Antiviral activity in L1210 cells of liposome-encapsulated (2'-5')oligo(adenylate) analogues

Evidence is available for a role of a (2'-5')(A)n-activated endoribonuclease (RNase L) in the antiviral activity of interferon for several RNA viruses. (2'-5')(A)n and their analogues might thus provide an interesting alternative to exogenous interferons or their inducers in antiviral chemotherapy. In addition, the evaluation of the activity of (2'-5)(A)n as mediators of interferon's biological activities or as cell growth regulators requires biochemical studies using agonists or antagonists of the system. Non-disruptive techniques for the introduction of (2'-5')(A)n and their analogues into cell lines or tissues are required for these studies since these highly charged compounds are cell impermeable. (2'-5')(A)n oligomers and analogues of increased stability towards phosphodiesterases were derived by chemical modification of their 2' end and encapsulated in protein-A-bearing liposomes. The specific delivery of liposome contents into L1210 mouse leukemic cells was achieved with the help of monoclonal antibodies directed against the appropriate class I major histocompatibility complex-encoded proteins expressed by these cells. This intracellular delivery led to transient inhibition of protein synthesis and an antiviral activity, both compatible with activation of RNase L. This activity was enhanced for the analogues designed to resist degradation, with respect to the natural product.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of ribonuclease L by (2'-5')(A)4-poly(L-lysine) conjugates in intact cells

Molecular hybrids were synthesized by coupling (2'-5')(A)n oligoadenylates or 2-5A, an intracellular mediator involved in antiviral activity of interferons (IFNs), with poly(L-lysine) used as a membrane carrier. (2'-5')(A)n in its free form was not taken up by cells, probably because of its ionic character. Conjugation with the polypeptide carrier overcame this problem and enabled its pharmacological properties to be developed. The alpha-glycol group of individual (2'-5')(A)n oligomers was oxidized by periodate oxidation and conjugated by an amino reductive reaction to poly(L-lysine), Mr 14 000, in a molar ratio of 5:1. These hybrid molecules left the biologically active 5' end moiety of the (2'-5')(A)n molecule unchanged, and in particular its triphosphate group, and stabilized the molecule by increasing its resistance to phosphodiesterase hydrolysis. A dose-dependent inhibition of virus growth was observed on concomitant incubation of (2'-5')(A)n-poly(L-lysine) conjugates with vesicular stomatitis virus infected L1210 cell cultures. This was a result of the activation of the (2'-5')(A)n-dependent endoribonuclease (RNase L) by intracellularly delivered (2'-5')(A)n as in some IFN-treated virus-infected cells. Indeed, (2'-5')(A)n-poly(L-lysine) conjugates bind RNase L effectively as can be seen from their ability to compete with authentic (2'-5')(A)n in a cell-free radiobinding assay. Moreover, (2'-5')(A)n-poly(L-lysine) conjugates promote transient inhibition of protein synthesis and a characteristic cleavage pattern of ribosomal RNAs in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorothioate analogues of (2'-5')(A)4: agonist and antagonist activities in intact cells

Metabolically stable phosphorothioate tetramer analogues of (2'-5')(A)n with Rp and/or Sp chirality in the 2'-5'-phosphodiester linkages constitute a new class of antiviral agents since they mimic the effects of interferons. Three of the diastereomeric 5'-monophosphates (i.e., pRpRpRp, pSpRpRp, and pRpSpSp) bind to and activate RNase L from extracts of HeLa cells. However, the pSpSpSp (2'-5')-(A)4-phosphorothioate is unique in that it binds to, but cannot activate, RNase L to cleave rRNA. When microinjected into the cytoplasm of HeLa cells followed by virus infection, the pRpRpRp, pSpRpRp, and pRpSpSp (2'-5')(A)4-phosphorothioates demonstrate antiviral activity, as does (2'-5')(A)4ox-red, an active (2'-5')(A)n analogue. When microinjected simultaneously with (2'-5')(A)nox-red, an active the pSpSpSp (2'-5')(A)4-phosphorothioate inhibits activation of RNase L in HeLa cells, thereby blocking direct protection of vesicular stomatitis virus. The agonist and antagonist properties of pRpRpRp and pSpSpSp, respectively, are transient probably as a consequence of the hydrolysis of the 5'-monophosphate and formation of the less active (2'-5')(A)4-phosphorothioate cores. The possible use of these (2'-5')(A)4-phosphorothioates as tools for dissecting the biological significance of the (2'-5')(A)n system or in antiviral chemotherapy is discussed.     

Detection of (2'-5')oligoadenylate binding proteins by nondenaturing polyacrylamide gel electrophoresis and affinity blotting onto nitrocellulose

A latent endoribonuclease, RNase L, binds to and is activated by (2'-5')oligoadenylates ((2'-5')(A)n, n = 2-15). Binding to a labeled derivative of (2'-5')(A)n, [32P](2'-5')(A)3pCp, is detected as a protein-ligand complex observed following nondenaturing polyacrylamide gel electrophoresis. One major binding complex and two minor binding complexes are readily seen in cytoplasmic extracts from Ehrlich ascites tumor cells, murine tissue extracts and rabbit liver tissue extracts. At least one of the more rapidly migrating complexes appears to be a proteolytic degradation product of the larger [32P](2'-5')(A)3pCp binding protein. Cell and tissue extracts containing [32P](2'-5')(A)3pCp binding activity can be immobilized onto nitrocellulose filters and [32P](2'-5')(A)3pCp binding activity detected using a simple, rapid, economical affinity blot assay. Detection of [32P](2'-5')(A)3pCp binding proteins following electrophoresis on nondenaturing polyacrylamide gels and the affinity blot assay significantly improve and simplify the analysis of (2'-5')(A)n binding proteins.     

Synthesis of (2'-5')oligoadenylate and activation of an endoribonuclease in interferon-treated HeLa cells infected with reovirus.

Treatment with interferon protected HeLa cells from infection with reovirus. This virus apparently activated an antiviral mechanism that was detected by the presence of (2'-5')oligoadenylate [(2'-5')An] in intact cells. The (2'-5')An was previously shown to activate an endoribonuclease, RNase L. We measured (2'-5')An by a sensitive competition-binding assay in cells infected at different multiplicities and for different lengths of time. Nanomolar concentrations of (2'-5')An were detected in cells infected at a multiplicity of greater than 5 after 2 h of infection, the time at which the infecting virions were uncoated. The level of (2'-5')An increased up to 6 h postinfection but declined afterward. To establish whether viral mRNAs were cleaved by RNase L, we analyzed the RNA extracted from infected cells by a highly specific hybridization assay on Northern blots. Full-sized reovirus mRNAs were detected in control infected cells, but not in interferon-treated infected cells, at 6 h postinfection. At this time, a nuclease activity could be detected in these cells by demonstration of cleavage of rRNA, degradation of cellular mRNA, and polysome breakdown in the presence of emetine. Since this inhibitor freezes ribosomes, cleavage of mRNA between ribosomes could only be accounted for by an endonuclease, presumably RNase L.     

Synthesis of (2'-5')(A)n from ATP. Characteristics of the reaction catalyzed by (2'-5')(A)n synthetase purified from mouse Ehrlich ascites tumor cells treated with interferon

The treatment of Ehrlich ascites tumor cells with mouse interferon increases the level of the latent enzyme (2'-5')(A)n synthetase. If activated by double-stranded RNA, this catalyzes the synthesis from ATP of a series of 2'-5'-oligoadenylates: (2'-5')(A)n where n extends from 2 to about 15. We isolated (2'-5')(A)n synthetase in a homogeneous state. In the presence of double-stranded RNA, the purified enzyme can convert the large majority (about 97%) of the ATP into (2'-5')(A)n and pyrophosphate, although it does not cleave the pyrophosphate. The stoichiometry of the reaction can be formulated as: (n + I) ATP leads to (2'-5') pppA(pA)n + n pyrophosphate. Added pyrophosphate does not inhibit the synthesis of (2'-5')(A)n. The extent of the reverse reaction, i.e. the pyrophosphorolysis of (2'-5')(A)n, was below the level of detection under our conditions. The affinity of the enzyme for ATP is low: the rate of the reaction increases by about 10% when the concentration of ATP is increased from 5 mM to 10 mM. The optimal concentration of double-stranded RNA increases with the concentration of the enzyme. As tested at 0.4, 2, and 10 micrograms/ml of enzyme concentrations, close to maximal (2'-5')(A)n synthesis can be obtained if reovirus double-stranded RNA or poly(I) . poly(C) are used at about half the concentration (in w/v) of the enzyme. The plot of the reaction rate versus enzyme concentration is sigmoidal. It remains to be seen if this reflects on a cooperative behavior of the enzyme.     

Subcellular distribution of 2'',5''-oligoadenylate synthetase in differentiating Friend leukemia cells

The occurrence of distinct (2'-5')(A)n-synthetase activities has recently been documented in cytoplasmic and nuclear extracts of several interferon (IFN)-treated cell lines. Since a role has been proposed for (2'-5')(A)n synthetase in the control of cell growth and differentiation, we examined the subcellular distribution of (2'-5')(A)n-synthetase activity both in IFN-treated undifferentiated Friend leukemia cells (FLCs) and during dimethyl-sulfoxide (DMSO)-induced erythroid differentiation of FLCs. Both the nuclear and cytoplasmic (2'-5')(A)n activities were modulated to the same extent by IFNs and DMSO. No evidence for a causal relationship between enzyme activation and FLC differentiation was found.     

3'-Fluoro-3'-deoxy analogs of 2-5A 5'-monophosphate: binding to 2-5A-dependent endoribonuclease and susceptibility to (2'-5')phosphodiesterase degradation

Analogs of 2-5A trimer 5'-monophosphate (2'-5')pA3,p5'A2'p5'A2'p5'A containing 9-(3-fluoro-3-deoxy-c-D-xylofuranosyl)adenine (AF) or 3'-fluoro-3'- deoxyadenosine (AF) at different positions of the chain have been synthesized. All of them were compared with (2'-5')pA3 and (2'-5')pA2 (3'dA) by (i) their ability to bind to 2-5A-dependent endoribonuclease(RNase L) of mouse L cells and of rabbit reticulocyte lysates and (ii) their susceptibility to the degradation by the (2'-5')phosphodiesterase activity. The results of this study suggest that the oligonucleotide conformation is important for its biochemical properties.     

3'-(N-hydroxyimino)-2',3-dideoxynucleosides and their derivatives: synthesis, broad spectrum antiviral properties and synthetical application for the preparation of other nucleoside analogues.

A series of 3'-(N-hydroxyimino)-2',3'-dideoxynucleosides bearing different nucleic bases has been prepared. In vitro antiviral activity studies showed that among these compounds the thymine derivative possesses significant activity against HIV, HSV, EBV and HBV. Conveniently 5'-protected 3'-(N-hydroxyimino)-2',3'-dideoxythymidine was further used as a synthon for the preparation of other nucleoside analogues.     

Analogs of (2'-5')oligo(A). Endonuclease activation and inhibition of protein synthesis in intact cells

Synthetic analogs of (2'-5')oligo(A) were assayed for endonuclease activation in cell extracts and for inhibition of protein synthesis in intact cells. The analogs are triadenylates: (i) methylated in the terminal 3'-OH; (ii) methylated at all three 3'-OH groups; (iii) with different numbers of phosphate groups at the 5' terminus or with a methylene group between the beta- and gamma-phosphate. Only 5'-phosphorylated monomethylated analogs activate an endonuclease in cell extracts and are powerful inhibitors of protein synthesis in intact cells. The analogs with only one 5'-terminal phosphate may require addition of another phosphate for activity since the kinase inhibitor 2-aminopurine prevents endonuclease activation by this compound but not by the di- and triphosphate-terminated triadenylates. These results suggest that two terminal phosphates and one or two free 3'-OH are required for endonuclease activation and inhibition of protein synthesis. The monomethylated analogs are more active than (2'-5')pppA3 because of their resistance to degradation by cellular enzymes. Accordingly, the monomethylated analogs cause a prolonged inhibition of protein synthesis in human fibroblasts treated with nanomolar concentrations of these compounds.     

Gene structure and expression of the 5'-(CGG)(n)-3'-binding protein (CGGBP1)

The human nuclear 5'-(CGG)(n)-3'-binding protein (CGGBP1) influences the expression of the fragile X mental retardation (FMR1) gene by specifically interacting with the 5'-(CGG)(n>5)-3' repeat in its 5' untranslated region. Here, we show that CGGBP1 binds to 5'-(CGG)(n)-3' repeats with n>or=5 and to interrupted repeats. The genomic and mRNA organization of the human and murine CGGBP1 genes was studied and the human gene was mapped to chromosome 3p. Due to alternative polyadenylation, mRNAs of 1.2 and 4.5 kb are transcribed at varying ratios in human and murine cells and in embryonic, fetal, and adult tissues. The human and the murine genes, including promoters and large parts of the untranslated regions, are highly conserved. A sequence of 235 nucleotides 5' upstream of CGGBP1 is essential for promoter activity in transfection experiments. Complete in vitro methylation inactivates the promoter, which is unmethylated in human cells as shown by bisulfite genomic sequencing.     

3'-Deoxy-3'-fluoro analogs of core trimers of 2-5A: effect on lytic activity of human NK-lymphocytes]

The effect of core trimers, (2'-5')-analogues of oligoadenylic acid containing 9-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)adenine (AF) and 3'-deoxy-3'-fluoroadenosine (AF) in various positions of the oligomer chain, on the lytic activity of human natural killer cells (NK cells) was studied in three different ways. The cellular cytotoxicity was determined using a highly sensitive nonradioactive approach employing a chelate europium-diethylenetriamino-pentaacetic acid complex (Eu-DTPA). It was shown that all fluorodeoxyanalogues enhance the lytic activity of intact NK lymphocytes, which follows from the lysis rate constant k2. At the same time, the substitution of either the central adenosine fragment or (to a greater extent) the 5'-terminal residue of (2'-5')A3 with AF causes a decrease in the number of active NK cells, which, unlike the case of the natural core trimer, leads to a loss of the capacity to increase the activity of NK. By contrast, isomeric ribo-analogues. (2'-5')(AF)A2 and (2'-5')A(AF)A, and trimers with the 2'(3')-terminal nucleotide substituted by AF or AF increased the activity of NK cells with an effectiveness close to or higher than the natural trimer (2'-5')A3. Inasmuch as isomeric xylo- and ribo-3'-deoxy-3'-fluoroanalogues of (2'-5')A3 are stereochemically modified oligomers, the data unambiguously suggest that the spatial structure of these trimers affects the increase in the lytic activity of NK cells.     

Cordycepin analogs of ppp5'A2'p5'A2'p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.     

Nerve growth factor induces changes in (2'-5')oligo(A) synthetase and 2'-phosphodiesterase activities during differentiation of PC12 pheochromocytoma cells

Treatment of rat pheochromocytoma cell line PC12 with Vipera lebetina (snake) nerve growth factor (NGF) induces a rapid increase (from 5 to 25-fold) in the level of (2'-5')oligo(A) synthetase activity and a simultaneous decrease (from 2 to 5-fold) in the activity of 2'-5' A degrading enzymes--2'-phosphodiesterases (2'-PDE). These changes in the enzyme activities led to the significant increase in the intracellular concentration of 2'-5' A. We have found that the serum starvation of PC12 cells causes a 1.5 to 2.0-fold increase in the level of 2'-5' A-synthetase activity, but the activities of 2'-PDE and the intracellular concentration of 2'-5' A remain unaltered. These results show that NGF modulates the activity of (2'-5')oligo(A) enzymes and intracellular concentration of 2'-5' A during the neural differentiation of PC12 cells.     

Antiviral activity of a chemically stabilized 2-5A analog upon microinjection into HeLa cells

2-5A[ppp(A2'p)n5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'-5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase-resistant analog of 2-A, in which the 2'-terminals adenosine residue is replaced by 2-(9-adenyl)-6-hydroxy-methyl-4-hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2-5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2-5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2-5A itself.     

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

Synthesis and biological activity of tubercidin analogues of ppp5'A2'p(5'A2'p)n5'A

A series of tubercidin (7-deazaadenosine) analogues of 2-5A of the general formula p5'(c7A)2'p[5'(c7A)-2'p]n5'(c7A) (n = 0-5) were prepared by lead ion catalyzed polymerization of the 5'-phosphoroimidazolidate of tubercidin. Through the corresponding imidazolidates, these oligonucleotide 5'-monophosphates were converted to the 5'-triphosphates. All reported structures were corroborated by enzyme digestion and 1H or 31P nuclear magnetic resonance. When evaluated for its ability to bind to the 2-5 A-dependent endonuclease of mouse L cells, the tubercidin analogue of trimeric 2-5A, namely, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), and the corresponding tetramer were bound as effectively as 2-5A itself; nonetheless, it and the corresponding tetramer, ppp5'-(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), failed to stimulate the 2-5A-dependent endonuclease as judged by its inability to inhibit translation in extracts of mouse L cells programmed with encephalomyocarditis virus RNA and to give rise to ribosomal RNA cleavage in the same cell system under conditions where 2-5A showed activity at 10(-9) M. The trimer, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), was an antagonist of 2-5A action in the L cell extract. In the lysed rabbit reticulocyte system, both the trimeric and tetrameric tubercidin 2-5A analogues were bound to the 2-5A-dependent endonuclease as well as 2-5A, but in this case, the tetramer triphosphate, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), was just as potent an inhibitor of translation as 2-5A tetramer triphosphate. Moreover, this inhibition was prevented by the established 2-5A antagonist p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical synthesis and biological activities of analogues of 2',5'-oligoadenylates containing 8-substituted adenosine derivatives.

The synthesis of sequence-specific 2'-5'-oligonucleotides and analogues of 2'-5' linked oligoadenylates containing 8-substituted adenosine derivatives [8-hydroxypropyladenosine (AHPr) and 8-hydroxyadenosine (AOH)] is reported. The reaction of 5'-phosphoroimidazolidate of 8-substituted adenosines under conditions of lead ion catalyst did not give the corresponding 2'-5' oligoadenylates containing pAHPr and pAOH. When these reactions were carried out in the presence of uranyl ion (UO2(2+] in place of lead ion as a catalyst, the desired 2'-5' oligoadenylates were obtained. The p5'AHPr2'p5'AHPr2'p5'AHPr and p5'AOH2'p5'AOH2'p5'AOH, p5'A2'p5'A2'pAOH were slightly resistant to snake venom phosphodiesterase. The both circular dichroism and 1H-NMR spectra studies were used to characterize the modified 2'-5' oligoadenylates. Further, the biological activity evaluations of 8-substituted analogues of 2-5A are also described.