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its ligand, could also act as an oncogenic down the pathway from PDGF to myc. protein. A recent report in Nature The next years will see us working out demonstrates homology between the these pathways. We shall soon fit many erbB oncogene protein and the receptor pieces of this puzzle together. for epidermal growth factor25. The subsequent elements in the path- References way are the intracellular transducers of 1 Stehelin, D., Varmus, H. E., Bishop, J. M. and Vogt, P. K. (1976) Nature 260, 170-173 signal. Examples of this, such as the 2 Coffin, J. M., Varmus, H. E., Bishop, J. M., normal version of the src- or ras-encoded Essex, M., Hardy, W. D., Martin, G. S., proteins, would seem to act by receiving Rosenberg, N. E., Scolnick, E. M., Weinberg, R. A. and Vogt, P. K. (1981) J. Virol. 40, growth-stimulatory signals and passing 953-957 them on to downstream targets. The abnormal, oncogenic versions of these 3 Shilo, B. Z. and Weinberg, R. A. (1981) Proc. Natl Acad. Sci. USA 78, 6789-6792 proteins would appear to act auton- 4 Gallwitz, D., Donath, C. and Sander, C. omously, stimulating downstream targets (1983) Nature 306, 704-706; DeFeo-Jones, D., without receiving the normally required Scolnick, E. M., Koller, R., Dhar, R. (1983) stimuli from an upstream source. The Nature 306, 706-709 experimental justification of such a 5 Shih, C. et al. (1979) Proc. Natl Acad. Sci. USA 76, 5714-5718 model, eagerly sought, is still lacking. 6 Krontiris, T. G. and Cooper, G. M. (1981) That such interacting pathways exist Proc. Natl Acad. Sci. USA 78, 1181-1184 is clear. The most dramatic demonstra7 Der, C. J., Krontiris, T. and Cooper, G. M. tion of this comes from recent work (1982) Proc. Natl Acad. Sci. USA 79, 3637-3640 showing that extracellular PDGF can 8 Parada, L. F., Tabin, C. J., Shih, C. and strongly stimulate the expression of the Weinberg, R. A. (1982) Nature 297, 474-478 normal myc gene 26. Thus, the homo9 Santos, E., Tronick, S., Aaronson, S. A., logue of the sis oncogene is able to reguPulciani, S. and Barbacid, M. (1982) Nature late transcription of the normal homo298, 343--347 logue of a second oncogene. Quite pos- 10 Goldfarb, M., Shimizu, K., Perucho, M, and sibly, other proto-oncogene-encoded Wigler, M. (1982) Nature 296, 404-409 proteins intervene to pass the signals 11 Collen, M. S. and Erikson, R. L. (1978) Proc.

Viroids T. O. Diener Viroids, unencapsidated, covalently closed circular RNA molecules consisting of 246 to 371 nucleotides, cause several important diseases of higher plants. The complete primary and most likely secondary structure of several viroids has been determined. With the demonstration that cloned viroid cDNA is infectious, structure-function correlations have become possible by introduction into plants of artificially modified cDNAs. Although the viroid concept had been introduced almost 5 years earlier1, doubts about its correctness still lingered in many a biochemist's mind at the beginning of 1976. This is not surprising because, initially, all molecular properties of these unorthodox plant pathogens could be determined only by bioassay on indicator plants and not by conventional biophysical/biochemical techniques. Most biochemists and molecular biologists were not at ease with such an unusual approach and many considered evidence obtained'in this fashion unconvincing, if not unacceptable. In plant virology, however, the viroid concept had gained rather rapid acceptance. Not only were plant virologists T. O. Diener is at the Plant Virology Laboratory, Agricultural Research Center, US Department of Agriculture, Beltsville, M D 20705, USA.

more accustomed to evaluating evidence obtained indirectly by virtue of a pathogen's biological activity, but some were already working with diseases whose inciting agents turned out to be viroids. Thus, the original work with the potato spindle tuber viroid (PSTV) became rapidly and independently confirmed and extended to other diseases, and some other plant virologists recognized the potential significance of the viroid and entered the newly emerging field. Consequently, viroid research accelerated substantially and, by 1976, four additional diseases were known to be caused by viroids and still another was suspected (see Ref. 2). At that time, the healthy spirit of competition that had already developed among viroid investigators stood in stark contrast with the skepticism and disbelief frequently

Natl Acad. Sci. USA 75, 2021-2024 12 Cooper, G. M. (1982) Science 218, 801-806 13 Land, H., Parada, L. F. and Weinberg, R. A. (1983) Science 222, 771-778 14 Ellis, R. W., Lowy, D. R. and Scolnick, E. M. (1982) in Advances in Viral Oncology, pp. 107-126, Raven Press 15 van den Eisen, P. J., de Pater, S., Houweling, A., vander Veer, A. and van der Eb, A. (1982) Gene 18, 175--185 16 Rassoulzadegan, M. et al. (1982) Nature 300, 713-718 17 Land, H., Parada, L. F. and Weinberg, R. A. (1983) Nature 304, 596-602 18 Newbold, R. F. and Overell, R. W. (1983) Nature 304, 648--651 19 Ruley, H. E. (1983) Nature 304, 602~06 20 Berk, A. J. et al. (1979) Cell 17, 935-944; Jones, N. and Shenk, T. (1979) Cell 17, 683--689; Jones, N. and Shenk, T. (1979) Proc. Natl Acad. Sci. USA 76, 3665-3669 21 Ross, E. M. and Gilman, A. G. (1980) Annu. Rev. Biochem. 49. 533-564 22 Doolittle, R. F., Hunkapiller, M., Hood, L., Devare, S., Robbins, K. C., Aaronson, S. A., Antoniades, H. N. (1983) Science 221,275-276 23 Waterfield, M., Scrace, G., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermaker, B., Heldin, C.-H., Huang, J. and Deuel, T. (1983) Nature 304, 35-39 24 DeFalco, J. and Todaro, G. J. (1978) Proc. Natl Acad. Sci. USA 75, 4001-4005 25 Downward, J. et al. Nature 307, 521-527 26 Kelly, K., Cochran, B. H., Stiles, C. D. and Leder, P. (1983) Cell 35, 603--610

expressed in the scientific community at large. The viroid in 1976

Unequivocal recognition of a viroid as a physical entity was achieved first with PSTV 3. Relatively pure viroid preparations soon became available, permitting, for the first time, the application of routine biophysical/biochemical analyses to the viroid. This led to rapid advances in our knowledge. Determination of the thermal denaturation properties of PSTV 3 showed that, contrary to some earlier speculations, viroids are single-stranded RNA molecules. On the basis of their appearance in electron micrographs4, it became known that viroids could assume a hairpin-like, quasi-double-stranded conformation. One of the questions frequently asked at that time concerned the identity of the different viroids isolated. Did each consist of a unique nucleotide sequence or were they all one and the same RNA species? Because of their similar biological properties, PSTV and the citrus exocortis viroid (CEV) could well have been identical molecules, but the first RNA fingerprints of viroids conclusively showed that each was a unique and distinct species of low molecular weight RNA s .

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Fig. 1. Electron micrograph depicting denatured viroid (PSTV) molecules. Both circular and linear molecules are visible (US Department of Agriculture).

No helper viruses could be identified in viroid-infected plants 1, thus viroids, despite their very limited information content, are replicated autonomously in susceptible cells. Because each viroid is a single distinct species of low molecular weight RNA, viroids were known to represent simple genetic systems whose biological properties must be determined by their particular nucleotide sequences. These properties of viroids evidently clashed with the then widely held, though mostly unspoken, belief that an autonomously replicating viral entity required genetic information equivalent to a minimum of mol. wt about 1 x 106 (which corresponds to the genome size of the smal!est independently replicating viruses known). How could an RNA roughly one-tenth this size subjugate a host cell to enforce its own replication and how could such a small molecule interfere with the cell's metabolism to produce the diseases that brought the viroids to our attention in the first place? Does the viroid exert its action via a polypeptide translated from the RNA sequence? By 1976, viroids had been tested as potential mRNAs in various cell-free protein synthesizing systems and by injection into Xenopus laevis oocytes. In none of these systems were the viroids translated nor did they interfere with the translation of genuine mRNAs (for a review see Ref. 2). These

findings further deepened the mystery of viroid function, implying, as they did, that viroids are replicated entirely by pre-existing (or activated) host enzymes. In 1976, one of the most frequently discussed questions was whether viroids were transcribed from RNA or DNA templates. Prima facie, the finding that actinomycin D inhibits viroid replication6 seemed to suggest that the latter might be the case. Indeed, claims soon were made that viroid-specific DNA sequences exist in viroid-infected, and even in uninfected, ceils. Although these claims were later shown to be in error, for a while they pointed viroid research in the wrong direction (for a detailed discussion, see Ref. 7). This then was the status of viroid research at the beginning of 1976. The original tenets of the viroid concept, as it had been advanced in 1971, had been amply confirmed, but detailed knowledge of their molecular structure was absent. The viroid today Have any of the questions that were prominent in 1976 been answered? Has the enigma of viroid function been solved? To begin with, five additional diseases of cultivated plants have been identified as viroid-incited problems and some progress has been made in understanding

the ecology and epidemiology of viroid diseases. Circumstantial evidence indicates that viroid diseases of crop plants have arisen (and do presently arise) by chance transfers of viroids from wild plant reservoirs2'7. The most spectacular advances in our knowledge of viroids, however, have been achieved in the elucidation of their molecular structure. The following milestones might be mentioned: electron microscopy of denatured viroids led to the important discovery that many, if not most, viroid molecules have a covalently closed circular structure 8'9 (Fig. 1), the first such RNAs found in nature; intensive thermodynamic and biophysical studies of viroid structure (for a review, see Ref. 10) were crowned by the determination of the complete nucleotide sequence and probable secondary structure of P S T V 11. PSTV thus became the very first pathogen of a eukaryotic organism for which the complete molecular structure had been established. This achievement is all the more remarkable as only 7 years had elapsed since the first recognition of the viroid*. Since then, the primary and most probable secondary structures of seven other viroids have been elucidated. With the growing library of viroid *(See TIBS, August 1979 (Vol. 4), p. 179.)

135

T I B S - A p r i l 1984 30

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TPMV Fig. 2. Nucleotide sequence and most probable secondary structure of a typical viroid (tomato planta macho viroid), illustrating the quasi-double-stranded rodlike shape o[" the molecule, in which short base-paired regions alternate with mismatched single-stranded loops.

sequences, certain regularities have become apparent. All viroids are able to assume a characteristic, highly basepaired, rod-like secondary structure, in which short base-paired regions alternate with short single-stranded loops (Fig. 2). On the basis of their nucleotide sequences, viroids may be divided into 3 groups: (1) the PSTV group, to which belong all viroids studied, except coconut cadang-cadang (CCCV) and avocado sunblotch (ASBV) viroids. Any two viroids of the PSTV group display extensive regions of sequence homology (50-80%). All contain a highly conserved central region, and all display a more or less extensive polypurine stretch (Fig. 3); (2) CCCV, which contains the conserved central region, but only a short stretch of contiguous purines and little sequence homology (aside from the central region) with the viroids of the PSTV group; and (3) ASBV, which differs from all other known viroids by containing only a small portion of the central conserved region and by displaying little sequence homology with any of the others. ASBV also differs from other viroids by its lower thermodynamic stability 1°. With PSTV, a number of strains are known that differ in the severity of symptoms they induce in host plants. Sequence determination disclosed that these strains differ from one another by only three or four nucleotide exchanges ~2. A detailed description of the 'comparative anatomy' of viroids at the molecular level is beyond the scope of this article; the reader is referred to pertinent reviews 7'~2. Knowledge of a growing number of nucleic acid sequences has made possible comparisons of viroid sequences with those of various viral and cellular RNAs; and computerassisted searches for nucleotide homologies have become a favorite enterprise in viroid research. Comparisons among viroids, it is hoped, may identify specific regions (nucleotide sequences) that are responsible for particular biological properties; and sequence comparisons with cellular or viral RNAs may shed light on viroid origin (see below). It is interesting to note that, in contrast to many other biological systems, viroids

are unique in that, today, our knowledge of their molecular properties far outstrips our knowledge of their basic biological characteristics, such as their mode of transmission in nature, natural host range, and epidemiological and ecological parameters. During the last year or two, a shift of emphasis in viroid research has been discernible. Undoubtedly, this shift was brought about by our extensive knowledge of the molecular structure of viroids which leaves relatively little new ground to be covered. Today, more and more emphasis is given to functional aspects of the viroid problem and particularly to the elucidation of structure-function correlations. Also, whereas in earlier years separation of viroids from cellular constituents and viroid purification were foremost in the minds of investigators, today the trend seems to go in the opposite direction. Clearly, purified viroids consist exclusively of RNA, are devoid of protein or other cellular components, and yet display all of the biological properties of the pathogens that had been studied previously in cruder preparations. The possibility nevertheless exists that, in situ, viroids are present as complexes with cellular constituents and that specific association with host components may be important biologically. Such association, for example, may confer additional stability to the pathogens, thereby cP PSTV TPMV CSV

Cc//Auc[~ CUUGAGG[gCCUGUG61JGCUCACCUGACCCUC-CAG GCAG CGG AACUAAA CUCGUGGUUCCUGUGGUUCACACCUGACC UCCUGAGCA CGGGAUC~CCL"JGUGGUUCCUGUGGUACACACCUGACC UCCUGACCA UGGGA CUUA CUUGUC4~UUCCUGUGGUGCACUCCUGACCCUGCUGCUL"3

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facilitating cell-to-cell and plant-to-plant movement. A recent re-investigation of the subcellular location of viroids has confirmed earlier results, indicating that the majority of viroids is present in the nucleus of infected cells, but has disdosed, in addition, that the viroid is mostly associated with the nucleolar fraction obtained from purified nuclei 13. Furthermore, the viroid appears to exist in the nucleolus in the form of a proteinnucleic acid complex 13. Still in the foreground of viroid investigations is the mechanism of viroid replication. Knowledge of the nucleotide sequences of several viroids has strengthened the earlier conclusion that viroids are not translated. Although A U G initiation codons are present in some viroid sequences (or in those of the complementary strands), in other viroids (or their complements) none are present. Also, open reading frames in different viroids drastically differ one from the other, leading to the conclusion that, if translation is involved in viroid replication, a number of different replication mechanisms must be involved. In view of their close structural similarities, this appears unlikely. Efforts to detect viroid-specific polypeptides also have not been productive, except to show that the concentrations of certain host proteins significantly increase in viroidinfected plants. GAA~GA~ GG CGGCC// GAAAAC~OA AGG CGGCUC GAAAAGA~GA At"IG CGGC C G AAAGAAAAA GA AAUG ACGC

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ACCUCGUCUCCUUCCUt~JCGCUGCUGGC UCC ACAUCCGAUCG UCC.CU GA AC-CC-CCUCC.CCCCCU CUIJUUCA C CCL"JCCUWd CL"3CC.GGUGUCCL"3CCUC S CGCCCGCAGGACC A CCCCUCCCCCCCU UUCCGC L ~ O ~ C GUt"3UCA C CC19JCCUt~ CV~CC~Gt,JUCCUUCCUCUG CC GUC GA C A cccucccccccuucocut~c¢c ucacCcuuc C.ULrUUCA C CCDUCCDUU AGUUUCCUUCCUC UCCUGGAGAGGUCL, JCUGCCCUAC.CCCC.GUCL~JC G A A C C W C CU 350

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~ C A A C UG AAOC~JCAACCCC AAACCGC UUUU 03 UAUAUCUUCACU GCUCUCC GGC,CGAGGGL~ A J U ~ C C C U ~ C C C U AGADU GG~J~CCU ~ C A A C UG AAGCU CCCGAGAA CCC,C t~JIFu~CUC UAUCt~ ACUU G CUUCGGGGCC, A G G G U G ~ q ~ U A G C C ~ C C C,CAG IFJ GGUUCCU GCUU¢.A~AC.A~C UG AACCU CCC~AC.CG CCGC UI"mUCUC UAUCUU CCU GG C U C C G G G G C ~ C C C U GG~ACCCUU~CCCU ~ C ~ AAC.C~CAAC GC C'0~fuUUUUCCA AUCUU CUUUAG CACC C.C.C.CUA~(X:AGU ~ C ~ O C , . d & P . C C UUAG~IPJt) G%"J~=(:CU

Fig. 3. Comparison of the nucleotide sequences of 5 viroids (TASV, tomato apical stunt viroid; CEV, citrus exocortis viroid; PSTV; TPMV, tomato planta macho viroid; and CSV, chrysanthemum stunt viroid), illustrating homologous and unique sequences. Boxed areas contain the conserved central region, adjacent dinu¢leotides and the polypurine region of the five viroid~.

TIBS-

136 Although details of the viroid replication process are still unknown, some progress has been made since 1976. The identification of viroid-complementary R N A sequences in viroid-infected cells 14 and later of full-length complementary PSTV molecules is strongly suggested that viroids are replicated by transcription from R N A templates. The finding that viroid replication is sensitive to 10 -s M et-amanitine suggested that (normally) DNA-dependent R N A polymerase II may be involved in this process 12 and, indeed, in an in vitro system, purified R N A polymerase II accepts the viroid as a template, transcribing full-length linear viroid complements 12. However, whether this occurs in vivo is uncertain, because other enzymes, such as an RNA-dependent R N A polymerase from plant tissue, can do likewise 12. The detection by several groups of longer-than-unit length viroid forms in infected cells (often present as oligomeric duplex molecules) suggests that some sort of rolling-circle-type mechanism is involved in viroid replication 16. Details of these processes evidently need to be determined. Undoubtedly, the availability of specific hybridization probes, consisting of cloned recombinant c D N A s 15, will be useful in these endeavors. Another aspect of the viroid enigma, the biochemistry of pathogenesis, has not progressed beyond speculation. Although several possible mechanisms have been suggested 1'2'7'12, none are based on experimental evidence or are capable of explaining why, in certain hosts, viroids replicate normally without discernible detrimental effects o n ' t h e host. Knowledge of the nucleotide sequences of several viroids has suggested possible relationships between viroids and other RNAs or certain DNAs. It has been noted, for example, that the lower portion of the central conserved viroid

region contains a region that is homologous to the 5' end of a small nuclear R N A , U1 (Refs 17 and 18), which is believed to be involved in the splicing process. It has, therefore, been suggested that viroids may have originated from intervening sequences or introns 17. The finding of covalently closed introns, some the size of viroids, has added plausibility to these speculations. More recently, a comparison of the primary structures of five viroids has revealed striking sequence similarities with the ends of transposable genetic elements and with those of retroviral proviruses in particular 19, suggesting that viroids may represent remnants of such elements, whose replication has become independent of a D N A phase.

The viroid tomorrow

At present, viroid research is still in its 'log phase' of growth. Several new and exciting approaches to the solution of old problems have become possible recently. Foremost among these is our newly gained capacity to precisely determine structure-function correlations. With the construction of c D N A clones that represent the complete PSTV sequence and the demonstration that these cDNAs are infectious in tomato 2°, this approach has been vastly facilitated. Precise modifications of viroid sequences, such as nucleotide substitutions, deletions, or insertions can now be accomplished readily through manipulation of cDNAs and their biological effects determined. Thus, in vitro mutagenesis ('reverse genetics') is now feasible and several problems of viroid research have become amenable to experimentation. It is likely that such studies will yield results with significance far transcending viroid research p e r se shedding light on old and still largely unsolved problems of host-pathogen interactions in general, such as the

A p r i l 1984

molecular bases of host specificity and pathogenesis. Finally, these studies may lead to a practical use of viroids as vectors in plant recombinant D N A technology.

References 1 Diener, T. O. (1971) Virology 45, 411-428

2 Diener, T. O. (1979) Viroids and Viroid Diseases, 252 pp., WileyInterscience 3 Diener, T. O. (1972) Virology 50, 4 Sogo, J. M., Koller, T. and Diener, T. O. (1973) Virology 55, 70-80 5 Dickson, E., Prensky, W. and Robertson, H. D. (1975) Virology 68, 309-316 6 Diener, T. O. and Smith, D. R. (1975) Virology 63, 421-427 7 Diener, T. O. (1983) Adv. Virus Res. 28, 241-283 8 McClements, W. (1975) Ph.D. Thesis, University of Wisconsin 9 S~inger,H. L., Klotz, G., Riesner, D., Gross, H. J. and Kleinschmidt, A. K. (1976) Proc. Nail Acad. Sci. USA 73, 3852-3856 10 Riesner, D., Steger, G., Schumacher, J., Gross, H. J., Randles, J. W. and S~inger, H. L. (1983) Biophys. Struct. Mech. 9, 145-170 11 Gross, H. J., Domdey, H., Lossow,C., Jank, P., Raba, M., Alberty, H. and S~inger,H. L. (1978) Nature 273, 203-208 12 S~inger, H. L. (1982) in Nucleic Acids and Proteins in Plants, Vol. II (Parthier, B. and Boulter, D., eds), pp. 368-454, Spfinger-Vedag 13 Schumacher, J., S~inger, H. L. and Riesner, D. (1983) EMBO Journal 2, 1549-1555 14 Grill, L. K. and Semancik, J. S. 0978) Proc. Natl Acad. Sci. USA 75, 896-900 15 Owens, R. A. and Cress, D. E. (1980) Proc. Natl Acad. Sci. USA 77, 5362-5306 16 Owens, R. A. and Diener, T. O. (1982) Proc. Natl Acad. Sci. USA 79, 113-117 17 Diener, T. O. (1981) Proc. Natl Acad. Sci. USA 78, 5014-5015 18 Gross, H. J., Krupp, G., Domdey, H., Raba, M., Jank, P., Lossow, C., Alberty, H., Ramm, K. and S~inger,H. L. (1982) Eur. J. Biochem. 121, 249-257 19 Kiefer, M. C., Owens, R. A. and Diener, T. O. (1983) Proc. Natl Acad. Sci. USA 80,

6234--6238 20 Cress, D. E., Kiefer, M. C. and Owens, R. A. (1983) Nucleic Acids Res. 11, 6821-6835