Abstract

The effect of the 5'-dephosphorylated 2',5'-adenylate trimer and its 2',5'-trimer core analogs on the inhibition of tobacco mosaic virus (TMV) replication was determined in tobacco leaf discs, protoplasts, and whole tobacco plants, using infectivity tests and enzyme-linked immunosorbent assays. A structure-activity-metabolic stability-toxicity analysis of the 2',5'-adenylate trimer core molecule in TMV-infected Nicotiana glutinosa was determined. Modification at either the 6-amino position of the adenylate residues (i.e. inosinate trimer core) or at the 2' terminus (i.e. A-A-ara-A or A-A-Tu) inhibited replication of TMV. Modification of the 3'-hydroxyl group of the adenylate residues to 3-deoxyribose (i.e. the 2',5'-cordycepin trimer core) inhibited TMV replication better than the 2',5'-adenylate trimer core molecule. With enzyme-linked immunosorbent assays, there was complete inhibition of TMV replication by 200 nM 2',5'-adenylate trimer core for 60 h and by 200 nM 2',5'-cordycepin trimer core for 96 h. The amount of 2',5'-oligonucleotides associated with the leaves was determined using 2',5'-[3H]cordycepin trimer core; 1 X 10(-12) mol/cm2 of plant leaves inhibited TMV replication by 99%. No 2',5'-phosphodiesterase activity was detected in TMV-infected and noninfected leaf extracts. Therefore, the 2',5'-trimer cores were potent inhibitors of TMV replication at nanomolar concentrations, i.e. at 1000-fold lower concentration than that required in mammalian systems.

Highlights

  • INHIBITION OF TOBACCO MOSAIC VIRUS REPLICATION IN TOBACCOMOSAIC VIRUS-INFECTED LEAF DISCS, PROTOPLASTS, AND INTACT TOBACCO PLANTS*

  • The effect of the 5’-dephosphorylated 2’,5’-adenylate trimer and its 2’,5’-trimer core analogs on the inhibition of tobacco mosaic virus (TMV) replication was determined in tobacco leaf discs, protoplasts, and whole tobacco plants, using infectivity tests and enzyme-linkedimmunosorbentassays

  • Because of the natural occurrence of the 2’,5‘-oligoadenylate core in mammalian cells, our laboratory and other laboratories have studied the effect of core compounds on cellular processes inanimal cells, tumor growth in whole animals, and inhibition of virus replication in plants [15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31].* For example, we have demonstrated that the 2’,5’-oligoadenylate cores and 2‘,5‘-cordycepin trimer core inhibit transformation of Epstein-Barrvirus-infected lymphocytes [20] by inhibiting the synthesis of the Epstein-Barr virus-induced nuclear antigen[23], augment natural killer cell activity,* inhibit TMVreplication in tobacco plants [28,29], and inhibit chondrosarcoma growth in animals [25]

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Summary

DISCUSSION

Core in TMV-infected or uninfected N. glutinosa when the amount of cell-free extract was doubled suggests that thereis. A substance inhibiting virus repli- replication (Tables Iand 11).The increased inhibition of TMV cation has been reported to be released from TMV- replication by the 2‘,5‘-cordycepintrimer core may be attribinfected protoplasts of the cultivar in which the infection in uted to increased stability to othe2r ‘,5‘-adenylate degradative the intact planits localized [34]. This study demonstratesthe potency of 2’,5’-adenylate trimer core and its 2’,5‘-analogs and their ability to inhibit TMV replication in infected protoplasts, TMV-infected leaf discs, and the whole plant. 1000-fold difference could be explained by the metabolic stability of 2‘,5‘-adenylate trimer core in plants due to lack of 2’,5’-phosphodiesterase activity (Table VI) These observations are in contrastto reports that 2’,5’-cores actas prodrugs in some mammalian systems due to degradation by esterases [24, 31].

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