The initiation of papaya mosaic virus assembly

VIROLOGY

90,

54-59

(1978)

The Initiation

of Papaya Mosaic Virus Assembly

M. ABOUHAIDAR

AND J. B. BANCROFT

Department of Plant Sciences, The University of Western Ontario, London, Ontario, Canada N6A 5B7 Accepted June 6, 1978 Initiation complexes were obtained by reacting papaya mosaic virus protein with its RNA at 1 or 25”. The encapsidated fragments of RNA extracted from the initiation complexes contained m’GpppGp and were rich in adenosine. Initiation occurs within about the first 200 nucleotides from the 5’-terminus of the virus RNA. INTRODUCTION

tein in 0.01 M Tris, pH 8, to a final RNA to In this paper, we specify the location at protein ratio of 1:2 (w/w) before incubation which the reconstitution of papaya mosaic at 25’ for 20 sec. Such reactions will stop without the addition of NaCl to 0.1 M. The virus begins on its RNA. We show that, unlike TMV (Zimmern and Wilson, 1976; low-temperature initiation complex was obOhno et al., 1977; Butler et al., 1977; Lebeu- tained by mixing precooled protein immerier et al., 1977), the process is initiated at diately after adjusting the pH to 8 with or near the 5’ end of the RNA which is concentrated Tris buffer finally diluted to terminated by methylated guanosine 0.01 M to precooled RNA (1 mg/ml) in (AbouHaidar and Bancroft, 1978). We also water so that the final RNA to protein ratio show that the initiation segment is rela- was 1:20 (w/w). The reaction was allowed to continue for 30 min at 1” before its tively rich in adenosine. termination with 0.1 M NaCl. MATERIALS AND METHODS Terminated reaction mixtures were inPreparation of virus, RNA, andprotein. cubated with RNase Tl (Sankyo Ltd., Tokyo) at 1 unit enzyme/25 pg RNA for 1 hr Virus was purified from papaya (Carica papaya L.) as described by Erickson and at 37” to degrade unencapsidated RNA. Bancroft (1978a) or with 0.02 M borate, pH EDTA to lo-” M was added to the solutions 7.5, as the extraction buffer (Purcifull and and the nucleoprotein particles were sediHiebert, 1971). “‘P-labeling was done by the mented by centrifugation at 36,000 rpm in a Beckman 40 rotor for 7 hr. Pellets were procedure of AbouHaidar and Bancroft (1978). RNA was obtained from the virus resuspended in 0.5 ml 0.01 1M Tris, lo-” A4 EDTA, pH 8. RNA was extracted from the after treatment with guanidine (Reichmann short nucleoprotein particles with the and State-Smith, 1959) or by extraction phenol mixture or the particles were dissowith 2 volumes of water-saturated phenol/chloroform (v/v) for 5 min at 70”. ciated in 5 M urea containing 4% (w/v) Virus coat protein was isolated by the acetic SDS and 0.5% mercaptoethanol at 70” for 1957) 5 min before loading on polyacrylamide acid method (Fraenkel-Conrat, gels. slightly modified (Erickson and Bancroft, Fractionation of initiation fragments. 1978a). The fragments of PMV-RNA, recognized Initiation complex formation and RNA extraction. Initiation complexes were made and encapsidated by limiting quantities of PMV-protein at pH 8.0, were fractionated at 25 and lo (Erickson and Bancroft, 1978b). For the formation of the 25” com- by electrophoresis on a slab polyacrylamide plex, “2P-labeled RNA at 2 to 5 mg/ml in gel of three different concentrations (6, 8, water was preincubated at 25” for 2 min. It and 10%). Electrophoresis was carried out was mixed with similarly preincubated pro- at constant current (20 mA) at room tem54 0042~6822/78/0901-0054$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

PMV

perature until the bromphenol blue marker had migrated l-2 cm from the bottom of the gel. The electrophoretic buffer was 0.1 MT&borate, 2.5 x low5 M EDTA, pH 8.3, containing 7 M urea. Fractionated products were detected by autoradiography using Cronex DuPont films. The different bands on the polyacrylamide gels which corresponded to different sizes of RNA were cut from the gel and electrophoresed into tubing sealed with a circle of DEAE-cellulose paper. The RNA fragments were then eluted from the DEAE-cellulose discs by triethylamine bicarbonate (TEABC). Cap and base ratio determinations. In order to test for the presence of the “cap” in the initiation site, RNA extracted from the nucleoprotein particles or recovered from the different bands on polyacrylamide gels was digested by a mixture of RNases A, Tl, and T2 and fractionated by electrophoresis (AbouHaidar and Hirth, 1977 and AbouHaidar and Bancroft, 1978). For the determination of base composition, RNA fragments were digested with RNases under the conditions described above. The hydrolysis products were fractionated by electrophoresis on Whatman paper No. 1 or No. 3 in 0.5% pyridine, 5% acetic acid (v/v), pH 3.5, for 45 min until the blue marker, xylene-cyan01 FF, had migrated about 12 cm from the origin. The different 3’nucleotides were localized by autoradiography and counted in a scintillation counter Beckman LS-230 in a toluene-based scintillation mixture. Electron microscopy. For electron microscopy, preparations of nucleoprotein particles were dispersed on Formvar-carbon-coated grids and negatively stained with 1% uranyl acetate or platinumshadowed at low angle using the Kleinschmidt and Zahn (1959) technique. Length measurements of stained particles were made as described by Erickson and Bancroft (1978b). RESULTS

Physical Properties plex

of the Initiation

55

INITIATION

Com-

The number and weight averages of nucleoproteins made at lo were 49 and 66 nm, respectively (Bancroft and Erickson,

197813). Those formed under protein-limiting conditions at 25”, which is the temperature used for reconstitution, were 49 and 66 nm, respectively (Fig. 1, A and B). The short particles, apart from their length, look like the virus (Fig. 1C). Examination of shadowed particles not treated with RNase show that RNA protrudes from one end of the particles (Fig. 1D). The short particles assembled at 25” sedimented at 55 S, (Fig. 2A) which corresponds to a weight average of about 50 run. An estimate of the RNA to protein ratio in the RNase-treated initiation complex obtained at 25” compared with that of the virus and its protein was obtained by equilibrium centrifugation in CsCl (Fig. 2B). Values of 1.299, 1.302, and 1.286 g/cm3, respectively, were obtained. Rebinding

of Initiation

Complex

RNA

It was necessary to prove that the RNA in the initiation complex would, if isolated, serve in initiation. Consequently, particles initiated at 25” were treated with phenol/chloroform at 70” to extract the protected RNA fragments. The fragments were encapsidated by protein to give short nucleoprotein particles similar in length distribution to those from which the RNA was obtained. Presence of m7GpppGp Complex RNA

in the Initiation

Since the 5’-terminus of PMV-RNA is methylated (AbouHaidar and Bancroft, 1978), it is possible to determine if initiation of the protein helix starts at or near the 5’ end of the RNA at 1 and 25”. Initiation complex 32P-RNA encapsidated either in the cold or at 25” contained m7GpppGp (Fig. 3, A and B), signifying that encapsidation was indeed initiated at or near the 5’ end of the RNA in both cases. The lengths of initiated particles vary at 25” particularly and it was deemed worthwhile to determine if the cap could be detected in the shortest protected pieces of RNA which could be isolated. If it could not, then it would be conceivable that initiation started at a location internal to the cap and that some elongation proceeded toward the 5’ end of the RNA in addition

56

ABOUHAIDAR

LENGTH

CLASS

(nm)

AND

BANCROFT

LENGTH

CLASS (nm I

FIG. 1. Size and appearance of initiation complex particles made at 25”. A, number average; B, weight average of 176 particles; C, RNase-treated particles negatively stained with 1% uranyl acetate; D, platinumshadowed preparation of particles before treatment. with RNase showing protruding tails. Bar represents 100 nm.

to the normal process which proceeds toward the 3’-terminus. Consequently, initiation comblex RNA was fractionated on polyacrylamide gels after disruption of the initiation particles at 70” in the urea-SDS mixture. The different bands (l-17) isolated from the gel were digested with RNases. Each contained a radioactive species which migrated with the same electrophoretic mobility as the m7GpppGp used as the control (Fig. 3C). The shortest fragment of RNA, which was present in sufficient quantity to be detected by autoradiography, had an estimated size, in comparison

with tRNA used as a marker, of about 200 nucleotides. Base Ratio of Initiation

Complex RNA

The base composition of RNA obtained from initiation complexes made at 1 and 25” was determined. The results in Table 1 show that both contained RNA which had 42% adenosine as compared to 33% adenosine in the virus RNA. The high content of adenosine in the initiation RNA was car pensated for mainly by a decrease in guanine. These results, in combination with

PMV

57

INITIATION DISCUSSION

The short nucleoproteins formed by PMV protein and RNA at 1” or under limiting amounts of coat protein at 25” contain special fragments of RNA necessary for the formation of an initiation complex. The fragment which reacts with PMV protein does not occur in the RNAs of viruses unrelated to PMV (Erickson et al., 1978). Although short, the initiation complexes seem to be constructed like the virus. The difference in density between the short particles and virus could result from the magnified effect that would, in a small nucleoprotein, arise from the content of RNA being halved in the first turn of the protein helix in polar initiation, but not necessarily in internal initiation (Zimmern and Butler, 1977). A difference may also derive from FIG. 2. Schlieren diagrams of RNase-treated initiribonuclease-induced “fraying” at the ends ation complex particles made at 25”. A, sedimentation of the encapsidated RNA as found with pattern-the fastest sedimenting material (55 S) is the TMV (Zimmern, 1977). It is unlikely that initiation complex and the slower (14 S) is unreacted the RNA is simply bound to the external protein, taken 12 min after a speed of 29,500 rpm was surfaces of the subunits, inasmuch as the reached. Sedimentation is to the right. B, CsCl-grafragments of RNA are protected from the dient (average density, 1.300 g/cm”) pattern of, left to action of ribonuclease. right, PMV protein, initiation complex, PMV after The presence of m7GpppGp in the initi22% hr at 44,770 rpm. ation complexes clearly shows that the 5’ end of PMV RNA is protected early during those in which the presence of the cap was an assembly process initiated at either 1 or demonstrated, show that the identity of the 25”, and that the elongation kinetics deRNA used in initiation at 1 or 25” is the scribed by Erickson and Bancroft (1978b) are the result of encapsidation toward the same. In order to specify the location of the 3’ end of the RNA. Fractionation of the adenosine residues, fragments of RNA were fragments encapsidated at 25” resulted in fractionated as before by polyacrylamide the detection of a capped oligonucleotide of would gel electrophoresis and 8 bands, ranging in about 200 bases. This oligonucleotide size from about 1000 (band 1) to 200 (band be encapsidated by five or six turns of the 8) nucleotides were eluted. The base ratios, protein helix taking four or five nucleotides shown in Table 1, suggest that the region per subunit and close to eight subunits per richest in adenosine is located near the cap turn of the helix. This result strongly sugsince the relative amount of adenosine in- gests that initiation must occur within creased with a decrease in fragment size. about 20 nm of the length of the virus from the 5’ end of its RNA, inasmuch as the The reason for the decrease in the absolute values of adenosine compared with those pitch of the protein helix is about 3.6 nm found in the complete initiation fragment (Erickson et al., 1976). A similar result was population is not understood but could be found for tobacco rattle virus (AbouHaidar related to a systematic error resulting from and Hirth, 1977). The presence of the cap counting at low levels. In spite of this, the in the initiation fragment does not preclude bidirectional growth if initiation occurs conclusion about the location of adenosine is not invalidated because the relative val- only near the cap. In such a case, there ues form a clear pattern. would have to be rapid growth in the 5’

58

ABOUHAIDAR

AND

BANCROFT

A

UP

c+p AP

B

7

5

9

73

FIG. 3. Electrophoretogram of a total digest with RNases Tl, T2, and A of the different fragments of RNA encapsidated by partial assembly between PMV-RNA and PMV-protein. A, digestion products of fragments obtained from the assembly between PMV-RNA and PMV-protein at pH 8.0 at lo. B, digestion products of the fragments of RNA obtained by partial assembly at pH 8.0 at 25”. C, hydrolysis products of the RNA fragments encapsidated by PMV-protein in the same conditions as in B. The fragments of RNA were fust fractionated by electrophoresis on a polyacrylamide slab gel (6, 8, 10%) then recovered from the gel and digested with the nucleases. Electrophoresis was carried out on DEAE-cellulose paper (Whatman DE-81) in 0.5% pyridine, 5% acetic acid buffer, pH 3.5. 0 is the origin, B is the position of the blue marker xylene-cyan01 FF, C is the m’GpppGp marker.

direction and presumably rapid but limited growth in the 3’ direction as well to account for the 50-60-nm length of the energetically distinct initiation phase particles (Erickson and Bancroft, 1978a and b). It is probably simplest at this stage to think of unidirectional growth toward the 3’ end of the RNA occurring in two thermodynamically distinct steps. The location of initiation is different from that of TMV, which is found in a region between 750 and 2000 nucleotides from the 3’-OH end of the RNA, subsequent growth proceeding rapidly toward

the 5’ end and slowly toward the 3’-OH end (Zimmern, 1977). The difference in modes of initiation and growth may conceivably be related to the ionic differences between TMV and PMV required for assembly in u&o, but preliminary results suggest that this is not so (unpublished). An interesting feature of the initiation complex RNA is its adenosine content which is about 1.3 times greater than in complete PMV-RNA. Presumably, the extra adenosine is involved in recognition of protein for the RNA. Polyadenylic acid

77

59

PMV INITIATION TABLE I BASE COMPOSITION OF THE INITIATION COMPLEX RNA AND FRAGMENTS FROM IT Molar per cent Material” PMV-RNA (controbh Initiation RNA, 1”’ Initiation RNA, 250” Fragments of initiation RNA, 25” 1 2 3 4 5 6 7 8

CP 24.0

AP 33.0

GP 22.0

UP 21.0

22.0 24.0

42.0 42.8

17.4 15.2

18.6 18.0

25.6 25.0 25.4 24.2 22.6 21.8 23.5 22.6

35.0 34.0 34.5 36.6 39.2 39.0 39.0 41.5

19.2

20.2 22.5 22.0

18.5 18.1

18.0

21.2

17.8 17.2 16.5 17.2

20.4 22.0 21.0 18.7

n RNA was digested with RNases Tl, T2, and A and residues were fractionated by electrophoresis on Whatman 3MM paper in 0.5% pyridine, 5% acetic acid, pH 3.5. * Average of three different preparations (cf., 23.4; 33.8; 20.7; 22.1 for Cp;Ap;Gp;Up (PurcifuII and Hiebert, 1971)). ’ Average of two different preparations. ” Average of three different preparations. ’ Fragments isolated from a polyacrylamide gel in three layers (6, 8, and 10%) in the presence of 7 M urea. The sizes of the RNA fragments decrease from about 800-1000 nucleotides (band 1) to about 206 nucleotides (band 8).

work was supported in part by a grant from the National Research Council of Canada. REFERENCES ABOUHAIDAR, M., and BANCROFT, J. B. (1978). The structure of the 5’-terminus of papaya mosaic virusRNA. J. Gen. Viral., 39,559-563. ABOUHAIDAR, M., and HIRTH, L. (1977). 5’-Terminal structure of tobacco rattle virus RNA: Evidence for polarity of reconstitution. Virology 76, 173-185. BUTLER, P. J. G., FINCH, J. T., and ZIMMERN, D. (1977). Configuration of tobacco mosaic virus RNA during virus assembly. Nature 266,217-219. ERICKSON, J. W., ABOUHAIDAR, M., and BANCROFT, J. B. (1978). The specificity of papaya mosaic virus assembly. Virology 90,60-66. ERICKSON, J. W., and BANCROFT, J. B. (1978a). The self-assembly of papaya mosaic virus. Virology 90, 36-46.

ERICKSON, J. W., AND BANCROFT, J. B. (1978b). The kinetics of papaya mosaic virus assembly. Virology 90,47-53.

ERICKSON, J. W., BANCROFT, J. B., and HORNE, R. W. (1976). The assembly of papaya mosaic virus protein. Virology 72, 514-517. FRAENKEL-CONRAT, J. (1957). Degradation of tobacco mosaic virus with acetic acid. Virology 4, 1-4. KLEINSCHMIDT, A., and ZAHN, R. K. (1959). Uber Desoxyribonuleinsie Molekiilen in Protein Mischfiien. Z. Naturforsch. 14b, 770-779. LEBEURIER, G., NICOLAIEFF, A., and RICHARDS, K. E. (1977). An inside-out model for the self-assembly of tobacco mosaic virus. Proc. Nat. Acad. Sci. USA 74,149-153.

OHNO, T., SUMITA, M., and OKADA, Y. (1977). Location of the initiation site on tobacco mosaic virus RNA involved in assembly of the virus in uitro. Virology 78,407-414.

serves as a nucleating agent for protein under reconstitution conditions but, unfortunately, is not unique in this property (Erickson et al., 1978). Nevertheless, a high content of adenosme also occurs in the TMV-RNA recognition site (Zimmern, 1977) and it is possible that a high adenosine content will turn out to be a general feature in the initiation of tubular viruses. A more detailed idea of the composition of the initiating region may be gained from nucleotide sequencing which is underway.

PURCIFULL, D. E., and HIEBERT, E. (1971). Papaya mosaic virus. C.M.I./A.A.B. No. 56. REICHMANN, M. E., and STACE-SMITH, R. (1959). Preparation of infectious ribonucleic acid from potato virus X by means of guanidine hydrochloride denaturation. Virology 9, 710-712. ZIMMERN, D. (1977). The nucleotide sequence at the origin for assembly on tobacco mosaic virus RNA. Cell l&463-482. ZIMMERN, D., and BUTLER, P. J. G. (1977). The isolation of tobacco mosaic virus RNA fragments containing the origin for viral assembly. Cell 11,

ACKNOWLEDGMENTS We wish to thank Mr. J. W. Erickson for discussion and Mr. R. Johnston for technical assistance. This

ZIMMERN, D., and WILSON, T. M. A. (1976). Location of the origin for viral reassembly on tobacco mosaic virus RNA and its relation to stable fragment. FEBS Lett. 71, 294-298.

455-462.