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Human placenta contains a high level of 2',5'-oligoadenylate (2-5A) synthetase activity of the 100-kD form of the enzyme. About 20% of the placental 2-5A synthetase activity was found to be cytosolic, whereas the remaining 80% was released by 0.5 M KCl in the presence of detergent. Most of the enzyme activity was localized in trophoblast cells, which also contain a high level of 2-5A-dependent RNase L activity. The purified trophoblast 100-kD 2-5A synthetase was shown to be activated by human immunodeficiency virus type 1 (HIV-1) 5' RNA 1-311 and 1-707, which both contain the TAR and primer binding site (PBS) structured regions. These two HIV-1 RNAs activated human trophoblast 2-5A synthetase at the same level as poly(I).poly (C), a standard highly efficient activator of the enzyme, and at the same optimal concentration. On the contrary, HIV-1 RNA 311-618, a poorly structured region missing TAR and PBS, was shown to be a poor activator of the enzyme. The specific cellular location of the 2-5A synthetase and its efficient activation by HIV 5' RNA favors the idea that the trophoblast 2-5A system negatively controls HIV replication in trophoblasts.

A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s).

RNase L activated by 2-5A (a series of 2'-5'-linked adenylic oligoribonucleotides) is a key enzyme of the interferon system. To study RNase L (endonuclease L) in intact cells independently of intracellular 2-5A and of its activity, we have developed polyclonal antibodies against RNase L. RNase L from mouse spleen was purified on a column of 2-5A-Sepharose and used to immunize rabbits in co-injection with polyadenylic-polyuridylic acid as adjuvant. Antibodies were purified by chromatography on Affi-Gel blue and 2-5A-Sepharose-immobilized RNase L. These polyclonal antibodies immunoprecipitate the 80- and 40-kDa forms of RNase L in mouse spleen. In Western blot, only the 80-kDa form of RNase L is recognized by these antibodies. These purified antibodies were used to localize RNase L in the cytoplasm of intact mouse NIH 3T3 cells by immunofluorescence. The cytoplasmic localization of RNase L was confirmed by its 2-5A binding activity after cellular fractionation.

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.

RNase L is considered as the major if not unique target of (2'-5')(A)n and therefore as an important intracellular mediator of interferon action. It behaves as an 185-kDa species in various cell extracts when analyzed by gel filtration. SDS/PAGE analysis of the polypeptides covalently labelled with a (2'-5')(A)4-3'-[32P]pCp probe reveals a single 80-kDa species, thus attesting a multimeric form of the 185-kDa protein. At variance with such data, mouse spleen extracts reveal an additional 40-kDa polypeptide with (2'-5')(A)n-dependent ribonucleolytic activity. This seemingly new form of RNase L migrates as a 40-kDa polypeptide when analyzed under native or denaturing conditions. It bears some structural similarity with the larger-molecular-mass RNase L as revealed by partial proteolysis. It is probably not generated through proteolytic degradation of the 185-kDa RNase L during extract preparation, although its physiological significance is unknown. Indeed various protease inhibitors do not significantly alter the ratio of 40-kDa and 185-kDa (2'-5')(A)n-dependent ribonucleases; moreover, the (2'-5')(A)n-binding capacity of the 40-kDa polypeptide is more stable than that of the 185-kDa one.

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)

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.

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)

Metabolically stable analogues of (2'-5')oligo(adenylate), (2'-5')(A)n, might constitute a new class of antiviral agents as they mimic some of the effects of interferons. 2'-O-phosphoglyceryl derivatives of (2'-5')(A)n oligomers, (2'-5')(A)n-PGro have been synthesized by chemical modification of their terminal ribose residue. Such analogues are resistant to degradation by phosphodiesterases but remain sensitive to phosphatase activity, at least in cell-free extracts. In line with its increased stability, (2'-5')(A)n-PGro has a powerful antiviral activity against an RNA virus when microinjected with micropipettes into the cytoplasm of intact cells. This antiviral activity remains transient however, possibly as a consequence of degradation in intact cells. Since (2'-5')(A)n and its derivatives do not easily cross cell membranes, their possible use in antiviral chemotherapy is tightly linked with the development of vectors suitable for their administration in vivo.

This short review summarizes available evidence for (i) growth regulatory properties of exogenous as well as recently described autocrine IFNs, (ii) down-regulation of cellular oncogene expression with emphasis on c-myc and (iii) the possible involvement of the IFN-regulated 2-5A pathway at these levels. Initially described as a part of the IFN-induced antiviral mechanism, this double-stranded RNA-activated pathway leads to the preferential degradation of viral mRNAs in IFN-treated virus-infected cells probably through localized activation at the site of virus replication. Such mechanisms could be involved in the regulation of the stability of rapidly turning over mRNAs as for instance c-myc mRNA in IFN-treated cells. Whatever the elegance of the concept, however, experimental evidence is essentially circumstantial; tools developed in our group to strengthen the demonstration are briefly described.