Paul F. Torrence

The 2–5 A system: Modulation of viral and cellular processes through acceleration of RNA degradation

Abstract 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-clependent 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.

AIDS dementia: Synthesis and properties of a derivative of 3′‐azido‐3′‐deoxythymidine (AZT) that may become ‘locked’ in the central nervous system

In an attempt to provide a derivative of 3′‐azido‐3′‐deoxythymidine (AZT) which might be sequestered in the central nervous system and release AZT, the dihydropyridine ester 5′‐(1,4‐dihydro‐1‐methyl‐3‐pyridinylcarbonyl)‐3′‐deoxythymidine, was synthesized in a three step sequence. This material showed potent anti‐HIV‐1 activity in MT‐4 cells most probably by hydrolysis to the parent nucleoside, AZT. This dihydropyridine derivative of AZT could be easily oxidized to a positively charged pyridinium derivative of AZT in rat brain cytosol. In turn the pyridinium form could be hydrolyzed, non‐enzymatically, to AZT.

Effect of Cytosine Arabinoside, lododeoxyuridine, Ethyldeoxyuridine, Thiocyanatodeoxyuridine, and Ribavirin on Tail Lesion Formation in Mice Infected with Vaccinia Virus

Summary Mice infected intravenously with vaccinia virus develop characteristic lesions over the entire tail surface. This experimental virus infection presents a highly sensitive and reliable model for evaluating the antivaccinia activity of antiviral compounds. Ara-C (1 -β-D-arabinofuranosylcytosine), ribavirin (1-β-D-ribofuranosyl-1,2,4-tria-zole-3-carboxamide), IUdR (5-iodo-2′-deoxyuridine) as well as two novel analogs of IUdR, EtUdR (5-ethyl-2′-deoxyuridine), and NCSUdR (5-thiocyanato-2′-deoxyuri-dine), were found to inhibit the formation of vaccinia tail lesions, when administered in-traperitoneally once daily for 7 days starting immediately after virus infection. The order of (decreasing) activity was: ara-C > IUdR > NCSUdR > ribavirin > EtUdR. Various drug combinations, involving IUdR + ara-C, NCSUdR + ara-C, NCSUdR + IUdR, NCSUdR + ribavirin, etc., were evaluated, but none proved more efficacious than either compound administered alone. This investigation was supported by grants from the F.G.W.O. (Fonds voor Geneeskundig Wetenschappel-ijk Onderzoek) and the K.U.L. (Katholieke Universi-teit Leuven) “Fonds Derde Cyclus.” M. L. was supported by a postdoctoral fellowship of the K.U.L. “Fonds Derde Cyclus.” We thank Dr. H. A. Jolley (Cyanamid International Co., Pearl River, New York) and Dr. F. Dehaen (Cyanamid Benelux, Lederle Laboratories Division, Brussels) for the generous supply of ribavirin.

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

ABSTRACT The antiviral activity of a new series of thymidine analogs was determined against vaccinia virus (VV), cowpox virus (CV), herpes simplex virus, and varicella-zoster virus. Several compounds were identified that had good activity against each of the viruses, including a set of novel 5-substituted deoxyuridine analogs. To investigate the possibility that these drugs might be phosphorylated preferentially by the viral thymidine kinase (TK) homologs, the antiviral activities of these compounds were also assessed using TK-deficient strains of some of these viruses. Some of these compounds were shown to be much less effective in the absence of a functional TK gene in CV, which was unexpected given the high degree of amino acid identity between this enzyme and its cellular homolog. This unanticipated result suggested that the CV TK was important in the mechanism of action of these compounds and also that it might phosphorylate a wider variety of substrates than other type II enzymes. To confirm these data, we expressed the VV TK and human TK1 in bacteria and isolated the purified enzymes. Enzymatic assays demonstrated that the viral TK could efficiently phosphorylate many of these compounds, whereas most of the compounds were very poor substrates for the cellular kinase, TK1. Thus, the specific phosphorylation of these compounds by the viral kinase may be sufficient to explain the TK dependence. This unexpected result suggests that selective phosphorylation by the viral kinase may be a promising new approach in the discovery of highly selective inhibitors of orthopoxvirus replication.

The solid-phase synthesis of 2'-5'-linked oligoriboadenylates containing 8-bromoadenine

To increase the accessibility of 8-bromo-2′,5′-oligoadenylates, we developed a synthesis of 2′-5′-linked oligoriboadenylates containing varying numbers of 8-bromoadenosine residues based on the use of a CPG-LCA solid support and the phosphoramidite approach. Although N6benzoyl protection was satisfactory for incorporation of nonmodified adenine residues into 2′,5′-oligonucleotides, the effective incorporation of 8-bromoadenine into such 2′,5′-linked oligomers required use of a non acyl protecting group. Amidine protection of the purine exocyclic amino function proved compatible with all aspects of the phophoramidite approach and with the hydroxyl protection groups employed.

TARGETING RNA FOR DEGRADATION WITH A (2',5')-OLIGOADENYLATE ANTISENSE CHIMERA

Abstract A novel method is described to selectively cleave RNA by harnessing the 2-5A-dependent ribonuclease.

Interferon inducers: general survey and classification

Publisher Summary This chapter outlines various classes of inducers of interferon. Different inducers may lead to the production of different interferons from the same cell culture. When human foreskin fibroblast cultures FS-4 are induced with poly(I) . poly(C), only F (fibroblast) interferon can be detected. Certain inducers are active only in vivo , whereas others are active both in vivo and in vitro . A complete classification of interferon inducers by chemical structure is currently not possible. There is simply too great a variation in structural types represented (polyanions of diverse structure, neutral molecules, basic molecules, proteins, polysaccharides, living organisms). The ideal interferon inducer for use in animals and man should be free from significant toxicity, noninfective, nonantigenic, free of teratogenic or carcinogenic potential, of acceptable cost, orally active, and possess efficacy of sufficient intensity and duration. Interferon inducers have, and continue to play, a major role in understanding the interferon system in particular and eukaryotic gene expression in general.

Activation of human and mouse 2-5A synthetases and mouse protein P1 kinase by nucleic acids: Structure-activity relationships and correlations with inhibition of protein synthesis and interferon induction

The natural occurrence of fully double-stranded RNA (dsRNA) of high molecular weight in viruses, virus-like particles in fungi, as intermediates in the replication of RNA and DNA viruses has suggested some possible biological role for such molecules [ 11. Interest in dsRNA as a possible regulatory signal has been generated by the observations that dsRNA is a potent interferon inducer [2] and a powerful inhibitor

Effect of pyrimidine and ribose modifications on the antiviral activity of synthetic polynucleotides.

Homo- and copolynucleotides derived from 3-methyluridine, 5,6-dihydrouridine and 2′-azido-2′-deoxyuridine (dUz) were prepared and evaluated for antiviral activity either as single strands or as double strands when complexed with complementary homopolynucleotides. The synthetic materials were not as active as (A)n · (U)n or (I)n · (C)n in inducing the interferon system. The antiviral activity of (A)n · (dUz)n was not affected by treatment with high concentrations of pancreatic RNase A, whereas this activity was totally abolished when (A)n · (dUz)n was digested with human serum.

Oligo(2′–5′)adenylate synthetase in human lymphoblastoid cells

The enzyme oligo(2′–5′)adenylate synthetase, when activated by double-stranded RNA, polymerizes ATP into the novel oligonucleotide (2′–5′)ppp(Ap)nA. We describe conditions for assay of this enzyme in crude extracts of a human lymphoblastoid cell line, Namalwa. The production of (2′–5′)ppp(Ap)nA by Namalwa extracts was 3–5 times greater than the production by extracts of interferon pretreated mouse L cells, and 700 fold higher than the production by extracts of untreated mouse L cells. The relatively high level of oligo(2′–5′)adenylate synthetase in Namalwa cells was not attributable solely to their constitutive secretion of low levels of interferon. Analysis of the size distribution of the oligomers formed at different times suggested that the enzyme can add ATP to a free pppApA. Infection by Newcastle disease virus or treatment with interferon raised the apparent synthetase levels only marginally. Experiments that employed antibody to interferon suggested that the interferon must be externalized from the NDV-infected cell to induce maximal synthetase levels.