5’-Dephosphorylated 2’,5’-Adenylate Trimer and Its Analogs INHIBITION OF TOBACCO MOSAIC VIRUS REPLICATION IN TOBACCO MOSAIC VIRUS-INFECTED LEAF DISCS, PROTOPLASTS, AND INTACT TOBACCO PLANTS*

The effect of the 5’-dephosphorylated 2’,5’-adenyl-ate 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 en- zyme-linked immunosorbent assays. A structure-activ-ity-metabolic stability-toxicity analysis of the 2’,5’-adenylate trimer core molecule in TMV-infected Ni- cotiana glutinosa was determined. Modification at either the 6-amino position of the adenylate residues (i.e. inosinate trimer core) or at the 2‘ terminus (Le. 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’,5f-cordycepin trimer

Protection is conferred to virus-infected mammalian cells by the formation of interferon which induces the synthesis of at least two enzymes. One of these enzymes is the 2',5'adenylate synthetase which in the presence of certain doublestranded RNAs converts ATP to a series of unique 2',5'oligonucleotides (1-7). In addition to the synthesis of the 5'triphosphate 2',5'-linked oligoadenylates, Knight et al. (8) have reported on the intracellular accumulation of the 5'dephosphorylated 2',5'-adenylate molecules (referred to here as 2'3'-adenylate cores') in mouse L-cells following treatment with interferon. They concluded that because the amount of cores seemed to be independent of the concentration of the 2',5'-oligoadenylate 5'-triphosphate molecule, 2',5'-oligoadenylate cores may play a separate role in the inhibition of DNA and cellular reactions. Because of the rapid hydrolysis of the naturally occurring 2',5'-oligoadenylate molecule in cells, there have been reports on the enzymatic and chemical synthesis of the 5'-triphosphates and corresponding cores in attempts to increase the metabolic stability and retain inhibition of protein synthesis (9-13). In addition, Rappoport et al. (14) have reported that the 5"diphosphate of 3',5'adenylate molecule activated the 2'3' -An-dependent endonuclease to hydrolyze mouse L-cell rRNA. This laboratory has recently reported that the 2',5'-cordycepin tetramer 5'triphosphate analog complexes with and activated the 2',5'-An-dependent endonuclease to hydrolyze vesicular stomatitis virus mRNA and inhibits protein synthesis (10).
Recently, Reichman et al. (29) and Orchansky et at. (33) reported that human recombinant leukocyte interferon inhibited TMV replication in tobacco leaf discs. Furthermore, the antiviral factor isolated from TMV-infected leaves of Nicotiana glutinosa and human leukocyte interferon induced double-stranded RNA-dependent synthesis of oligoadenylates from ATP in plants, producing nucleotides with antiviral activity (29).
In this study, we report that five 2',5'-adenylate trimer core analogs inhibit TMV replication in TMV-infected tobacco leaf discs, TMV-infected protoplasts, and in whole plants. This study as well as previous studies (28-29) indicate that the antiviral factor (29) or the inhibitor of viral replication (34,35), released following TMV infection in N. glutinosa plants or protoplasts, may inhibit viral replication via polymerization of ATP to form oligoadenylates.

DISCUSSION
Viral diseases occurring in the animal kingdom and in the plant kingdom present serious problems. Therefore, it is important to understand how the animal and plant cell are protected from virus infection. In the animal cell, interferon is synthesized in response to virus infection which induces the synthesis of 2',5'-linked oligoadenylates. In the plant cell, an antiviral factor has been isolated from N. glutinosa leaves infected with TMV (41). A substance inhibiting virus replication has also been reported to be released from TMVinfected protoplasts of the cultivar in which the infection in the intact plant is localized (34). These inhibitors may be the "protectors" of plants from virus infection. Reichman et al. (29) and Orchansky et al. (33) demonstrated that some human interferon preparations inhibited TMV replication in tobacco leaf discs. Application of the 2',5'-adenylate trimer core to TMV-infected tobacco leaf discs also inhibited TMV replication (28,29,42). TMV, antiviral factor, or interferon induces an enzyme capable of converting ATP to putative oligoadenylates with the ability to inhibit TMV replication (29). These oligoadenylates inhibit protein synthesis when added to either lysates from rabbit reticulocytes or wheat germ extracts via a mechanism different from the 2',5'-A,dependent endonuclease a~tivation.~ Previous reports indicate that 2',5'-adenylate trimer core and 2',5'-cordycepin trimer core protect plant cells against viral infection (28,29). This study demonstrates the 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. In all experiments, the 2',5'-trimer cores were potent inhibitors of TMV replication at nanomolar concentrations. It is not yet clear whether passive equilibrium, uptake, binding, or a combination of uptake and binding is A structure-activity-metabolic stability-toxicity relationship of the 2',5'-adenylate trimer core in TMV-infected N. glutinosa was determined. Replacement of the 6-amino group of the adenylate residues in 2',5'-adenylate trimer core with a 6-hydroxyl group (i.e. inosinate trimer core) resulted in a derivative that inhibited TMV replication. When the 2'terminal adenylate was replaced by either ara-A or tubercidin (i.e. A-A-ara-A or A-A-Tu), the resulting trimer cores also inhibited TMV replication. When the 3'-hydroxyl of 2',5'adenylate trimer core was replaced by a hydrogen atom (i.e. the cordycepin trimer core), TMV replication was inhibited better than that observed with the naturally occurring 2',5'adenylate trimer core. None of these oligoadenylate analogs were toxic to N. glutinosa. Structural modification of the ribose on the 2"terminal nucleotide in the 2',5'-oligoadenylate molecule results in analogs that are considerably more stable to hydrolysis by the 2',5'-phosphodiesterase in the mammalian system (16, 20, 22); however, 2',5'-phosphodiesterase activity was not detected in N. glutinosa. The fact that there is no increase in hydrolysis of 2',5'-[3H]adenylate trimer core in TMV-infected or uninfected N. glutinosa when the amount of cell-free extract was doubled suggests that there is no 2',5'-phosphodiesterase activity as determined under these assay conditions (Table VI). In contrast to the leaf discs and intact plants, where the degradation products of 2',5'-adenylate trimer core and analogs did not inhibit TMV replication (Table I11 and Fig. l ) , in protoplasts a different inhibitory mechanism appears to exist because 3',5'-adenylate trimer core, AMP, and adenosine showed some inhibition of TMV replication (Tables I and 11). The increased inhibition of TMV replication by the 2',5'-cordycepin trimer core may be attributed to increased stability to other 2 ',5'-adenylate degradative enzymes in plants or the inhibition of RNA, DNA, or protein synthesis. The concentrations of 2',5'-adenylate trimer core and trimer analogs required for the inhibition of TMV replication in plants are 1000-fold lower than the concentrations required for the inhibition of transformation of Epstein-Barr virus-infected lymphocytes, the inhibition of tumor growth, the antimitogenic effect, and the antiproliferative effects reported in mammalian systems (16,17,(19)(20)(21)(24)(25)(26)(27). The 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 contrast to reports that 2',5'-cores act as prodrugs in some mammalian systems due to degradation by esterases (24, 31). This does not appear to be the case in all mammalian cell systems. For example, we have reported that 2',5'-cordycepin core trimer is taken up intact by human lymphocytes (27).
We have previously reported the induction of a plant oligoadenylate synthetase which converted ATP into oligoadenylates when immobilized on poly(r1) .poly(rC) (28, 29, 39). The plant oligonucleotides inhibited TMV replication. More recently, we have shown that these same oligoadenylates, following purification by high pressure liquid chromatography, inhibit protein synthesis in lysates from rabbit reticulocytes; however, we could not detect any 2',5'-A,-dependent endonuclease or 2',5'-phosphodiesterase activity in uninfected or TMV-infected p l a n k 4 Similarly, Cayley et al. (43) reported that the 5'-triphosphate of the 2',5'-oligoadenylate or its binding proteins were not present in tobacco plants.

2',5'-Adenylates Inhibit
TMV Replication cepin trimer core, 2',5'-A-A-Tu, and 2',5'-A-A-ara-A. Earlier studies also showed that the 5"triphosphate of the 2',5'adenylate trimer molecule, when applied to TMV-infected leaf discs, also inhibited TMV replication (29). There is the possibility that 2',5'-trimer cores inhibit TMV replication via the activation of plant discharging factor (39) to discharge aminoacylated TMV RNA, which inhibits viral protein synthesis. Further studies on the effect of 2',5'-adenylate molecule on plant protein, RNA and DNA synthesis, activation of discharging factor, viral coat protein synthesis, TMV RNA synthesis, and virus assembly are in progress. Abrasion of the plant leaf epidermis with a TMV/carborundum solution containing very dilute solutions of the 2',5'-adenylate core or 2',5'-analogs results in inhibition of TMV replication without any toxicity to the plants. This novel approach to the inhibition of viral replication may have a great potential in the control of plant virus infections.
Irhibltlon of TIN replication by 2*.5*-trimr cores with increaslnq time. we 2',5'-adenylate trimer core and the 2*,5'-cordycepln trimer core i n infected also tested the kinetics of the inhibition of TIN-replication by the leaf discs. Tobacco leaf discs Inoculated with TIN vere treated for an additrimer core. The discs were then washed and the infection was allowed t o t i m a l hr with 200 nM 2',5*-admylate trimer core or 200 nu 2*,5'-cordycepin proceed. Virus repllcatlon was totally inhibited for 60 hr by 2',5*-adenylate trimer m r e and the inhibitory effect gradually diminished frm 60 t o 96 hr. Hwever, TIN inhibition by the 2' 5'-cordycepin trimer core was noted f o r 96 hr cowared to the naturally occurring adenylate core may be due to increased sta- (Fig. 2). The extended inhibitor; activity of the 2'.5*-cordycepin trimer core mechanism.
billty of the analog, an effect of Increased wtake, or a different inhibitory intact plants.

ELISA limit
,ooo "_ " " " " " " " " -"""""-" bInhibition of TFN replication was determined 68 hr after Infection and calculated as in Table 1. fmtnote b. P a r t i a l l y p u r i f i e d AVF ( 5 0 ng p r o t e i n h l l prepared as described i n Materia 5 and Methods was added t o W -m t a i n i n g solution a t a dilution o f 1 x lo-+. of the infecting Solution was a6sociated w i i h the leaves (Table 5). merefore, when 2*,5'-cordycepln trimer core in the infecting solution i s a t a concentration Of 2 x M. m l y 2 x 10-l2 mles are a 0 iated with each I x c. 2 of leaf. About 5 0 % of the 2 x 10-12 m l e s of 2*,5*-fSHfjcordycepin trimer core remalned associated with the leaves after extensive washing. No tritium was mic spread of the nucleotides.
c e l l i l n e s g y ; p , q leaves. Tte'Gay;Ge& done i s &scribed in kt&;ii-and Adenylate trimer core was hydrolysed to adenosine and RIP by the 2',5*-phosphodiester present in EPT or Raji cells (Table 61. mbrever, and nm-infected plant leaf extracts ( Table 6 )