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P F Torrence

Publications and source records attributed to P F Torrence.

At least 37 records · Page 2Linked to original sources
PubMed1998

The 2-5A system: modulation of viral and cellular processes through acceleration of RNA degradation.

The 2-5A system is an RNA degradation pathway that can be induced by the interferons (IFNs). Treatment of cells with IFN activates genes encoding several double-stranded RNA (dsRNA)-dependent synthetases. These enzymes generate 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A) from ATP. The effects of 2-5A in cells are transient since 2-5A is unstable in cells due to the activities of phosphodiesterase and phosphatase. 2-5A activates the endoribonuclease 2-5A-dependent RNase L, causing degradation of single-stranded RNA with moderate specificity. The human 2-5A-dependent RNase is an 83.5 kDa polypeptide that has little, if any, RNase activity, unless 2-5A is present. 2-5A binding to RNase L switches the enzyme from its off-state to its on-state. At least three 2',5'-linked oligoadenylates and a single 5'-phosphoryl group are required for maximal activation of the RNase. Even though the constitutive presence of 2-5A-dependent RNase is observed in nearly all mammalian cell types, cellular amounts of 2-5A-dependent mRNA and activity can increase after IFN treatment. One well-established role of the 2-5A system is as a host defense against some types of viruses. Since virus infection of cells results in the production and secretion of IFNs, and since dsRNA is both a frequent product of virus infection and an activator of 2-5A synthesis, the replication of encephalomyocarditis virus, which produces dsRNA during its life cycle, is greatly suppressed in IFN-treated cells as a direct result of RNA decay by the activated 2-5A-dependent RNase. This review covers the organic chemistry, enzymology, and molecular biology of 2-5A and its associated enzymes. Additional possible biological roles of the 2-5A system, such as in cell growth and differentiation, human immunodeficiency virus replication, heat shock, atherosclerotic plaque, pathogenesis of Type I diabetes, and apoptosis, are presented.

Adenine Nucleotides↗
PubMed1998

Targeting RNase L to human immunodeficiency virus RNA with 2-5A-antisense.

In an attempt to develop a lead for the application of 2-5A-antisense to the targeted destruction of human immunodeficiency virus (HIV) RNA, specific target sequences within the HIV mRNAs were identified by analysis of the theoretical secondary structure. 2-5A-antisense chimeras were chosen against a total of 11 different sequences: three in the gag mRNA, three in the rev mRNA and five in the tat mRNA. 2-5A-antisense chimera synthesis was accomplished using solid-phase phosphoramidite chemistry. These chimeras were evaluated for their activity in a cell-free assay system using purified recombinant human RNase L to effect cleavage of 32P-labelled RNA transcripts of plasmids derived from HIV NL4-3. This screening revealed that of the three 2-5A-antisense chimeras targeted against gag mRNA, only one had significant HIV RNA cleavage activity, approximately 10-fold-reduced compared to the parent 2-5A tetramer and comparable to that reported for the prototypical 2-5A-anti-PKR chimera, targeted against PKR mRNA. The cleavage activity of this chimera was specific, since a scrambled antisense domain chimera and a chimera without the key 5'-monophosphate moiety were both inactive. The 10 other 2-5A-antisense chimeras against tat and rev had significantly less activity. These results imply that HIV gag RNA, like PKR RNA and a model HIV tat-oligoA-vif RNA, can be cleaved using the 2-5A-antisense approach. The results further imply that not all regions of a potential RNA target are accessible to the 2-5A-antisense approach.

Adenine Nucleotides↗
PubMed1997

A study of the interferon antiviral mechanism: apoptosis activation by the 2-5A system.

The 2-5A system contributes to the antiviral effect of interferons through the synthesis of 2-5A and its activation of the ribonuclease, RNase L. RNase L degrades viral and cellular RNA after activation by unique, 2'-5' phosphodiester-linked, oligoadenylates [2-5A, (pp)p5' A2'(P5'A2')]n, n >=2. Because both the 2-5A system and apoptosis can serve as viral defense mechanisms and RNA degradation occurs during both processes, we investigated the potential role of RNase L in apoptosis. Overexpression of human RNase L by an inducible promoter in NIH3T3 fibroblasts decreased cell viability and triggered apoptosis. Activation of endogenous RNase L, specifically with 2-5A or with dsRNA, induced apoptosis. Inhibition of RNase L with a dominant negative mutant suppressed poly (I).poly (C)-induced apoptosis in interferon-primed fibroblasts. Moreover, inhibition of RNase L suppressed apoptosis induced by poliovirus. Thus, increased RNase L levels induced apoptosis and inhibition of RNase L activity blocked viral-induced apoptosis. Apoptosis may be one of the antiviral mechanisms regulated by the 2-5A system.

3T3 Cells↗
PubMed1997

Synthesis and properties of second-generation 2-5A-antisense chimeras with enhanced resistance to exonucleases.

In order to stabilize 2-5A-antisense chimeras to exonucleases, we have synthesized chimeric oligonucleotides in which the last phosphodiester bond at the 3'-terminus of the antisense domain was inverted from the usual 3',5'-linkage to a 3',3'-linkage. The preparation of such analogues was accomplished through standard phosphoramidite chemistry with the use of a controlled pore glass solid support with a nucleoside attached through its 5'-hydroxyl, thereby permitting elongation at the 3'-hydroxyl. The structures of such terminally inverted linkage chimeras of the general formula pA4-[pBu]2-(pdNn3'-3'dN) were corroborated by a combination of snake venom phosphodiesterase digestion in the presence or absence of bacterial alkaline phosphatase. Most characteristically, the presence of the 3'-terminal-inverted phosphodiester linkage produced an unnatural dinucleotide of general composition dN3'p3'dM. These structures could be confirmed by independent synthesis and fast atom bombardment mass spectroscopy (FAB). 2-5A-Antisense chimeras of this structural class, pA4-[pBu]2-(pdNn'3-3'dN), were 5-6-fold more stable than their unmodified congeners, pA4-[pBu]2-(pdN)n, to degradation by a representative phosphodiesterase from snake venom. In 10% human serum, the new 2-5A-antisense chimeras, pA4-[pBu]2-(pdNn3'-3'dN), possessed a half-life that was 28-fold longer than that of the unmodified chimeras. These results provide entry to a second generation of 2-5A-antisense chimeras.

Adenine Nucleotides↗
PubMed1997

Correlation of selective modifications to a 2',5'-oligoadenylate-3',5'-deoxyribonucleotide antisense chimera with affinity for the target nucleic acid and with ability to activate RNase L.

The use of an antisense oligonucleotide to address a specific targeted RNA sequence and subsequent localized activation of the 2-5A-dependent RNase (RNase L) to effect selective RNA degradation is a new approach to the control of gene expression called 2-5A-antisense. The previously reported biological activity of the 2-5A:AS chimeric oligonucleotide [p5'(A2'p)3A-antiPKR1], directed against nucleotides 55-73 of the coding sequence of the PKR mRNA, has been used as a point of reference to examine the effect of introducing mismatches into the chimeric oligonucleotide, altering the chain length of the antisense domain of the chimeras, removal of the 5'-monophosphate moiety, shortening the 2',5'-oligoadenylate domain, and substitution of 3',5'-linked 2'-deoxyadenosine nucleotides for the 2-5A domain. The general formula for the novel chimeric oligonucleotides is p5'(A2'p)3A2'p(CH2)4p(CH2)4p(5'N3'p)mN, where N is any nucleoside and m is any integer. When the biological activity of these new chimeric oligonucleotides was compared to that of the parent chimera, 2-5A-aPKR, for their ability to effect target PKR RNA cleavage in a cell-free and in an intact cell assay, it was determined that there was a close correlation between the activity of 2-5A-antisense chimeras and their affinity (Tm) for a targeted nucleic acid. In addition, there was also a close correlation between activity of the 2-5A-antisense chimeras and their ability to activate the 2-5A-dependent RNase L.

Adenine Nucleotides↗
PubMed1997

Targeting RNA decay with 2',5' oligoadenylate-antisense in respiratory syncytial virus-infected cells.

Treatment of human cells with 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) results in the selective cleavage of targeted RNA species by 2-5A-dependent RNase L. Here we show that 2-5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2-5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2-5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2-5A linked to antisense, 2-5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2-5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2-5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.

Adenine Nucleotides↗
PubMed1996

Neighboring group catalysis in the design of nucleotide prodrugs.

An approach is described for potential application to the delivery of polar nucleosides and nucleotides across lipophilic membranes, namely, nucleotide prodrugs based on salicyl phosphate. 3'-Azido-3'-deoxythymidine (AZT) and 3'-deoxythymidine (ddT) were chosen as models. For the synthesis of prototype compounds 1 and 2, the approach was first to react either methyl salicylate (for 1) or phenyl salicylate (for 2) with phosphorus oxychloride in dry methylene chloride at 0 degree C with the addition of triethylamine as acid scavenger. The resulting intermediate phosphorodichloridate was reacted immediately with excess nucleoside under the same conditions. The control model compound 3 was prepared by reaction of phenyl phosphorodichloridate and excess nucleoside in pyridine/methylene chloride at 0 degree C to give 3 in 82% yield. The synthesis of triester 7 involved reaction of alpha-(chloroacetyl)salicyl chloride with 2,3,4,6-tetra-O-benzyl-D-glucopyranose to give [[(2,3,4,6-tetra-O-benzyl-D-glucopyranosyl)-oxy]carbonyl]-2- (1-chloroacetoxy)benzene (4) which was dechloroacetylated to 5,2,3,4,6-tetra-O-benzyl-D-glucopyranosyl salicylate. Phosphorylation of 5 with phosphorus oxychloride provided the phosphorodichloridate which was directly converted to 6 by reaction with dideoxythymidine. Removal of benzyl groups by catalytic hydrogenation gave compound 7, bis(2',3'-dideoxythymidin-5'-yl) D-glucopyranosyl phosphate. The AZT prodrug triesters, 1 and 2, underwent much more rapid hydrolysis than the triester 3, most probably due to the formation of an acyl phosphate complex from the attack on phosphorus of the salicylate carboxylate. The hydrolysis of the less lipophilic 7 was significantly slower than that of 1 or 2. Both pig liver esterase and rat brain cytosol were able to effect the cleavage to dinucleotide or mononucleotide of prodrug forms 2 and 7, much more rapidly than either 3 or 1, suggesting that the esterase-like enzymatic activity of rat brain was similar to that of pig liver esterase. This study suggests the possibility of use of salicylic acid-based prodrugs for nucleotides, subject to specific refinements in the choice of carboxylate- and phosphoric acid ester-protecting groups.

Animals↗
PubMed1996

Synthesis and characterization of composite nucleic acids containing 2', 5'-oligoriboadenylate linked to antisense DNA.

Composite nucleic acids, known as 2-5A antisense chimeras, cause the 2-5A-dependent ribonuclease (RNase L) to catalyze the specific cleavage of RNA in cell free systems and in intact cells. Such 2-5A antisense chimeras are 5'-monophosphorylated, 2,'5'-linked oligoadenylates covalently attached to antisense 3',5'-oligodeoxyribonucleotides by means of a linker containing two residues of 1,4-butanediol phosphate. Here we report a fully automated synthesis of 2-5A antisense chimeras on a solid support using phosphoramidite methodology with specific coupling time modifications and their subsequent purification by reverse-phase ion-pair and anion exchange HPLC. Purified 2-5A antisense chimeras were characterized by [1H]NMR and [31P]NMR, MALDIMS, and capillary gel electrophoresis. The synthetic 2',5'-linked oligoadenylate showed no phosphodiester isomerization to 3',5' during or after synthesis. In addition, we have developed facile methodologies to characterize the chimeras using digestion with various hydrolytic enzymes including snake venom phosphodiesterase I and nuclease P1. Finally, Maxam-Gilbert chemical sequencing protocols have been developed to confirm the entire sequence of these chimeric oligonucleotides.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Synthesis and biological activities of a phosphorodithioate analog of 2',5'-oligoadenylate.

To enhance the resistance of 2-5A (pppA2'p5'A2'p5'A) to degradation by exo- and endonucleases, a phosphorodithioate analog was synthesized using a solid-phase phosphite triester approach with N6-benzoyl-5'-O-dimethoxytrityl-3'-O-t-butyldimethylsilyladenosine 2'-[S-(beta-thiobenzoylethyl)-pyrrolidinophosphorothioamidit e]. 5'-Monophosphorylation was accomplished with 2-[2-(4,4'-dimethoxytrityloxy)-ethylsulfonyl]ethyl-(2-cyanoe thyl)-(N,N- diisopropyl)-phosphoramidite. The resulting product, p5'A2'(s2p)- 5'A2'(s2p)5'A, was approximately 10-fold less effective as an activator of purified human recombinant 2-5A-dependent RNase than was 2-5A itself. This loss of activation ability was related directly to the loss of binding ability of the phosphorodiothioate analog. As predicted, p5'A2'(s2p)5'A2' (s2p)5'A was stable to snake venom phosphodiesterase and the nucleolytic activities of both human lymphoblastoid CEM cell extracts and human serum, under conditions that led to facile degradation of parent 2-5A. This nuclease stability permitted the observation of the CEM cell extracts and human serum phosphatase activity which led to 5'-dephosphorylation of p5'A2'(s2p)5'A2'(s2p)5'A.

Adenine Nucleotides↗
PubMed1995

Catalytic cleavage of an RNA target by 2-5A antisense and RNase L.

2-5A antisense (2-5A-AS) molecules are chimeric oligonucleotides that cause 2-5A-dependent RNase (RNase L) to catalyze the selective cleavage of RNA in human cells. These composite nucleic acids consist of a 5'-monophosphorylated, 2',5'-linked oligoadenylate known as 2-5A (an activator of RNase L) covalently attached to antisense 3',5'-oligodeoxyribonucleotides. Here, we characterize the targeted cleavage of the double-stranded RNA-dependent protein kinase (PKR) mRNA by purified, recombinant human RNase L. A 2-5A-AS chimera, which contains complementary sequence to PKR mRNA, and unmodified 2-5A, which causes general RNA decay, were about 20- and 40-fold more active, respectively, than 2-5A-AS chimeras in which the DNA domains are not complementary to sequences in PKR mRNA. Directed cleavage was efficient because each 2-5A-AS chimera targeted many RNA molecules. Moreover, RNase L caused the catalytic cleavage of the RNA target (kcat of approximately 7 s-1). The precise sites of PKR mRNA cleavage caused by 2-5A-AS were mapped, using a primer extension assay, to phosphodiester bonds adjacent to the 3' terminus of the chimera binding site (5' on the RNA target) as well as within the chimera's oligonucleotide binding site itself. The selectivity of this approach is shown to be provided by the antisense arm of the chimera, which places the RNA target in close proximity to the RNase.

Base Sequence↗
PubMed1995

Effect of interferon and 2',5'-oligoadenylates on rotavirus RNA synthesis.

Based on the antiviral effect of interferon on rotavirus replication the inhibitory effect of 2',5'-oligoadenylates on MRNA and double-stranded RNA synthesis was studied using an in vitro assay. The chemically synthesized oligonucleotides were used to determine several characteristics of the inhibitory effect, such as chain length, presence of phosphate residues at the 5'-end, and the 2',5'-phosphodiester bond itself. In vitro transcription was inhibited by oligos with 5 or more adenine residues at a final concentration of 100 microM or greater. This result makes rotavirus transcriptase different from other viruses in which the inhibitory effects are associated with dinucleotides and trinucleotides. The inhibitory effect was increased when the oligo contained a phosphate residue at the 5'-end; in this case, inhibition was also seen at lower oligo concentrations as well as at shorter oligo chain length. The study of the kinetics of inhibition showed that the inhibition by p(A2'p5')(3)3A was competitive with a Ki value of 256 microM. The effect of the oligonucleotides on the in vitro viral RNA replication showed that the 2',5'-oligoadenylates were not able to significantly inhibit the in vitro rotavirus RNA synthesis. The lack of inhibition in the in vitro assay was very peculiar since RNA transcription and replication involves the viral RNA polymerase, VP1.

Adenine Nucleotides↗
PubMed1994

Blockage of NF-kappa B signaling by selective ablation of an mRNA target by 2-5A antisense chimeras.

Activation of 2-5A-dependent ribonuclease by 5'-phosphorylated, 2',5'-linked oligoadenylates, known as 2-5A, is one pathway of interferon action. Unaided uptake into HeLa cells of 2-5A linked to an antisense oligonucleotide resulted in the selective ablation of messenger RNA for the double-stranded RNA (dsRNA)-dependent protein kinase PKR. Similarly, purified, recombinant human 2-5A-dependent ribonuclease was induced to selectively cleave PKR messenger RNA. Cells depleted of PKR activity were unresponsive to activation of nuclear factor-kappa B (NF-kappa B) by the dsRNA poly(I):poly(C), which provides direct evidence that PKR is a transducer for the dsRNA signaling of NF-kappa B.

Adenine Nucleotides↗
PubMed1994

Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase.

2-5A-dependent RNase (RNase L), a unique endoribonuclease that requires 5'-phosphorylated 2',5'-linked oligoadenylates (2-5A), functions in the molecular mechanism of interferon action. Because this enzyme is present at very low levels in nature, characterization and analysis have been limited. The molecular cloning of human, 2-5A-dependent RNase cDNA has facilitated its expression to high levels in insect cells by infecting with recombinant baculovirus. To determine the properties of the enzyme in the absence of other proteins, the recombinant 2-5A-dependent RNase was purified to homogeneity. The purified enzyme migrated as a monomer upon gel filtration in the absence of activator and showed highly specific, 2-5A-dependent RNase activity. The precise activator requirements were determined by stimulating the purified enzyme with a variety of 2',5'-linked oligonucleotides. The activated enzyme was capable of cleaving poly(rU) and, to a lesser extent, poly(rA), to sets of discrete products ranging from between 4 and 22 nucleotides in length. Reduced rates of 2-5A-dependent RNA cleavage were observed even after removal of ATP and chelation of divalent cations. However, optimal RNA cleavage rates required the presence of either manganese or magnesium and ATP.

Adenosine Triphosphate↗
PubMed1993

Synthesis and pharmacokinetics of a dihydropyridine chemical delivery system for the antiimmunodeficiency virus agent dideoxycytidine.

In order to explore the possibility that a dihydropyridine/pyridinium redox chemical delivery system might enhance significantly the brain uptake of the anti-HIV agent dideoxycytidine (DDC), we prepared a DDC derivative which bore the 1,4-dihydro-1-methyl-3-pyridylcarbonyl moiety at both the cytidine exocyclic amino moiety and the sugar 5'-hydroxyl function; namely, 5',4N-bis-[(1,4-dihydro-1-methyl-3-pyridinyl)carbonyl]-2',3'- dideoxycytidine (2). In cell-free extracts of rat brain tissue, compound 2 was readily converted to free DDC by stepwise oxidation and hydrolysis of the dihydropyridyl groups. Time-dependent plasma and brain concentrations of DDC and 2 were determined following iv administration of 2 (49.3 mg/kg) to rats. Compound 2 could be detected in brain, reaching peak concentrations of 7.7 +/- 2.9 nmol/g at 15 min. Low levels of DDC also were detected with a peak concentration of 1.4 +/- 0.5 nmol/g at 240 min after injection. The brain/plasma concentration integral of compound 2 was 0.95 whereas that for DDC in brain as a ratio of combined DDC and compound 2 levels in plasma was 0.24. Despite this, brain concentrations remained low and not significantly different from those achieved following administration of DDC alone.

Animals↗
PubMed1993

Targeting RNA for degradation with a (2'-5')oligoadenylate-antisense chimera.

Antisense oligonucleotides hold considerable promise both as research tools for inhibiting gene expression and as agents for the treatment of a myriad of human diseases. However, targeted destruction of RNA has been difficult to achieve in a versatile, efficient, and reliable manner. We have developed an effective strategy for cleaving unique RNA sequences with 2-5A-dependent RNase, an endoribonuclease that mediates inhibitory effects of interferon on virus infection and is activated by 5'-phosphorylated 2'-5'-linked oligoadenylates known as 2-5A [pn5' A2'(p5' A2')mp5'A], resulting in the cleavage of single-stranded RNA predominantly after UpUp and UpAp sequences. To direct 2-5A-dependent RNase to cleave unique RNA sequences, p5' A2' p5' A2'p5'A was covalently linked to an antisense oligonucleotide to yield a chimeric molecule (2-5A:AS). The antisense oligonucleotide component of 2-5A:AS bound a specific RNA sequence while the accompanying 2-5A component activated 2-5A-dependent RNase, thereby causing the cleavage of the RNA in the targeted sequence. This strategy was demonstrated by inducing specific cleavage within a modified human immunodeficiency virus type 1 vif mRNA in a cell-free system from human lymphoblastoid cells. Because 2-5A-dependent RNase is present in most mammalian cells, the control of gene expression based on this technology--including therapies for cancer, viral infections, and certain genetic diseases--can be envisioned.

Adenine Nucleotides↗
PubMed1992

A new and potent 2-5A analogue which does not require a 5'-polyphosphate to activate mouse L-cell RNase L.

In order to explore the possibility of supplanting the requirement of a 5'-triphosphate moiety for the activation of the 2-5A-dependent endonuclease (RNase L) of mouse L-cells, two new tetrameric analogues of 2-5A were synthesized. The first tetramer, obtained by both a modified prebiotic synthetic approach as well as a phosphite triester solid phase oligonucleotide synthesis method, was p5'A2'p5'A2'p5'(br8A)2'p5'(br8A). The second oligonucleotide was derived from the former by a sequence involving periodate oxidation, reaction with n-hexylamine, and cyanoborohydride reduction, resulting in conversion of the 2'-terminal adenosine residue to 9-(3'-aza-4'-hexyl-1',2',3',4'-tetradeoxyhexopyranos-1(1)-yl)-8-++ +bromoadenine. Both of these oligomers, bearing only 5'-monophosphate groups, were found to be as potent as 2-5A itself as activators of the RNase L of mouse L-cells.

Adenine Nucleotides↗