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.     

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.     

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.     

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.     

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.     

In vitro antiviral activity of poly (A-U) and ellipticines.

The role of N2-methyl-9-hydroxy-ellipticine (NMHE) and N2,N6-dimethyl-9-hydroxy-ellipticine (DMHE) in modulating the antiviral activity of poly (A-U) was examined using a human foreskin fibroblast-vesicular stomatitis virus (HSF-VSV) bioassay in which the concentration of poly (A-U) was fixed at 0.05 mM or 0.2 mM while the NMHE or DMHE concentration was varied to produce variable NMHE (or DMHE)/ribonucleotide ratios ranging from 1:16 to 2:1. Poly (A-U), NMHE and DMHE tested individually were not efficacious antiviral agents. When the poly (A-U) was combined with the NMHE or DMHE, the antiviral activity of the poly (A-U) was potentiated 16- to 20-fold a NMHE (or DMHE)/ribonucleotide ratios in the region of 1/4. Poly (A-U), NMHE and DMHE induce beta-IFN. The interferon-inducing activity of the NMHE (or DMHE)/poly (A-U) combination was equal to the sum of the interferon-inducing activity of the poly (A-U) alone and the NMHE (or DMHE) alone. The direct viral inactivation study demonstrated that NMHE, DMHE, poly (A-U) and the NMHE (or DMHE)/poly (A-U) combinations did not inactivate VSV at concentrations near the 50% viral inhibitory dose. Photomicrographs of HSF cells incubated with NMHE alone or with a NMHE/poly (A-U) combination suggest that poly (A-U) affects the subcellular distribution of the NMHE by steering the NMHE to the nucleolus. These observations suggest that modulation of a nuclear process may be responsible for the enhanced antiviral activity.     

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.     

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.     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

CD of the synthetic RNA duplexes poly[r(A-T)] and poly[r(A-U)] in salt and ethanolic solutions.

Synthetic RNA poly[r(A-T)] has been synthesized and its CD spectral properties compared to those of poly[r(A-U)], poly[d(A-T)], and poly[d(A-U)] in various salt and ethanolic solutions. The CD spectra of poly[r(A-T)] in an aqueous buffer and of poly[d(A-T)] in 70.8% v/v ethanol are very similar, suggesting that they both adopt the same A conformation. On the other hand, the CD spectra of poly[r(A-T)] and of poly[r(A-U)] differ in aqueous, and even more so in ethanolic, solutions. We have recently observed a two-state salt-induced isomerization of poly[r(A-U)] into chiral condensates, perhaps of Z-RNA [M. Vorlícková, J. Kypr, and T. M. Jovin, (1988) Biopolymers 27, 351-354]. It is shown here that poly[r(A-T)] does not undergo this isomerization. Both the changes in secondary structure and tendency to aggregation are different for poly[r(A-T)] and poly[r(A-U)] in aqueous salt solutions. In most cases, the CD spectrum of poly[r(A-U)] shows little modification of its CD spectrum unless the polymer denatures or aggregates, whereas poly[r(A-T)] displays noncooperative alterations in its CD spectrum and a reduced tendency to aggregation. At high NaCl concentrations, poly[r(A-T)] and poly[r(A-U)] condense into psi(-) and psi(+) structures, respectively, indicating that the type of aggregation is dictated by the polynucleotide chemical structure and the corresponding differences in conformational properties.     

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.     

Sequence-dependant polymorphism of DNA duplex in solutions

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.     

Determination of base pairing in ribonucleic acid by Fourier-transform infrared spectrometry: yeast ribosomal 5S ribonucleic acid

Fourier-transform infrared (FT-IR) spectra of yeast ribosomal 5S RNA have been acquired at several temperatures between 30 and 90 degrees C. The difference spectrum between 90 (bases unstacked) and 30 degrees C (bases stacked) provides a measure of base stacking in the RNA. Calibration difference spectra corresponding to stacking of G-C or A-U pairs are obtained from "reference" FT-IR spectra of poly(rG) X poly(rC) minus 5'-GMP and 5'-CMP or poly(rA) X poly(rU) minus 5'-AMP and 5'-UMP. The best fit linear combination of the calibration G-C and A-U difference spectra to the 5S RNA (90-30 degrees C) difference spectrum leads to a total of 25 +/- 3 base pairs (17 G-C pairs + 8 A-U pairs) for the native yeast 5S RNA in the absence of Mg2+. In the presence of Mg2+, an additional six base pairs are detected by FT-IR (one G-C and five A-U). FT-IR melting curve midpoints show that A-U and G-C pairs melt together (65 and 63 degrees C) in the presence of Mg2+ but A-U pairs melt before G-C pairs (47 vs. 54 degrees C) in the absence of Mg2+.     

Poly(I:C) Induces Antiviral Immune Responses in Japanese Flounder (Paralichthys olivaceus) That Require TLR3 and MDA5 and Is Negatively Regulated by Myd88

Polyinosinic:polycytidylic acid (poly(I:C)) is a ligand of toll-like receptor (TLR) 3 that has been used as an immunostimulant in humans and mice against viral diseases based on its ability to enhance innate and adapt immunity. Antiviral effect of poly(I:C) has also been observed in teleost, however, the underling mechanism is not clear. In this study, we investigated the potential and signaling mechanism of poly(I:C) as an antiviral agent in a model of Japanese flounder (Paralichthys olivaceus) infected with megalocytivirus. We found that poly(I:C) exhibited strong antiviral activity and enhanced activation of head kidney macrophages and peripheral blood leukocytes. In vivo studies showed that (i) TLR3 as well as MDA5 knockdown reduced poly(I:C)-mediated immune response and antiviral activity to significant extents; (ii) when Myd88 was overexpressed in flounder, poly(I:C)-mediated antiviral activity was significantly decreased; (iii) when Myd88 was inactivated, the antiviral effect of poly(I:C) was significantly increased. Cellular study showed that (i) the NF-κB activity induced by poly(I:C) was upregulated in Myd88-overexpressing cells and unaffected in Myd88-inactivated cells; (ii) Myd88 overexpression inhibited and upregulated the expression of poly(I:C)-induced antiviral genes and inflammatory genes respectively; (iii) Myd88 inactivation enhanced the expression of the antiviral genes induced by poly(I:C). Taken together, these results indicate that poly(I:C) is an immunostimulant with antiviral potential, and that the immune response of poly(I:C) requires TLR3 and MDA5 and is negatively regulated by Myd88 in a manner not involving NK-κB. These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish.     

Effect of 5-alkyl substitution of uracil on the thermal stability of poly [d(A-r5U)] copolymers.

The thermal transition of poly[d(A-r5U)] polydeoxynucleotides (where r was a hydrogen atom, or a methyl, ethyl, n-propyl, n-butyl or n-pentyl group) was studied by measuring the derivative melting profiles of the polymers in the range of 0.01--0.36 M K+, at pH 6.8. According to the Tm values, polydeoxynucleotide analogues show lower thermal stability than poly[d(A-T)] at any counterion concentration applied. At a given salt concentration, Tm of the alkyl analogues decreased as the number of carbon atoms (n) in the r substituent of poly[d(A-r5U)] increased. 1/Tm plotted against against 1/n yielded a linear relationship. Cooperativity of the melting of all poly[d(A--U)] analogues decreased with the increase of salt concentration in the solution. This change depended again on 5-substitution of the uracil moiety of poly[d(A-U)]. Smallest decrease was observed in the case of poly[d(A--U)] whereas largest decrease was shown by poly[d(A-pe5U)] (pe=pentyl group).     

The antiviral activity of 9-beta-D-arabinofuranosyladenine is enhanced by the 2',3'-dideoxyriboside, the 2',3'-didehydro-2',3'-dideoxyriboside and the 3'-azido-2',3'-dideoxyriboside of 2,6-diaminopurine

The 2',3'-dideoxyriboside (ddDAPR), 2',3'-didehydro-2',3'-dideoxyriboside (ddeDAPR) and 3'-azido-2',3'-dideoxyriboside (AzddDAPR) of 2,6-diaminopurine have been previously recognized as potent inhibitors of human immunodeficiency virus replication. These compounds are also potent inhibitors of adenosine deaminase and inhibit the deamination of 9-beta-D-arabinofuranosyladenine (araA). ddDAPR, ddeDAPR and AzddDAPR markedly potentiate the antiviral activity of araA against herpes simplex virus type 1 (HSV-1), type 2 (HSV-2) and vaccinia virus (VV). When used at a concentration of 20 micrograms/ml, which had by itself no antiviral effect, ddDAPR, ddeDAPR and AzddDAPR increased the ability of araA to suppress HSV-1, HSV-2 and VV yield by several orders of magnitude. The maximum antiviral effect was obtained with the combinations of ddDAPR or ddeDAPR with araA concentrations of 1 and 10 micrograms/ml.     

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.