)3 tz 8+ STRUCTURES OF VIROIDS VIRUSOIDS AND SATE LLI TES Jim Hasel-of f B. Sc. (Hons ) for Gene Technology Adelaide University Centre ' South Aus traÌ i a. Thesis submltLed to the university of Adelaide in fulfil-lment of the requirements for the degree of Doctor of PhilosoPhY. May, 1 983. CON TENTS S TATEMEN T A C KNOV\¡LED GEMEN TS SUMMARY CHA PTE R 1 TNTRODUCTION A. Viroids 1 B. Vl rusoids 4 C. Aims 5 CHAPTER 2 RNA SESUENCE DETERMINATION IN TROD U C TI ON I o MATERIALS ME THOD S A. Isol-ation and sequence determination of linear viroid or virusoid fragments A-1 AnalYtical RNase digests 9 A-2 PreParatlve RNase digests 11 A-3 5'-32P fabell-ing of RNA fragments 11 A-4 3'-32P l-abel1ing of RNA fragments i) Synthesis of Ir'-3'rldpcp 12 ri ) 3'-32P l-abeì,1i-ng 12 h-)AE Polyacrylamide geI fractlonation 13 A-6 Sequence determination of RNA fnagments using the partial enzymic cfeavage technique 14 A-7 Sequence determination of RNA fragments using the dideoxynucleotide chaln termj-nation technique i) PhosPhatase treatment 16 ii ) PolYadenYì-ation 16 ij-i) Reverse transcriPtion 1T A-B Bolyacnylamide gel elecLrophoresis 18 B. Sequence determination using cloned viroid or virusoid sequences B-1 Synthesls and cl-oning of viroid and virusoid ds cDNA i) LinearizaLion and polyadenyl-ation 1g ii ) First strand cDNA sYnthesis 20 iii ) Second strand cDNA sYnthesis 20 iv ) Restrj-ctlon enzyme cleavage and fractionation 20 v) M1 3 cloning 21 B-2 Sequence determination of recombinant phage M1 3 ¿t B-3 Sequence determination of RNAs using cfoned DNA Prlmers i) PneParation of Primer ¿¿ ii ) RNA-DNA hYbnidizatlon ¿J iii ) Reverse transcriPtion 23 RESULTS and DISCUSSION A. Techniques 24 B Partial- enzymic cleavage of viroid and virusold RNAs 24 c Partial enzymic cleavage of radiol abel-l-ed RNA fnagments 25 D Dideoxynucl-eotide chain termination sequencing of RNA fragments 25 ¿õ E Cloning of vinoid and virusoid sequences F Sequence determination using cfoned viroid or vinusoid sequences - 27 G Detenmination of complete vinoid or virusoid primanY structunes 2B CHAPTER 3 CHRYSANTHEMUM STUNT V]ROID INTRODUCTI ON 2g MATER]ALS 30 ME THOD S A. Primary structure determination 30 B. Secondary structure determination 3',I RESULTS A. Sequence determination 31 B. Primary sequence and secondary sLructure 33 DISCUSS]ON A. Homology between CSV and PSTV 33 B. Replication of CSV and PSTV 34 C. Rel-ationship of this isolate of CSV to other viroid isol-ates 36 CHAPTER 4 COCONUT CADANG-CADANG VIROID INTRODUCT] ON 39 M ETHOD S A. Isolation of the ccRNAs 41 B. Sizing of the ccRNAs 42 C. Fingerprinting of the ccRNAs 43 D. Sequence and structure deLermination of the ccRNAs 44 RESULTS ANd DTSCUSSION A. Sizing of the ccRNAs 46 B. Fingerprinting of the ccRNAs 47 C. Sequence and structure determination of the ccRNAs 48 D. ccRNAs differ in size but not sequence complexitY 50 E. Varlation in sequence between different ccRNA isoÌates 52 F. Structural simitarities between ccRNAs and viroids 53 G . Re plication of ccRNAs 55 H . ccR NA s l-ow variants and the time course of infection 56 I. Origin of cadang-cadang disease 5B CHAPTER 5 VELVET TOBACCO MOTTLE V]RUS AND SOLANUM NODIFLORUM MOTTLE V]RUS INTRODUCTION 60 MATERIALS and METHODS A. Viruses and RNA 61 B. RNase Fingerprinting 62 C. BNA sequence analysis i-) Partial- enzYmic digestion 62 fj- ) Di deoxynucleotide chain termlnation 62 D. Synlhesis and cloning of double-strand cDNA 63 RESULTS A. RNase fingerprints of VTMoV and SNMV RNA 2 63 B. Primary structures of VTMoV and SNMV RNA 2 64 C. Secondary structures of VTMoV and SNMV RNA 2 66 D. Possibl-e polypeptide translation products from RNA 2 species and their compfements o/ DI SCUSS I ON 6B CHAFTER 6 SUBTERRANEAN CLOVER MOTTLE VIRUS INTRODUCTION 72 MATER]ALS 73 METHODS A. Synthesls and restriction endonucl-ease cleavage of ds cDNA 73 B. Fingerprlnting of SCMoV RNAs (¿1 C. Sequence determlnation of SCMoV RNA 2 and RNA 2I 74 RESU LT S A Analysis of SCMoV RNA 1 nucleotide sequences 75 B RNase fingerprinting of SCMoV RNAs 2 and FNAs 2t 76 C Sequence determination of SCMoV-A RNA 2 and RNA 2I 7B DI SCUSSI ON A. RelationshiPs between the v'arious isolates of SCMoV 79 B. Sequence homology between SCMoV RNA 2 and RNA 2I 79 C. Sequence homologY between SCMoV, VTMOV, SNMV and LTSV RNAs 2 BO D. Satellite RNA of TobRV B2 E.'sequence homoì-ogY between TobRV s atellite RNA and vlrusoids B4 CHAPTER 7 VIROIDS VIRUSOIDS AND SATELLITES INTRODUCTION B6 ME THOD S A. tsolation of RNA B7 B. Blot hybridization B7 RESULTS A. Analysls of VTMoV and SNMV RNA 2 sequences present in vinus and infected tissues 8B DI SCUSS I ON A. Multimers of VTMoV and SNMV RNA 2 8,9 B. A possible site for RNA processing 90 C. Vlroid, vlrusoid and satetlite RNAs 92 STAT EMEN T This thesis has not previously been submibted for an academic award at this or any other University, and is the original work of the author, except where due reference is made in the tex t . JIM HASELOFF AC KN Ol/tr LED GEMENTS I wish to thank Prof . VÙ. H. El1iot for permission to underLake these studies in the Department. I aÌso wlsh to thank my supervisor, Bob Syrnons for the advice and support provlded to me during the course of this wonk. In addition, I wish to express my appreciation to the following people: Dr . Pe ter Palukaitis , for purified CSV and arouslng my interest in viroids; Drs. Richard Fnanckl and John RandIes, for heJ-pf ul- discussions and interesting vinuses; Dr. Nizar Mohamed, Julita Imperial and Judith Rodrigue z, for providing ample amounts of ccRNAs; Dr. George BrueninS, f or unpubl-ished resul-ts and stimulaLing discussion 1n the J-ab; Dr. Detl-ev Riesner' f or providing unpubl-ished resufts and eintoPf; Dr. Al,l-an Gould, f or his help and dangerous sense of humour; Karf Gordon, for his red wine and socj-o-politics (more fike rosè), and scientific discussion; My fel-1ow viroid/virusoj-d infected co-workers, Dn. Peter Murphy, Jane Visvader and Paul- Keese, as weIf, as the other numerous faboratory and departmenlal lnmates; Jenny Rosey, Sharon Freund and Lisa Waters, for exceflent technical assj-stance and preparation of the figures for thls thesis; Mrs. To, for her care in typing this thesis; and finally my family, for putting up with this student. SUMMARY The work described in this thesis coticerrls the establishment and application of technj-ques for the rapid sequence determination of small circular RNAs such as those of viroids and virusoids. The determined sequences of chrysanthemum stunt viroid, the vari-ant RNAs of coconut cadang-cadang viroid and the virusoids of velvet tobacco mottle virus, solanum nodiflorum mottl-e virus and subterranean clover mottle virus are presented. The overall conclusions from the work are outlined briefly below. 1. Viroids contain highly conserved sequences central to their rod-like native structures. 2. Virusoids also contain highly conserved. sequences central to their rod-like native structures and share the pentanucleotid.e sequence GAAAC with that of viroids. 3. In addition the conserved. seguences of vírusoids are shared by the linear Rr\A of tobacco ringspot virus. Presumably the coInmon sequences of each cl-ass of RIIAs reflect common function, and perhaps suggest some functional similarity between viroids and virusoids. Sequence homology between virusoids and the satellite RNA of tobacco ringspot virus al-Iows pred.iction of sites for processing of these RNAs from multimeric RIJA intermediates of replication. CHAPTER 1 TNTRODUCTION A. Viroids Viroids constitute a unique class of infectious plant pathogens, and as such are a fairly recent dicovery. The viroid concept vJas first recognized when the infectlous agents of the spindJ-e tuber disease of potato (Diener: and Raymer, 1967 ) and the exocortis disease of citrus (Semancik and lltleathers, 1968 ) , which vrere thought to be viruses, I^i ere shown to possess unusual propertles : ( 1 ) phenol or other organic sofvents had no effect upon the lnfectivity of buffered extracts from lnfected plants; (2) no virus particles coul-d be isolated or dernonstrated by el-ectron microscopy; ( 3 ) the infectious agents displayed a resistance to nucfeases and an elutlon profile off cel-Ìufose columns simil-ar to double-stranded RNA; and (4 ) the inf ectious agents I^/ere always present in high speed supernatants, possessing sedimentation coefficients of 1 0- 1 5S. As a more detailed knowl-edge of the sizes and physlco-chemicaf properties of these two disease agents became availabl-e (Raymer and Diener' 1969; Diener and Raymer, 1969; Semancik and Weathers 1972a) , it became apparent that these two agents were the first of a new cfass of infectious nucÌeic acids (Diener, 1971b; Semanclk and Vrleathers, 1972b; Sånger, 1972); the Lerm virold I^Ias proposed (Diener, 1971b), and L the causal agents were renamed potato spindle tuber viroid ( pSfV ) and citrus exocortls viroid ( CEV ) . Since that t1me, viroids have been shown Lo be the causative agents of a further eight plant diseases, âhd are listed in Table 1 -1 . These viroids consj-st of infectious l-ow mol-ecul-ar weight RNA species which are unencapsidated, sì-ngJ-e sLranded, coval-ent1y-closed circul-ar moleçul-es with a hì-gh degree of intramol-ecul-ar base-pairing (Diener, 1972; Semancik et â1., 1975; Sången et âf., 1976). Physj-co-chemica] sludies of several- viroids (Henco et â1 .