TLR3 Activation of Intratumoral CD103+ Dendritic Cells Modifies the Tumor Infiltrate Conferring Anti-tumor Immunity.

An important challenge in cancer immunotherapy is to expand the number of patients that benefit from immune checkpoint inhibitors (CI), a fact that has been related to the pre-existence of an efficient anti-tumor immune response. Different strategies are being proposed to promote tumor immunity and to be used in combined therapies with CI. Recently, we reported that intratumoral administration of naked poly A:U, a dsRNA mimetic empirically used in early clinical trials with some success, delays tumor growth and prolongs mice survival in several murine cancer models. Here, we show that CD103+ cDC1 and, to a much lesser extent CD11b+ cDC2, are the only populations expressing TLR3 at the tumor site, and consequently could be potential targets of poly A:U. Upon poly A:U administration these cells become activated and elicit profound changes in the composition of the tumor immune infiltrate, switching the immune suppressive tumor environment to anti-tumor immunity. The sole administration of naked poly A:U promotes striking changes within the lymphoid compartment, with all the anti-tumoral parameters being enhanced: a higher frequency of CD8+ Granzyme B+ T cells, (lower Treg/CD8+ ratio) and an important expansion of tumor-antigen specific CD8+ T cells. Also, PD1/PDL1 showed an increased expression indicating that neutralization of this axis could be exploited in combination with poly A:U. Our results shed new light to promote further assays in this dsRNA mimetic to the clinical field.

Stimulation of the toll-like receptor 3 promotes metabolic reprogramming in head and neck carcinoma cells.

In this study, a possible link between the innate immune recognition receptor TLR3 and metabolic reprogramming in Head and Neck carcinoma (HNC) cells was investigated. The effects of TLR3 stimulation/knock-down were assessed under several culture conditions in 4 HNC cell-lines by cell growth assays, targeted metabolomics, and glycolysis assays based on time-resolved analysis of proton release (Seahorse analyzer). The stimulation of TLR3 by its synthetic agonist Poly(A:U) resulted in a faster growth of HNC cells under low foetal calf serum conditions. Targeted analysis of glucose metabolism pathways demonstrated a tendency towards a shift from tricarboxylic acid cycle (Krebs cycle) to glycolysis and anabolic reactions in cells treated with Poly(A:U). Glycolysis assays confirmed that TLR3 stimulation enhanced the capacity of malignant cells to switch from oxidative phosphorylation to extra-mitochondrial glycolysis. We found evidence that HIF-1α is involved in this process: addition of the TLR3 agonist resulted in a higher cell concentration of the HIF-1α protein, even in normoxia, whereas knocking-down TLR3 resulted in a lower concentration, even in hypoxia. Finally, we assessed TLR3 expression by immunohistochemistry in a series of 7 HNSCC specimens and found that TLR3 was detected at higher levels in tumors displaying a hypoxic staining pattern. Overall, our results demonstrate that TLR3 stimulation induces the Warburg effect in HNC cells in vitro, and suggest that TLR3 may play a role in tumor adaptation to hypoxia.

Two Oyster Species That Show Differential Susceptibility to Virus Infection Also Show Differential Proteomic Responses to Generic dsRNA.

Viral diseases are a significant cause of mortality and morbidity in oysters, resulting in significant economic losses. We investigated the proteomic responses of these two species of oysters to generic double-stranded RNAs (poly I:C and poly A:U). Analysis of proteomic data using isobaric tags for relative and absolute quantitaion (iTRAQ) indicated that there were significant differences in the proteomic responses of the two oyster species resulting from this treatment. Gene ontology analysis showed that several biological processes, cellular components, and molecular function were unique to the different data sets. For example, a number of proteins implicated in the TLR signaling pathway were associated with the Saccostrea glomerata data set but were absent in the Crassostra gigas data set. These results suggest that the differences in the proteomic responses to dsRNA may underpin the biological differences in viral susceptibility. Molecular targets previously shown to be expressed in C. gigas in response to OsHV1 infections were not present in our proteomic data sets, although they were present in the RNA extracted from the very same tissues. Taken together, our data indicate that there are substantial disparities between transcriptomic and proteomic responses to dsRNA challenge, and a comprehensive account of the oysters' biological responses to these treatments must take into account that disparity.

Effect of toll-like receptor 3 agonists on the functionality and metastatic properties of breast cancer cell model.

There exists compelling evidence that Toll-like receptor 3 (TLR3) agonists can directly affect human cancer cells. The aim of this study was to investigate anti-cancer effects of TLR3 agonist in human breast cell line. We assessed potential effects of poly (A:U) on human breast cell line (MDA-MB-231) on a dose-response and time-course basis. Human breast cell line MDA-MB-231 was treated with different concentrations of poly (A:U) and lipopolysaccharide (LPS). Then, the following assays were performed on the treated cells: dose-response and time-course cytotoxicity using colorimetric method; matrix metalloproteinase-2 (MMP-2) activity using gelatin zymography method; apoptosis using annexin-v flowcytometry method; and relative expression of TLR3 and MMP-2 mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) method. Following treatments, dose- response and time-course cytotoxicity using a colorimetric method, (MMP-2) activity (using gelatin zymography), apoptosis (using annexin-v flowcytometry method) assays and expression of TLR3 and MMP-2 genes (using PCR method) were performed. Cytotoxicity and flowcytometry analysis of poly (A:U) showed that poly (A:U) do not have any cytotoxic and apoptotic effects in different concentrations used. MMP-2 activity analysis showed significant decrease in higher concentrations (50 and 100 µg/ ml) between treated and untreated cells. Moreover, poly A:U treated cells demonstrated decreased expression of MMP-2 gene in higher concentrations. Collectively, our data indicated that human breast cancer cell line (MDA-MB-231) was highly responsive to poly (A:U). The antimetastatic effect of direct poly (A:U) and TLR3 interactions in MDA-MB-231 cells could provide new approaches in malignant tumor therapeutic strategy.

Direct effect of dsRNA mimetics on cancer cells induces endogenous IFN-ß production capable of improving dendritic cell function.

Viral double-stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN-ß autocrine loop, dsRNA-elicited IFN-ß production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN-ß at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte-derived DCs. IFN-ß produced by poly I:C-activated human cancer cells increased the capacity of monocyte-derived DCs to stimulate IFN-γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3(-/-) mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1(-/-) mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN-ß production and contribute to the antitumoral response.

A synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34(+) cells.

Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type-dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)-mediated very rapid apoptosis occurs within 1 hour in CD34(+) cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34(+) cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34(+) cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34(+) cells, neither exogenous IFN-α nor IFN-ß induces rapid apoptosis in CD34(+) cells and neutralization or blocking of type-I IFN receptor does not rescue CD34(+) cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TLR3, signaling triggers poly I:C-induced rapid apoptosis of human CD34(+) cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands.

Many cancer cells express Toll-like receptors (TLR) that offer possible therapeutic targets. Polyadenylic-polyuridylic acid [poly(A:U)] is an agonist of the Toll-like receptor TLR3 that displays anticancer properties. In this study, we illustrate how the immunostimulatory and immunosuppressive effects of this agent can be uncoupled to therapeutic advantage. We took advantage of two TLR3-expressing tumor models that produced large amounts of CCL5 (a CCR5 ligand) and CXCL10 (a CXCR3 ligand) in response to type I IFN and poly(A:U), both in vitro and in vivo. Conventional chemotherapy or in vivo injection of poly(A:U), alone or in combination, failed to reduce tumor growth unless an immunochemotherapeutic regimen of vaccination against tumor antigens was included. CCL5 blockade improved the efficacy of immunochemotherapy, whereas CXCR3 blockade abolished its beneficial effects. These findings show how poly(A:U) can elicit production of a range of chemokines by tumor cells that reinforce immunostimulatory or immunosuppressive effects. Optimizing the anticancer effects of TLR3 agonists may require manipulating these chemokines or their receptors.

Immunoadjuvant effects of polyadenylic:polyuridylic acids through TLR3 and TLR7.

Double-stranded RNA (dsRNA) is produced upon viral infection and can activate innate immunity. Polyinosinic:polycytidylic acids [poly(I:C)] is a synthetic mimetic of dsRNA and functions through an endosomal receptor, Toll-like receptor (TLR) 3 or cytosolic receptors. Another type of dsRNA, polyadenylic:polyuridylic acids [poly(A:U)], can also act as an immune adjuvant, but it remains unclear how it exhibits its adjuvant effects. Here, we have characterized the adjuvant effects of poly(A:U). Poly(A:U) could induce both IFN-alpha and IL-12p40 from murine bone marrow dendritic cells (DCs). Poly(A:U)-induced IFN-alpha production depended on a DC subset, plasmacytoid dendritic cell (pDC), and required TLR7. IL-12p40 was also produced by poly(A:U)-stimulated pDC in a TLR7-dependent manner. In addition to pDC, conventional dendritic cell (cDC) also produced IL-12p40 in response to poly(A:U). This IL-12p40 induction resulted from two cDC subsets, CD24(high) cDC and CD11b(high) cDC in a TLR3- and TLR7-dependent manner, respectively. In vivo injection of poly(A:U) with antigen led to clonal expansion of and IFN-gamma production from antigen-specific CD8(+) T cells. Consistent with the in vitro findings, TLR3 and TLR7 were required for the clonal T-cell expansion. Notably, TLR3, rather than TLR7, was critical for generating IFN-gamma-producing CD8(+) T cells. CD8(+) T-cell responses induced by poly(A:U) were independent of type I IFN signaling. Our results demonstrate that poly(A:U) functions as an in vivo immunoadjuvant mainly through TLR3 and TLR7.

Pancreatic autoimmunity induction with insulin B:9-23 peptide and viral mimics in the NZB mouse.

To create a new experimental model of diabetes, we used the New Zealand Black (NZB) mouse as a potential model. NZB mice were immunized with B:9-23 insulin peptide in IFA and the viral mimic, poly(A:U). No diabetes was observed but blood glucose was significantly higher in the B:9-23 peptide group compared to controls. Insulin autoantibodies (IAA) were only induced in groups given the B:9-23 peptide. B:9-23 alone induced peri-insulitis. We demonstrate insulin autoimmunity in the NZB mouse using the insulin peptide B:9-23 and viral mimics. The reason for the protection from diabetes despite the presence of autoimmunity is currently not established.

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.

The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT >> TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.

Polyadenylic-polyuridylic acid plus locoregional radiotherapy versus chemotherapy with CMF in operable breast cancer: a 14 year follow-up analysis of a randomized trial of the Fédération Nationale des Centres de Lutte contre le Cancer (FNCLCC).

With a median follow-up of 14 years, the combination of polyadenylic-polyuridylic acid plus locoregional radiotherapy (257 patients) has significantly improved disease-free survival (p = 0.03) and significantly reduced the incidence of metastases (p = 0.04) when compared to CMF alone (260 patients), in women with operable breast cancer. The trial does not, however, permit an appreciation of the respective role of radiotherapy and PolyAU in these results.

Enantiodifferentiating Z-E photoisomerization of cyclooctene sensitized by DNA and RNA.

DNA and RNA have been shown for the first time to function as chiral photosensitizers in aqueous solution, to effect the enantiodifferentiating photoisomerization of (Z)-cyclooctene (1Z), giving the chiral (E)-isomer in enantiomeric excesses (ee's) of up to 15%. In order to elucidate the effect of nucleotide sequence, enantiodifferentiating photoisomerization of 1Z was also performed using oligo and homopolynucleotides as chiral sensitizer. The -18.8% ee was observed by using d(T)15.d(A)15 as sensitizer, whereas sensitization by the poly(U).poly(A) duplex gave only racemic (E)-cyclooctene. From these results, oligothymidine sequence is essential for efficient enantioselective photoisomerization of 1Z.

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.

Ultrastructural nucleolar alterations induced by an ametantrone--poly r(A-U) complex.

The current study has documented changes in the ultrastructure as well as in the intranucleolar distribution of rDNA and rRNA in RT4 (human transitional cell bladder carcinoma) cell nucleoli following a 3-h exposure to toxic doses of 50 microM ametantrone (AMT), 200 microM poly (adenylate-uridylate) (poly r(A-U) or an AMT/poly r(A-U) combination with an AMT/polyribonucleotide ratio of 1:4 and a poly r(A-U) concentration of 200 microM. While the main nucleolar components (fibrillar center (F), dense fibrillar component (D), granular component (G) and interstices (I) can be discerned following all treatments, the nucleoli exhibit: compaction, segregation, a decrease in the number of F, an increase in the size of remaining F, margination of intranucleolar chromatin and retention of intranucleolar pre-rRNA and rRNA. The relative abilities of the test agents to induce nucleolar compaction are AMT/poly r(A-U) > poly r(A-U) > AMT > sham-treated, while the abilities of the test agents to induce the remaining nucleolar changes are AMT/poly r(A-U) > or = AMT > poly r(A-U) > sham-treated cells. Poly r(A-U) and the induced interferon induce nucleolar compaction, while AMT produces nucleolar segregation. These results are consistent with a model in which the poly r(A-U) and/or the AMT inhibit DNA transcription and rRNA processing as well as the release of nascent preribosomes from the nucleolus.

Phase I/IB study of polyadenylic-polyuridylic acid in patients with advanced malignancies: clinical and biologic effects.

The synthetic polynucleotide polyadenylic-polyuridylic acid (polyA:polyU) has shown antitumor activity in murine studies and human breast cancer. PolyA:polyU was evaluated in 25 cancer patients receiving weekly intravenous doses between 3 and 600 mg/m2. PolyA:polyU was well tolerated up to 600 mg/m2, with no doselimiting toxicity (all < grade 3). Side effects included mild elevation in temperature, fatigue, and mild hyperglycemia. No changes outside of the normal range in hematocrit, WBC count, platelet count, total bilirubin, or alkaline phosphatase were observed. Of 25 patients, 18 completed at least one cycle of 6 weeks, and 5 completed two cycles (median 6 weeks). Four patients had stable disease over 11-13 weeks of treatment, and no clinical responses were observed. At 24 h after the first treatment, there were no significant increases in biologic response (beta 2-microglobulin and neopterin in serum, or 2',5'-oligoadenylate synthetase in peripheral blood mononuclear cells). A small increase in beta 2-microglobulin was observed 24 h after the week 3 treatment (1.1-fold, p < 0.01). By the third week of treatment, 2-5A synthetase levels decreased slightly (to 80% of baseline, p < 0.01). No changes in cytokines IL-6, IL-12, tumor necrosis factor (TNF), or IL-2 receptor in serum were detected after 24 h of treatment. Thus, at these doses, polyA:polyU had no marked modulation on biologic responses in vivo, although this preparation significantly induced 2-5A synthetase in peripheral blood mononuclear cells in vitro. PolyA:polyU was well tolerated. An MTD was not reached but was greater than 600 mg/m2 on this weekly schedule.

Involvement of interleukin-6 and interferon-alpha in the poly(A).Poly(U)-induced 2',5'-oligoadenylate synthetase activity in the mouse monocyte-macrophage cell line, J774A1.

The synthetic polyribonucleotide poly(A).poly(U) induces 2',5'-oligoadenylate synthetase activity in the murine macrophage cell line J774A1. The possible role of several cytokines involved in macrophage activation (i.e., IL-1, IL-6, TNF, and IFN) was examined in the present study. It was first demonstrated that among the anticytokine antibodies, only monoclonal antibodies directed against IL-6 inhibited the induction of 2',5'-oligoadenylate synthetase by poly(A).poly(U) in a dose-dependent manner. Moreover, it was established that poly(A).poly(U) elicited IL-6 production in J774A1 cells in a time-and dose-dependent manner. Consequently, the effect of IL-6 on 2',5'-oligoadenylate synthetase activity was studied. IL-6 either alone or in combination with IL-1 and TNF did not induce 2',5'-oligoadenylate synthetase activity. IL-6 did not potentiate IFN-gamma-induced 2'-5'-oligoadenylate synthetase activity. In contrast, addition of IL-6 to the incubation medium potentiated the stimulation of 2'-5'-oligoadenylate synthetase activity by IFN-alpha. These results suggest that IL-6 is a necessary but not sufficient factor in the induction of 2'-5'-oligoadenylate synthetase activity in the J774A1 cell line by poly(A).poly(U).

Polyadenylic-polyuridylic acid enhances the natural cell-mediated cytotoxicity in patients with breast cancer undergoing mastectomy.

BACKGROUND: Surgical procedures suppress host antitumor defense mechanisms, which may increase the risk of metastatic tumor dissemination. We have evaluated the effects of the biologic response modifier polyadenylic-polyuridylic acid (PAPU) on natural cytotoxicity in patients with breast cancer undergoing operation. METHODS: PAPU (150 mg) or placebo was given intravenously during the perioperative period (preoperative, days -1 and 0; postoperative, days 1, 3, 5, 7, and 14). The function (chromium release assay) and number (flow cytometry) of natural killer (NK) cells were measured before operation (days -2 and -1), on the day of operation (day 0), and after operation (days 1, 2, 4, 6, and 18). RESULTS: Surgical procedures suppressed NK cell cytotoxicity in the placebo group on postoperative days 1 (p < 0.001), 4, 6, and 18 (p < 0.05), whereas inhibition on postoperative day 2 failed to reach significance. PAPU abolished this immunosuppression after operation. The NK cell activity was elevated when compared with the control group; it was significant (p < 0.05) on postoperative days 1, 2, 4, 6, and 18. Surgical procedures also reduced circulating NK cell numbers during the first postoperative week in the placebo group; the decrease was statistically significant on day 4. The decrease in NK cell numbers in the PAPU group was insignificant. CONCLUSIONS: PAPU prevented the decrease in the circulating number and cytotoxic activity of NK cells that occurred after operation and enhanced NK cell cytotoxicity. This may have important implications for patients with cancer undergoing major operation.

Synthetic polyribonucleotides: current role and potential use in oncological practice.

Polyadenylic-polyuridylic acid (PAPU) has a wide range of effects on various immunological cells and functions. It has been shown to enhance cellular and humoral immunity, in particular anti-cancer host defences, in both animals and man. In a small number of clinical trials (breast and stomach cancers) it appears to have a beneficial adjuvant effect, in terms of prolongation of disease-free and overall survival. Side effects with PAPU are minimal. The precise mode of action of PAPU is unclear and the most beneficial therapeutic regimen has yet to be established. PAPU, therefore, merits further careful consideration and evaluation. This article reviews its present status and considers further priority areas for investigation.

Effect of ethidium 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.