (or FAD or FMN)/ribonucleotide ratios ranging from 1/16 to 2/1. Riboflavin, FAD and FMN tested individually did not exhibit any antiviral activity, while poly r(A-U) alone exhibited antiviral activity. When poly r(A-U) was combined with riboflavin, FAD or FMN, the antiviral activity was potentiated seven- to twelve-fold at RFN (or FAD or FMN)/ribonucleotide ratios in the region of 1/4.     

Potentiation of the antiviral activity of poly r(A-U) by xanthene dyes

Ten xanthene dyes (XAN) are evaluated for their ability to potentiate the antiviral activity of poly r(A-U) using a human foreskin fibroblast-vesicular stomatitis virus bioassay in which the XAN is combined with 0.2 mM poly r(A-U) to produce a XAN/ribonucleotide ratio of 1/4. Four of the ten XANs tested in this study, rhodamine 123, rhodamine B, rhodamine 6G and sulforhodamine B, enhance the antiviral activity of poly r(A-U) 8- to 15-fold. The interferon-inducing activity of the four active XAN/poly r(A-U) combinations is equal to the sum of the activities of their constituents. These four XANs appear to potentiate the antiviral activity of the poly r(A-U) without superinduction of interferon. The direct viral inactivation study demonstrates that the XANs, poly r(A-U) and the XAN/poly r(A-U) combinations do not inactivate the VSV at concentrations near the 50% effective dose.     

Enhanced antiviral activity and altered subcellular distribution of magnesium/poly r(A-U) combinations.

When Mg2+ or ethidium bromide (EB) were combined with poly r(A-U) at a ligand/ribonucleotide ratio of 1/4, the antiviral activity of the Mg2+ and EB increased 136-fold and 154-fold. Eriochrome Blue SE was employed to visualize the subcellular distribution of Mg2+ following co-incubation of Human Foreskin Fibroblasts (HSF) with Mg2+ alone or with the Mg2+/poly r(A-U) combination. Phase contrast micrographs of these Mg(2+)-treated HSF cells as well as phase contrast and fluorescence micrographs of EB-treated or EB/poly r(A-U)-treated HSF cells illustrated that the Mg2+ (or EB)/poly r(A-U) combinations display altered subcellular distribution with the Mg2+ and EB being localized in the nucleoli and chromatin of the HSF cells. These results suggest that modulation of nuclear processes may be responsible for the enhanced antiviral activity.     

Effect of enthidium on the morphology, antiviral activity and subcellular distribution of Poly r(A-U)

When ethidium bromide (EB) is combined with poly r(A-U) at an EB/ribonucleotide ratio of 1/4, the antiviral activity of the EB increases 22-fold. The increased antiviral activity is not due to increased interferon induction, direct viral inactivation or host cell cytotoxicity. Phase contrast, confocal and fluorescence microscopic observations reveal an increase in the nucleolar accumulation of the EB and/or the poly r(A-U) in the EB/poly r(A-U)-treated fibroblasts. Ultrastructure of negatively stained and replica preparations demonstrated that EB-induced condensation of poly r(A-U). These results suggest the elevated antiviral activity may be related to the altered uptake and subcellular distribution of the EB/poly r(A-U) complex.     

Subcellular localization and antiviral activity of carminic acid/poly r(A-U) combinations

Carminic acid (CAR) enhances the antiviral activity of poly r(A-U) twelve-fold without increasing interferon induction, inactivating the vesicular stomatitis virus or inducing host cell cytotoxicity. Phase contrast photomicrographs of human foreskin fibroblasts (HSF) incubated with CAR alone, poly r(A-U) alone or with a CAR/poly r(A-U) combination illustrate that the CAR/poly r(A-U) combinations display altered subcellular distribution with the CAR being localized in the nucleoli and chromatin. Phase contrast and fluorescence photomicrographs of adriamycin (ADR)-treated and ADR/poly r(A-U)-treated HSF cells corroborate these findings. These results suggest that modulation of one or more nucleolar processes may be responsible for the enhanced antiviral activity.     

Antiviral activity of magnesium and magnesium/poly r(A-U) combinations against two RNA viruses.

Magnesium (Mg2+) potentiated the anti-vesicular stomatitis virus (VSV) activity of poly r(A-U) or poly r(G-C) and the anti-HIV-1 activity of poly r(A-U). Mg2+ did not affect the anti-VSV activity of poly (rI).poly (rC), poly (dA-dT).poly (dA-dT) or poly (dG-dC).poly (dG-dC). Modulation of one or more nuclear (nucleolar) processes of the host cell may be responsible for the synergistic antiviral activity.     

Antiviral Activity of Magnesium and Magnesium/poly r(A-U) Combinations Against two RNA Viruses

Abstract

Magnesium (Mg²⁺) potentiated the anti-vesicular stomatitis virus (VSV) activity of poly r(A-U) or poly r(G-C) and the anti-HIV-1 activity of poly r(A-U). Mg²⁺ did not affect the anti-VSV activity of poly (rI) ? poly (rC), poly (dA-dT) ? poly (dA-dT) or poly (dG-dC) ? poly (dG-dC). Modulation of one or more nuclear (nucleolar) processes of the host cell may be responsible for the synergistic antiviral activity.     

Enhanced efficacy of conditionally replicating herpes simplex virus (G207) combined with 5-fluorouracil and surgical resection in peritoneal cancer dissemination models

BACKGROUND: The therapeutic efficacy of G207, a replication-competent herpes simplex virus, for malignancies is increased when combined with certain chemotherapies, but the mechanism is unclear and the interaction between G207 and surgical resection has not been extensively studied. The goals of the current study were to examine the performance of combination treatments for peritoneal disseminated cancers and to explore the mechanism of effective combinations. METHODS: Hamsters and SCID and BALB/c mice harboring peritoneal dissemination of gallbladder, gastric or colon cancer cells were treated with G207, 5-fluorouracil (5FU), or surgical resection alone, or G207 combined with 5FU or surgery. Animal survival, antiviral immunity, intratumoral ribonucleotide reductase activity, and viral spread were compared between the groups. RESULTS: The combination of G207 and 5FU prolonged the survival of hamsters bearing peritoneal dissemination of gallbladder cancer compared with the controls, G207 alone and 5FU alone. 5FU did not suppress the production of neutralizing antibodies against G207, but increased ribonucleotide reductase activity and viral spread in subcutaneous gallbladder tumors. The enhanced efficacy of the combination treatment was also observed in immunodeficient mice with disseminated gastric cancer. Although surgical resection did not significantly prolong animal survival or increase the intratumoral activity of ribonucleotide reductase, long-term survivors emerged from groups of animals treated with surgical resection and G207 for gallbladder and colon disseminated cancers. CONCLUSIONS: These results indicate that the increased activity of ribonucleotide reductase in tumors mediated by 5FU and the decreased tumor burden resulting from surgical resection may enhance the therapeutic efficacy of oncolytic herpes virus for peritoneal disseminated cancer.     

Modification of duplex poly(G).poly(C) by platinum (II) compounds.

The modification of the double-stranded poly(G).poly(C) complex by cis-diamminedichloroplatinum(II) was studied by two modes: the action of cis-DDP on poly(G) before formation of the duplex with poly(C) and that on the prepared duplex. It was shown that in the latter case modification disordered the integrity of the duplex only negligibly at rb less than or equal to 0.05 and led to improved interferon-inducing and antiviral activity tested on mice infected by Influenza and Herpes viruses.     

Biophysical studies of the modification of poly(rG) . poly(rC) by cisplatin. Relations to the biological activity of the complex

The integrity of the double-stranded complex polyriboguanylic.polyribocytidylic acid [poly(rG).poly(rC)] modified by antitumour cis-diamminedichloroplatinum(II)(cis-DDP) was studied with the aid of differential pulse polarography and terbium fluorescence measurement. The modification was made to level corresponding to rb = 0.05 (rb is defined as the number of platinum atoms covalently bound per one nucleotide residue). Two modes of the modification of the polynucleotide complex were employed: The action of cis-DDP on poly(G) before formation of the complex with poly(C) and on the complex already formed from non-modified polynucleotides. It was shown that in the latter case modification disordered the integrity of the complex only negligibly. while in the former case the modification resulted in a noticeably more extensive disturbance of the double-stranded polynucleotide complex. Moreover, the modification of the complex (after its formation) at rb = 0.02 led to improved interferon-inducing and antiviral activity of poly(rG).poly(rC) tested on mice infected by influenza virus. It was suggested that the combined effects of interferon-inducing and antiviral activities of poly(rG).poly(rC) and antiviral activity of cis-DDP may result in an increased effect over and above what may be expected from the actions of the two modalities separately.     

Polynucleotides. XLV Synthesis and properties of poly(2''-azido-2''-deoxyinosinic acid).

Poly (2'-azido-2'-deoxyinosinic acid), [poly (Iz)], was synthesized from 2'-azido-2'-deoxyinosine diphosphate by the action of polynucleotide phosphorylase. Poly (Iz) has UV absorption properties similar to poly (I) and hypochromicity of 11% at 0.15M Na+ and neutrality. In solutions of high Na+ ion concentration, poly (Iz) forms a multi-stranded complex and its Tm at 1.0M Na+ ion concentration was 43 degrees. Upon mixing with poly (C), poly (Iz) forms a 1:1 complex having a Tm lower than that of poly (I)-poly (C) complex in the same conditions. The effect of substitution at the 2'-position of the poly (I) strand was discussed in relation to the interferon-inducing activity.     

Induction of interferon by polyribonucleotides containing thiopyrimidines.

The influence of thioketo substitution in pyrimidine bases of double-stranded polynucleotides on interferon induction was investigated. The stabilizing effect of 2-thioketo substitution was reflected in the increased interferon inducing activity of poly(A-s2U) over that of poly(A-U). Poly(A-s2U) and poly(I)-poly(s2C) were as effective as poly(I)-Poly(C) in rabbit cells. Poly(I)-poly(C) and poly(I)-poly(s2C) were compared in several animal species. No differences in biological effects were observed in rabbits and dogs. In rodents, poly(I)-poly(s2C) was less effective and less toxic.Poly(I)-poly(s2C) was highly resistant against degradation by human serum. Further investigations seem to be justified to elucidate whether this property offers any advantages for the potential clinical utilization of poly(I)-poly(s2C).     

Isolation and characterization of two enzymatic activities from chick embryos which degrade double-stranded RNA

Enzymatic activities capable of degrading double-stranded RNA have been solubilized from whole 9-day-old chick embryos and separated by ion exchange chromatography on DEAE-cellulose into two classes, designated nucleases DI and DII. Nuclease DI exhibits an absolute requirement for Mn2+ in the range of 5 to 10 mM. Monovalent cations, including K+, Na+, and NH4+, are inhibitory. The molecular weight of DI is 60,000 to 62,500 as estimated from sedimentation in sucrose density gradients. Following gradient fractionation, nuclease DI possesses the ability to degrade several substrates exhibiting a 250-fold preference for poly(rC) as compared to poly(rC)-poly(rG). The activity responsible for degrading double-stranded RNA functions as an endonuclease generating oligonucleotides with 5'-phosphate termini. Nuclease DII requires both monovalent and divalent cations. Optimal degradation of poly[r(A-U)] is seen at 75 to 100 mM salt and 0.5 to 1.0 mM MgCl2 or MnCl2. The molecular weight estimated from sucrose gradient sedimentation is in the range of 38,000 to 40,000. Nuclease DII acts endonucleolytically producing oligonucleotides terminating in 5'-phosphates. During the isolation and characterization of nucleases DI and DII, a third activity was detected which degrades single-stranded RNA substrates but which, in the presence of either DII or RNase H, significantly enhances the degradation of poly[r(A-U)] or poly(rA)-poly(dT) substrates.     

Dependence of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen photoaddition on the conformation of ribonucleic acid

The photoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) to different conformational states of RNA was studied. Poly(U), poly(A,U) (random copolymer), poly(A-U) (alternating copolymer), poly(A) . poly(U) (double stranded), and poly(U) . poly(A) . poly(U) (triple stranded) were reacted with HMT at different temperatures and salt concentrations. The conformation of the polymers was monitored by UV absorption and circular dichroism. It was found that the rate of HMT photoaddition changed dramatically at structural transitions in the RNA. The alternating copolymer poly(A-U) was found to have the highest rate of addition. Low salt and temperature produced maximal incorporation.     

Relationship between the Molecular Size of Poly I·Poly C and Its Biological Activity1

Seven polyinosinic·polycytidylic acid (poly I·poly C) preparations, ranging from 4.2 S to 21.2 S, prepared from various sizes of polyinosinate and polycytidylate, were examined for toxicity and interferon-inducing activity in mice. The increase in size of poly I·poly C was accompanied by increases both in the maximal amount of interferon produced and in the length of persistence of a high level of interferon in plasma. Toxicity of poly I·poly C was proportional to the molecular size within the range of 8 S to 16 S. The amount of interferon induced by 1/5 LD₅₀ of poly I·poly C depended on the size of the inducer, being increasingly lower with progressively smaller sizes. Next, activities of poly I·poly C in culture cells were examined. The resistance-inducing activity of poly I·poly C in primary chick embryo cells (CEC) increased with the size of the inducer (4.2 S to 11.6 S), whereas the activity in L cells was not so markedly dependent upon its molecular size as in CEC. In the presence of calf serum during induction of resistance the activity was lowered. The activities of preparations with small molecular sizes were affected by calf serum more markedly than those of large molecular sizes. The interferon-inducing activity in RK13 was not appreciably influenced by the size of poly I·poly C, especially in the presence of DEAE-dextran, while the activity in L cells was markedly dependent upon the size of the inducer. These results suggest that the influence of the molecular size of poly I·poly C upon the resistance-inducing and interferon-inducing activities varies among different kinds of cells, and alters in the presence of serum or DEAE-dextran.     

The ribonucleotide reductase R1 subunits of herpes simplex virus 1 and 2 protect cells against poly(I · C)-induced apoptosis

We recently provided evidence that the ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 (HSV-1 and -2) protect cells against tumor necrosis factor alpha- and Fas ligand-induced apoptosis by interacting with caspase 8. Double-stranded RNA (dsRNA) is a viral intermediate known to initiate innate antiviral responses. Poly(I · C), a synthetic analogue of viral dsRNA, rapidly triggers caspase 8 activation and apoptosis in HeLa cells. Here, we report that HeLa cells after HSV-1 and HSV-2 infection were quickly protected from apoptosis caused by either extracellular poly(I · C) combined with cycloheximide or transfected poly(I · C). Cells infected with the HSV-1 R1 deletion mutant ICP6Δ were killed by poly(I · C), indicating that HSV-1 R1 plays a key role in antiapoptotic responses to poly(I · C). Individually expressed HSV R1s counteracted caspase 8 activation by poly(I · C). In addition to their binding to caspase 8, HSV R1s also interacted constitutively with receptor-interacting protein 1 (RIP1) when expressed either individually or with other viral proteins during HSV infection. R1(1-834)-green fluorescent protein (GFP), an HSV-2 R1 deletion mutant protein devoid of antiapoptotic activity, did not interact with caspase 8 and RIP1, suggesting that these interactions are required for protection against poly(I · C). HSV-2 R1 inhibited the interaction between the Toll/interleukin-1 receptor domain-containing adaptor-inducing beta interferon (IFN-β) (TRIF) and RIP1, an interaction that is essential for apoptosis triggered by extracellular poly(I · C) plus cycloheximide or TRIF overexpression. TRIF silencing reduced poly(I · C)-triggered caspase 8 activation in mock- and ICP6Δ-infected cells, confirming that TRIF is involved in poly(I · C)-induced apoptosis. Thus, by interacting with caspase 8 and RIP1, HSV R1s impair the apoptotic host defense mechanism prompted by dsRNA.     

Effect of virazole on the antiviral activity of poly(G) X poly(C) and other polyribonucleotide interferonogens

The effect of virazole on the antiviral activity of poly (G) X poly (C), poly (G, A) X X poly (C) and poly(G, I) X poly (C) was studied in cell cultures and on mice. It was shown that virazole in concentrations not sufficient for significant inhibition of the development of vesicular stomatitis virus or Sindbis virus in chick embryo cell cultures markedly increased the antiviral effect and allowed decreasing the minimum effective doses of the synthetic polyribonucleotide complexes with respect to the above viruses. Combined administration of poly (G) X poly (C) and virazole to mice 1-2 or 24 hours after infection with tick-borne encephalitis virus provided a much more pronounced decrease in the death rate of the animals than the use of the interferonogen alone. Virazole per se was little active and had no significant effect on the intensity of interferonogenesis promoted by the use of poly (G) X poly (C). A possibility of successful therapy of viral infections with polyribonucleotide interferonogens in combination with virazole or other chemotherapeutic drugs with broad antiviral spectrum is discussed.