DNA Polymerase Beta Participates in Mitochondrial DNA Repair.

We have detected DNA polymerase beta (Polß), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polß in the mitochondria. Using Polß fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Polß directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Polß knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Polß mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Polß null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Polß caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Polß is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.

Loss of caspase-3 sensitizes colon cancer cells to genotoxic stress via RIP1-dependent necrosis.

Caspase-3 is the best known executioner caspase in apoptosis. We generated caspase-3 knockout (C3KO) and knockdown human colorectal cancer cells, and found that they are unexpectedly sensitized to DNA-damaging agents including 5-fluorouracil (5-FU), etoposide, and camptothecin. C3KO xenograft tumors also displayed enhanced therapeutic response and cell death to 5-FU. C3KO cells showed intact apoptosis and activation of caspase-7 and -9, impaired processing of caspase-8, and induction of necrosis in response to DNA-damaging agents. This form of necrosis is associated with HMGB1 release and ROS production, and suppressed by genetic or pharmacological inhibition of RIP1, MLKL1, or caspase-8, but not inhibitors of pan-caspases or RIP3. 5-FU treatment led to the formation of a z-VAD-resistant pro-caspase-8/RIP1/FADD complex, which was strongly stabilized by caspase-3 KO. These data demonstrate a key role of caspase-3 in caspase-8 processing and suppression of DNA damage-induced necrosis, and provide a potentially novel way to chemosensitize cancer cells.

DNA polymerase beta mediates protection of mammalian cells against ganciclovir-induced cytotoxicity and DNA breakage.

The efficacy of suicide herpes simplex virus-1 thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy is often limited by intrinsic resistance of tumor cells. Here we show that repair of GCV incorporated in DNA is a factor involved in GCV resistance. A protective role of DNA repair in GCV-induced cell killing is supported by the following findings: (a) GCV-exposed Chinese hamster ovary-HSVtk cells exhibited both reduced repair of GCV and cloning efficiency in the presence of a specific polymerase beta (beta-pol) inhibitor, prunasin; (b) DNA beta-pol-deficient mouse fibroblasts were more sensitive to the cytotoxic, apoptosis-inducing, and genotoxic (DNA breakage and chromosomal aberration-inducing) effects of GCV as compared with wild-type and beta-pol-complemented cell lines; (c) methoxyamine, an inhibitor of beta-pol-dependent short-patch base excision repair, sensitized wild-type and complemented beta-pol cells to GCV, whereas it had no effect on the sensitivity of beta-pol-null cells to GCV. Because methoxyamine-mediated sensitization of beta-pol wild-type and beta-pol-complemented cells to GCV did not reach the level of null cells, we suggest that both beta-pol-dependent short- and long-patch base excision repair are involved in protection of cells to GCV. Some implications for HSVtk/GCV gene therapy are being discussed.

Mammalian DNA beta-polymerase in base excision repair of alkylation damage.

DNA beta-polymerase (beta-pol) carries out two critical enzymatic reactions in mammalian single-nucleotide base excision repair (BER): DNA synthesis to fill the repair patch and lyase removal of the 5'-deoxyribose phosphate (dRP) group following cleavage of the abasic site by apurinic/apyrimidinic (AP) endonuclease (1). The requirement for beta-pol in single-nucleotide BER is exemplified in mouse fibroblasts with a null mutation in the beta-pol gene. These cells are hypersensitive to monofunctional DNA methylating agents such as methyl methane-sulfonate (MMS) (2). This hypersensitivity is associated with an abundance of chromosomal damage and induction of apoptosis and necrotic cell death (3). We have found that beta-pol null cells are defective in repair of MMS-induced DNA lesions, consistent with a cellular BER deficiency as a causative agent in the observed hypersensitivity. Further, the N-terminal 8-kDa domain of beta-pol, which contains the dRP lyase activity in the wild-type enzyme, is sufficient to reverse the methylating agent hypersensitivity in beta-pol null cells. These results indicate that lyase removal of the dRP group is a pivotal step in BER in vivo. Finally, we examined MMS-induced genomic DNA mutagenesis in two isogenic mouse cell lines designed for study of the role of BER. MMS exposure strongly increases mutant frequency in beta-pol null cells, but not in wild-type cells. With MMS treatment, beta-pol null cells have a higher frequency of all six base-pair substitutions, suggesting that BER plays a role in protecting the cell against methylation-induced mutations.

Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair.

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.

The lyase activity of the DNA repair protein beta-polymerase protects from DNA-damage-induced cytotoxicity.

Small DNA lesions such as oxidized or alkylated bases are repaired by the base excision repair (BER) pathway. BER includes removal of the damaged base by a lesion-specific DNA glycosylase, strand scission by apurinic/apyrimidinic endonuclease, DNA resynthesis and ligation. BER may be further subdivided into DNA beta-polymerase (beta-pol)-dependent single-nucleotide repair and beta-pol-dependent or -independent long patch repair subpathways. Two important enzymatic steps in mammalian single-nucleotide BER are contributed by beta-pol: DNA resynthesis of the repair patch and lyase removal of 5'-deoxyribose phosphate (dRP). Fibroblasts from beta-pol null mice are hypersensitive to mono-functional DNA-methylating agents, resulting in increases in chromosomal damage, apoptosis and necrotic cell death. Here we show that only the dRP lyase activity of beta-pol is required to reverse methylating agent hypersensitivity in beta-pol null cells. These results indicate that removal of the dRP group is a pivotal step in BER in vivo. Persistence of the dRP moiety in DNA results in the hypersensitivity phenotype of beta-pol null cells and may signal downstream events such as apoptosis and necrotic cell death.

Cells deficient in DNA polymerase beta are hypersensitive to alkylating agent-induced apoptosis and chromosomal breakage.

DNA polymerase beta (beta-pol), which is involved in base excision repair, was investigated for its role in protection of cells against various genotoxic agents and cytostatic drugs using beta-pol knockout mouse fibroblasts. We show that cells lacking beta-pol are highly sensitive to induction of apoptosis and chromosomal breakage by methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and methyl methanesulfonate and the cross-linking antineoplastic drugs mitomycin C and mafosfamide. The cross-sensitivity between the agents observed suggests that beta-pol is involved in repair not only of DNA methylation lesions but also of other kinds of DNA damage induced by various cytostatic drugs. Cells deficient in beta-pol were not hypersensitive to cisplatin, melphalan, benzo(a)pyrene diol epoxide, chloroethylnitrosourea, or UV light. Because both established and primary beta-pol knockout fibroblasts displayed the hypersensitive phenotype, which, moreover, was complemented by transfection with a beta-pol expression vector, the alkylating agent hypersensitivity can clearly be attributed to the beta-pol deficiency. The results demonstrate that beta-pol-driven base excision repair is highly important for protection of cells against cell killing due to apoptosis and induced chromosomal breakage and suggest that incompletely repaired DNA damage causes chromosomal changes and may act as a trigger of DNA damage-induced apoptosis.

Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.

DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.

Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells.

Mammalian cells possess two distinct pathways for completion of base excision repair (BER): the DNA polymerase beta (Pol beta)-dependent short-patch pathway (replacement of one nucleotide), which is the main route, and the long-patch pathway (resynthesis of 2-6 nucleotides), which is PCNA-dependent. To address the issue of how these two pathways share their role in BER the ability of Pol beta-defective mammalian cell extracts to repair a single abasic site constructed in a circular duplex plasmid molecule was tested in a standard in vitro repair reaction. Pol beta-deficient extracts were able to perform both BER pathways. However, in the case of the short-patch BER, the repair kinetics was significantly slower than with Pol beta-proficient extracts, while the efficiency of the long-patch synthesis was unaffected by the loss of Pol beta. The repair synthesis was fully dependent on PCNA for the replacement of long patches. These data give the first evidence that in cell extracts DNA polymerases other than Pol beta are specifically involved in the long-patch BER. These DNA polymerases are also able to perform short-patch BER in the absence of PCNA, although less efficiently than Pol beta. These findings lead to a novel model whereby the two BER pathways are characterized by different protein requirements, and a functional redundancy at the level of DNA polymerases provides cells with backup systems.

Impairment of proliferating cell nuclear antigen-dependent apurinic/apyrimidinic site repair on linear DNA.

Repair of apurinic/apyrimidinic (AP) sites by mammalian cell extracts was compared using circular and linear DNA substrates. Extracts prepared from DNA polymerase beta (polbeta)-proficient mouse fibroblasts repaired AP sites on both circular and linear DNA. However, extracts from the isogenic polbeta-knockout cells repaired AP sites on circular DNA but not efficiently on linear DNA. The circularity-dependent repair by the polbeta-knockout cell extract was completely inhibited by anti-proliferating cell nuclear antigen (PCNA) antibody but fully restored by addition of purified PCNA. Pretreatment of the linear DNA with AP endonuclease did not improve repair, indicating that impairment of AP site repair on linear DNA by polbeta-knockout cell extracts is not due to inefficiency of damage incision but rather to deficiency at the subsequent steps. These results indicate that AP sites can be repaired on circular DNA by the PCNA-dependent pathway in addition to the polbeta-dependent pathway and that the PCNA-dependent repair mechanism is poorly functional on linear DNA in vitro.

Requirement of mammalian DNA polymerase-beta in base-excision repair.

Synthesis of DNA by DNA polymerase-beta is distributive on single-stranded DNA templates, but short DNA gaps with a 5' PO4 in the gap are filled processively to completion. In vitro studies have suggested a role of beta-polymerase in different types of DNA repair. However, the significance of these studies to the in vivo role of beta-polymerase has remained unclear. Because genetic studies are essential for determining the physiological role of a gene, we established embryonic fibroblast cell lines homozygous for a deletion mutation in the gene encoding DNA polymerase-beta. Extracts from these cell lines were found to be defective in uracil-initiated base-excision repair. The beta-polymerase-deleted cells are normal in viability and growth characteristics, although they exhibit increased sensitivity to monofunctional DNA-alkylating agents, but not to other DNA-damaging agents. Both the deficiency in base-excision repair and hypersensitivity to DNA-alkylating agents are rescued following stable transfection with a wild-type beta-polymerase minitransgene. These studies demonstrate that beta-polymerase functions specifically in base-excision repair in vivo.

Inhibition of HIV-1 replication and activation of RNase L by phosphorothioate/phosphodiester 2',5'-oligoadenylate derivatives.

2',5'-Oligoadenylate (2-5A) derivatives have been designed to act distal to the human immunodeficiency virus-1 (HIV-1)-induced blockade in the 2-5A synthetase/RNase L antiviral pathway. Stereochemical modification of individual internucleotide linkages of the 2-5A molecule was accomplished by phosphoramidite and phosphotriester chemical syntheses. Phosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives revealed differences in the stereodynamics of activation of RNase L and inhibition of HIV-1 replication. The first and second internucleotide linkages are critical for activation of recombinant, human RNase L; A(Rp)ApA, A(Sp)ApA and ApA(Rp)A are agonists (IC50 = 2 x 10(-7), 2 x 10(-6) and 8 x 10(-6) M); ApA(Sp)A is an antagonist. The second and third internucleotide linkages are crucial for activation of murine RNase L; ApA(Rp)A, ApA(Rp)ApA, and ApApA(Rp)A are agonists (IC50 = 5 x 10(-7) M); ApA(Sp)A, ApA(Sp)ApA, and ApApA(Sp)A are antagonists. Inhibition of HIV-1-induced syncytia formation by the phosphorothioate/phosphodiester derivatives is specific for derivatives with substitution at the 2',3'-terminus. ApA(Rp)A, ApA(Sp)A, ApApA(Rp)A, and ApApA(Sp)A are potent inhibitors of HIV-1-induced syncytia formation (80-, 10-, 40-, and 15-fold more inhibitory, respectively, than solvent control). HIV-1 infection results in enhanced uptake and accumulation of ApA(Rp)A and ApA(Sp)A (7- and 10-fold, respectively). These stereochemically modified 2-5A derivatives are taken up preferentially by HIV-1-infected cells and show promise in anti-HIV-1 chemotherapy.

Upregulation of the 2-5A synthetase/RNase L antiviral pathway associated with chronic fatigue syndrome.

Levels of 2',5'-oligoadenylate (2-5A) synthetase, bioactive 2-5A, and RNase L were measured in extracts of peripheral blood mononuclear cells (PBMCs) from 15 individuals with chronic fatigue syndrome (CFS) before and during therapy with the biological response modifier poly(I).poly(C12U) and were compared with levels in healthy controls. Patients differed significantly from controls in having a lower mean basal level of latent 2-5A synthetase (P < .0001), a higher pretreatment level of bioactive 2-5A (P = .002), and a higher level of pretherapy RNase L activity (P < .0001). PBMC extracts from 10 persons with CFS had a mean basal level of activated 2-5A synthetase higher than the corresponding control value (P = .009). All seven pretherapy PBMC extracts tested were positive for the replication of human herpesvirus 6 (HHV-6). Therapy with poly(I).poly(C12U) resulted in a significant decrease in HHV-6 activity (P < .01) and in downregulation of the 2-5A synthetase/RNase L pathway in temporal association with clinical and neuropsychological improvement. The upregulated 2-5A pathway in CFS before therapy is consistent with an activated immune state and a role for persistent viral infection in the pathogenesis of CFS. The response to therapy suggests direct or indirect antiviral activity of poly(I).poly(C12U) in this situation.

HIV-1 reverse transcriptase: inhibition by 2',5'-oligoadenylates.

2',5'-Oligoadenylates (2-5A) and derivatives are noncompetitive inhibitors of primer/HIV-1 reverse transcriptase complex formation. The mechanism and specificity of this inhibitory action of 2-5A and 2-5A derivatives have been evaluated with 2-5A molecules modified in ribosyl moiety, chain length, extent of 5'-phosphorylation, and 2',5'-phosphodiester linkage. UV covalent cross-linking of preformed complexes of p66/p66 homodimer or p66/p51 heterodimer recombinant HIV-1 reverse transcriptase and the primer analog pd(T)16 allowed analysis of the initial step in HIV-1 reverse transcriptase-catalyzed DNA synthesis. Utilizing this primer binding assay, it is demonstrated that 2-5A and 2-5A derivatives inhibit the binding of pd(T)16 to HIV-1 reverse transcriptase. This inhibition is specific for the 2',5'-internucleotide linkage in that the corresponding 3',5'-adenylate derivatives do not exhibit inhibitory activity. Enhanced inhibitory properties were observed following modifications of the 2-5A molecule which result in an increase in hydrophobicity. Replacement of the D-ribosyl moiety of 2-5A with the 3'-deoxyribosyl moiety increased the inhibition of primer/HIV-1 reverse transcriptase complex formation 15-20%. 2',5'-Phosphorothioate substitution yielded the most effective inhibitors, with Ki's of 7-13 microM. In all cases, inhibition of primer/HIV-1 reverse transcriptase complex formation showed a preference for the 5'-triphosphate moiety. Nonphosphorylated derivatives were not inhibitory; 5'-monophosphate derivatives exhibited little or no inhibition. The inhibition of primer binding to HIV-1 reverse transcriptase correlated well with the inhibition of DNA-directed DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of nucleic acid binding in proteolytic domains of HIV-1 reverse transcriptase.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane and renatured after SDS-PAGE; the proteins are then probed with the primer analogues 32P-labeled d(T)16 or 32P-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 approximately equal to 300. By contrast, a C-terminal domain fragment termed p30(303 approximately equal to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical synthesis and biological characterization of phosphorothioate analogs of 2', 5'-3'-deoxyadenylate trimer.

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate (2-5A) dependent endoribonuclease (RNase L), four 2-5A trimer analogs were examined to evaluate the effect of chirality of phosphorothioate substitution on biological activity. The chemical syntheses and purification of the four isomers of P-thio-3'-deoxyadenylyl-(2'-5')-P-thio-3'- deoxyadenylyl-(2'-5')-3'-deoxyadenosine, by the phosphoramidite approach, is described. The isolated intermediates were characterized by elemental and spectral analyses. The fully deblocked compounds were characterized by 1H and 31P NMR and HPLC analyses. The 2',5'-(3'dA)3 cores with either Rp or Sp chirality in the 2',5'-internucleotide linkages will bind to but will not activate RNase L. This is in contrast to 2',5'-A3 core analogs with either RpRp or SpRp phosphorothioate substitution in the 2',5'-internucleotide linkages which can bind to and activate RNase L. There are also marked differences in the ability of the 2',5'-A3 analogs to activate RNase L following introduction of the 5'-monophosphate. For example, the 5'monophosphates of 2',5'-(3'dA)3-RpRp and 2',5'-(3'dA)3-SpRp can bind to and activate RNase L, whereas the 5'-monophosphates of 2',5'-(3'dA)3-RpSp and 2',5'-(3'dA)3-SpSp can bind to but can not activate RNase L.

Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding.

Properties of primer recognition by purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66 homodimer have been investigated. Earlier studies had shown that RNA-directed DNA synthesis catalyzed by HIV-1 RT proceeds by an ordered mechanism in which template-primer combines with the free enzyme to form the first complex in the reaction scheme, and it was also shown that primer alone is a competitive inhibitor of template-primer. In this study, enzyme-primer binding has been further characterized utilizing pd(T)8 and pd(T)16 as model primers and UV cross-linking to covalently trap the enzyme-primer complexes. Competition experiments with several authentic primers, including tRNA(3Lys), indicate that pd(T)n binds to the kinetically significant primer binding site of RT. Salt reversal experiments suggested that the free energy of pd(T)n binding to RT has a large nonelectrostatic component. Binding of pd(T)n to p66-RT is not affected by dNTPs and does not require the presence of template. The site of UV cross-linking of pd(T)16 was localized to the NH2-terminal half of p66 by use of V8 protease hydrolysis and microsequencing. Our results indicate that a polynucleotide binding site is in close proximity to residues in the peptide comprising amino acids 195 approximately 300. This region could be either a single-stranded template or single-stranded primer binding site; however, we have documented the specificity of binding with oligonucleotides that act as primer in the in vitro DNA synthesis reaction. Therefore, this d(T)16 binding site may be part of a primer-binding groove within the HIV-1 reverse transcriptase.

Heterogeneous nuclear RNA from hairy cell leukemia patients activates 2',5'-oligoadenylate synthetase.

Interferon treatment of cells induces double-stranded RNA (dsRNA)-dependent 2',5' oligoadenylate (2-5A) synthetase, an enzyme which has been implicated in the mechanism of growth arrest in tumour cells. Since interferon (IFN) can inhibit the growth of cells that are not infected with virus, natural non-viral dsRNAs should be present in these cells which can activate 2-5A synthetase. If such nuclear dsRNAs are associated with the mechanism of growth control, cells inherently sensitive to growth inhibition by IFN should contain significant levels of 2-5A synthetase-activating dsRNAs. We measured the ability of size fractionated nuclear dsRNAs isolated from patients with hairy cell leukemia (HCL) to activate purified 2-5A synthetase. Peripheral blood mononuclear cells from HCL patients were utilized because of the inherent sensitivity of these patients to IFN treatment. The heterogeneous nuclear RNA fraction from four out of five HCL patients showed high levels of 2-5A synthetase-activating dsRNAs. The 2-5A formed contained biologically active trimers, tetramers, pentamers and hexamers as demonstrated by HPLC analysis and their ability to activate RNase L. In contrast, the nuclear RNA fraction from three out of four healthy controls were unable to activate 2-5A synthetase. These results indicate that natural, nuclear dsRNAs inherently exist in IFN-sensitive cells and imply that these molecules may play a role in the inhibition of cellular growth.

Cordycepin analogues of 2',5'-oligoadenylate inhibit human immunodeficiency virus infection via inhibition of reverse transcriptase.

Analogues of 2',5'-oligoadenylates (2-5A), the cordycepin (3'-deoxyadenosine) core trimer (Co3) and its 5'-monophosphate derivative (pCo3), were shown to display pronounced anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. Treatment of HIV-1 infected H9 cells with 1 microM Co3 or pCo3 resulted in an almost 100% inhibition of virus production. The compounds were encapsulated in liposomes targeted by antibodies specific for the T-cell receptor molecule CD3. Substitution of one or two cordycepin units in Co3 or pCo3 decreased the antiviral activity of the compounds. pCo3 did not stimulate 2-5A-dependent ribonuclease L activity and displayed no effect on the amount of cellular RNA and protein. At a concentration of 10 microM the cellular DNA polymerases alpha, beta, and gamma were almost insensitive toward Co3 or pCo3. In contrast, these compounds reduced the activity of HIV-1 reverse transcriptase (RT) by 90% at a concentration of 10 microM if the viral RNA genome and the cellular tRNALys.3 was used as template/primer system; if the synthetic poly(A).(dT)10 was used as template/primer, no marked inhibition was observed. Dot-blot, gel-retardation, and cross-linking assays showed that Co3 or pCo3 interfere with the binding site of tRNALys.3 to RT. These results indicate that inhibition of RT at the level of initiation of the enzymic reaction is a novel approach to inhibit HIV-1 replication.