The self-assembly of papaya mosaic virus

90, 36-46

VIROLOGY

(1978)

The Self-Assembly

Department

of Plant

JOHN

W. ERICKSON

Sciences,

The

University

of Papaya Mosaic Virus AND of Western

Accepted

June

J. B. BANCROFT Ontario,

London,

Ontario,

Canada

N6A

5B7

6, 1978

The conditions for the in vitro reconstitution of papaya mosaic virus (PMV) from its isolated constituents are described and are related to the formation of coat protein subassembly products. PMV assembles best in 0.01 M, pH 8.0, Tris buffer at 25” at a protein -to RNA ratio of 2O:l (w/w). At lower pH levels, faulty, segmented particles are formed which are sensitive to ribonuclease. The assembly process at pH 8.0 is composed of two energetically distinct phases corresponding to helix initiation and elongation. Both reactions may be stopped by low levels of NaCl, whereas only elongation requires elevated temperatures. If the growth of elongating particles is stopped by lowering the temperature, long and thin “extended particles” are detected, if NaCl is used, the arrested particles terminate with a “brush” at one end only. Under virus assembly conditions, the coat protein exists in an equilibrium among several polymeric species, most notably 14 S and 25 S polymers. The latter is not required for reconstitution, The equilibrium mixture is very sensitive to changes in pH, ionic strength, temperature, and protein concentration. INTRODUCTION

We have been interested in the manner by which flexuous or sinuous viruses and their proteins self-assemble (Goodman et al., 1976; McDonald and Bancroft, 1977) and have chosen to investigate papaya mosaic virus (PMV) (Purcifull and Hiebert, 1971) which is a member of the potato virus X family (Harrison et al., 1971). PMV has all the attributes necessary for such a study; it is obtainable in high yields comparable to tobacco mosaic virus and brome mosaic virus; it is easy to purify and to assay physically and biologically; it can be disassembled by standard methods and its protein assumes a variety of polymeric structures, some of which have been briefly described (Erickson et al., 1976). We now wish, in this introductory paper, to describe methods by which PMV may be assembled from its isolated constituents and in so doing draw attention to some small polymers which the protein structure units may form. MATERIALS

AND

METHODS

Virus purification. PMV was grown in Carica papaya L. and purified as described (Erickson et al., 1976) but with some mod-

ifications. Upper leaves from greenhousegrown papaya trees, infected for several months to years, were harvested in 100-g lots and either homogenized directly or stored at -20” before use. Tissue was homogenized at 4’ with 2 vol (w/v) of 0.05 M sodium phosphate buffer, pH 8, containing 0.01 M EDTA. In some purifications, a 10% aqueous solution of Triton X-100 (Baker Chemicals) was blended in gradually (over about 1 min) with the homogenized tissue to yield a final concentration of 1%. If Triton was used, several drops of a 1:l mixture of butanol/chloroform were added at the start of the blending to prevent excessive foaming. For clarification, l/2 vol (v/v) of the butanol/chloroform mixture was added to the blender. The emulsion was separated by centrifugation in a GSA rotor (Sorvall) at 10,000 rpm for 10 min. The virus-containing upper aqueous phase was decanted and centrifuged in an SS-34 rotor (Sorvall) at 17,000 rpm for 30 min. The supernatant fluid was overlaid onto 7 ml of a 30% (w/v) sucrose solution containing 5 x 10m3 M EDTA, pH 8, and was then centrifuged in a Beckman 30 rotor at 27,000 rpm for 3% hr. The pellets were suspended in about l/ 10 vol (v/v) of 5 x 10m3M Tris, pH 8, and 36

0042-6822/78/0901-0036$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

PMV

SELF-ASSEMBLY

several crystals of sodium azide were added as preservative. PMV prepared in this manner was usually free of plant pigments, had an Azao/Azm ratio of about 1.4 and sedimented as a single species in the analytical centrifuge. If further cycles of differential centrifugation were required, PMV suspensions were first diluted to l-2 mg/ml. Yields without Triton were about 1.5 to 2 mg/g fresh weight tissue and with Triton 2 to 2.5 mg/g tissue. PMV concentrations were estimated assuming Etg= 2.85 (Hiebert, 1970). Protein from fresh virus prepared as described usually gave a single band migrating at the rate of coat protein in SDSpolyacrylamide gels. Preparation of PMV coat protein. PMV coat protein was prepared by acetic acid degradation (Fraenkel-Conrat, 1957) as described previously (Erickson et al., 1976) but with modifications. Two volumes of glacial acetic acid were added to concentrated virus on ice. Insoluble RNA was removed after 1 hr by centrifugation in an SS-34 rotor (Sorvall) at 10,000 rpm for 30 min. The supernatant liquid was dialyzed overnight against 20 vol of distilled HZ0 to a final concentration of about 3% and then centrifuged at 27,000 rpm for 5 hr in a Beckman 30 rotor to remove any residual virus. The supernatant solution was then dialyzed for 48 hr against several changes of distilled water to remove completely the acetic acid. The protein solution was then titrated slowly with 0.1 N NaOH to pH 9.5 to be certain that helices, which form during the long term dialysis as the solution pH goes through pH 4, are all disassembled. The resulting preparations, which were stored in water after about 10 min at pH 9.5, were devoid of virus-like particles as judged by electron microscopy and had typical protein spectra with Azm -/AZ50 nm between 2.0 and 2.5. Protein concentrations were estimated assuming E4ksnnn,= 1.0. Preparation of PMV RNA. PMV RNA was prepared by the guanidine hydrochloride method (Reichmann and State-Smith, 1959). One volume of 5 M guanidine HCl containing 1 x 10m3 M EDTA, pH 8.0, was added to concentrated virus in ice. The RNA precipitate was collected by centrifugation at 5,000 rpm for 1 min and was then

37

dissolved in sterilized, distilled HzO. Several water washings were usually required for quantitative recovery (about 5 mg RNA/100 mg virus). Successive washings were pooled and the RNA was precipitated with 2 vol of cold 95% ethanol to which a drop of 5 M NaCl was added to aid precipitation. PMV RNA preparations gave typical nucleic acid spectra with A260 dA2~ nm ratios of 2.0. RNA concentrations were estimated assuming EBgm = 25.0. Virus reconstitution. All solutions employed for reconstitution studies were prepared with sterile, deionized, glass-distilled HzO. PMV RNA, stored in ethanol at -2O”, was collected by centrifugation st 1500 g for 5 min in a clinical centrifuge and was dissolved in water and adjusted to 1 mg/ml. PMV coat protein, stored in water at 4’, was diluted to the appropriate concentration in water and equilibrated for 5 min at the desired temperature in a water bath. The pH of the reaction was preadjusted by addition, to the protein in water, of the appropriate 1.0 M buffer to a final concentration of 0.01 M and reconstitution was initiated about 30 set thereafter by addition of a small aliquot of RNA to the buffered protein solution. Protein and RNA concentrations of 1 mg/ml and 0.05 mg/ml, respectively, in 1 ml of 0.01 M Tris, pH 8, at 25” were usually used. For the reconstitutions in NaCl, the zero-time salt adjustments were made just before the pH adjustments by addition, to the protein in water, of the appropriate volume. of 2.0 M NaCl. Reconstitution reactions were usually stopped after 30 min by the addition of 4 M NaCl to a final concentration of 0.2 M before assay. Infectiuity assays. The infectivities of reconstitution mixtures, incubated after the NaCl treatment with 1 unit ribonuclease T1 (Calbiochem) per 50 pg RNA for 30 min at 37”, were assayed on Gomphrena globosa L., a local lesion host for PMV (Purcifull and Hiebert, 1970). The assays were arranged so that eight to ten half-leaf comparisons were made for each treatment against a suitable control. Infectivities are expressed as percentages of control infectivity. Electron microscopy. Reconstitution

38

ERICKSON

AND

mixtures were examined for morphology in the electron microscope both before and after the addition of salt. Samples treated with NaCl were diluted as required and pipetted onto carbon-coated Formvar grids and stained with 1% uranyl acetate. Untreated samples were pipetted directly, without dilution, onto grids and stained. Determination of weight average lengths was done as described by Erickson and Bancroft (1978). Turbidimetry. A Pye Unicam SP1800 recording spectrophotometer was employed to monitor the turbidity of various samples at 310 nm. For kinetic studies, solution additions to the cuvettes were made in the dark. Cuvette temperatures were controlled by means of a variable temperature, circulating water bath connected to a waterjacketed cuvette block with a thermistor connection. Analytical sedimentation. A Beckman model E analytical ultracentrifuge equipped with Schlieren optics was employed to analyze the polymorphism of PMV coat protein under a variety of experimental conditions. Sedimentation coefficients were corrected to water at 20”. RESULTS

Reconstitution The conditions necessary for the reconstitution of biologically active PMV were determined. The Effect ofpH The relative infectivities of ribonuclease T1-treated virus reconstituted at 25” from pH 6.0 to 10.0 in 0.01 M levels of suitable buffers show that the best pH for assembly lies between pH 8.0 and 8.5 (Fig. 1). The yield of virus at pH 8.0 to 8.25 was routinely high (>90%) as measured by rate densitygradient analyses, whereas this was not always true at pH 8.5, so the lower pH levels were used. The specific infectivity of virus made at these pH levels was about 2% that of natural virus. Nucleoprotein assembly at pH 5.0, 6.0, 7.0, and 7.5 also gave high yields but the particles were “kinked” or segmented (Fig. 2A) which probably accounts for their susceptibility to RNase T,. The amount of

BANCROFT

PH FIG. 1. Relative infectivities of nucleoprotein assembled at pH 5.0 (0.01 Msodium acetate), pH 6.0 and 7.0 (0.01 M 2-(N-morpholino)ethanesulfonate-HCl (MES)), pH 7.5, 8.0, and 8.5 (0.01 M Tris-HCl), pH 9.0, 9.5, and 10.0 (0.01 M glycine-NaOH) after incubation with 1 unit of RNase T1 per 50 pg RNA for 30 min at 37”. Such treatment completely inactivated naked RNA. The assembly conditions were: protein concentration, 1 mg/ml; RNA concentration, 0.05 mg/ ml; temperature, 25”; time, 30 min. Infectivities are expressed as percentages of that obtained at pH 8.0.

segmentation was greater at pH 5.0 to 6.0 than at pH 7.0 to 7.5. Particles made at pH 8.0 to 8.5 were mainly sinuous (Fig. 2B). No rods were visible at or above pH 9.5. The Effect of Temperature Virus assembly, as measured by infectivity resistant to RNase T1, was not efficient below about 25” in 0.01 M Tris buffer, pH 8.0 (Fig. 3A). However, partial rod growth did occur at lower temperatures as shown by turbidity and length measurements (Fig. 3B). Below about lo”, a length of about 50 nm (number average) was attained by particles in assembly mixtures. Complete rod growth required an elevated temperature. Thus, we infer that PMV assembly is comprised of two distinct processes: an early, temperature-dependent event followed by a cold-sensitive phase as shown in the Arrhenius plot (Fig. 4). These two processes are defined as initiation and elongation, respectively. Rod growth, once underway at a permissive temperature, could be stopped by cooling the assembly mixture to a nonpermissive temperature resulting in partially reconstituted rods called “extended particles” (Fig. 5). Extended particles, which are degraded by RNase A, are also detected after short incubation periods at 25”. No such structures have ever been obtained with PMV coat protein alone.

PMV

SELF-ASSEMBLY

FIG. 2. Electron micrographs of nucleoprotein assembled txperimental conditions. The bar represents 100 nm.

at pH

6.0 (A)

and

pH

8.0 (B).

See Fig.

1

40

ERICKSON

AND

BANCROFT

$0 * $i

75

2

25

iis

50 u

0

5

IO

5

10

15 20 “C

25

30

0 2

0-o

152025

30

“C

FIG. 3. A, the effect of temperature on the assembly of PMV as measured by infectivity. PMV protein and RNA at 1.0 and 0.05 mg/ml, respectively, were reacted in 0.01 h4 Tris, pH 8.0, for 30 min at various temperatures before treatment with RNase T, and assay. Infectivity is expressed relative to that obtained at 25”. B, the effect of temperature on the assembly of PMV as measured by rod length and turbidity. Assembly conditions were those described in A. Rod growth was measured by turbidity at 310 nm, U, and by weight average lengths of particles measured from electron micrographs, G--O. Turbidity and weight average lengths are expressed relative to those measured at 25” (0.020 ODzlonm and 280 nm, respectively).

log k logk

0

3.45

The effect of temperature on nucleoprotein formation is pH dependent. In contrast to reconstitution at pH 8.0, assembly at pH 6.0 occurred immediately at 0” as well as at 25”. The Effect of Protein

Concentration

The effect of protein concentration on reconstitution over the range 0.1-2 mg/ml at constant pH (0.01 M Tris, pH 8.0) and temperature (25’) is seen from Fig. 6. Virus assembly is equally efficient between 0.25 and 2 mg/ml starting protein concentration and is halved at 0.1 mg/ml.

1.5

#\ 1.2 3.35

FIG. 5. Electron micrograph of an extended particle. Protein and RNA at 1 and 0.05 mg/ml, respectively, were reacted at pH 8.0 (0.01 M Tris) for 30 min at 10’. The bar represents 100 nm.

355

3.65

l/T x IO3 FIG. 4. Arrhenius plot of the effect of temperature on the relative rate, k, of elongation for PMV assembly at pH 8.0 in 0.01 M Tris. Values for log k represent log (percentage weight average rod length/30 min) relative to 25” and were calculated from the experimental data presented in Fig. 3B.

The Effect of NaCl The presence of NaCl from 0.025 to 0.2 M inhibited the formation of infective PMV (Fig. 7). The same effect was observed even if the assembly reaction was allowed to proceed for 2 min before the addition of NaCl, suggesting that both initiation and elongation were inhibited by this salt within the times tested. Rod growth was stopped upon addition of the salt, resulting in structures with “brushes” localized at one end of the particles (Fig. 8). These brushes may result from salt-induced collapse of the ex-

41

PMV SELF-ASSEMBLY

tended particles which were never found in the presence of NaCl. At the end opposite the brushed end, presumed to be at or near the initiating region, convex-rounded ends are found, suggesting that the growing rod presents a concave surface at the elongating site. Stoichiometry In typical reconstitution

experiments,

a

protein to RNA ratio of 2O:l was used inasmuch as the RNA comprises about 5% of the weight of the native virus. The infectivity of assembled preparations increased linearly with increasing protein to RNA ratio, at constant RNA concentration, up to 2O:l (Fig. 9). Infective virus was found at the lower ratios (l:l-lO:l), suggesting that protein was preferentially incorporated into growing particles. do*

/

5 75 : t, 50 : $ 254

FF 01 05

1

2

mg/ml FIG. 6. The effect of protein concentration on the assembly of PMV at 25’. PMV protein at various concentrations was reacted with RNA at a constant protein to RNA ratio of 2O:l in 0.01 M Tris, pH 8.0, for 30 min before RNase T, digestion. The infectivity assays from 0.25 to 2.0 mg/ml protein were all made at a final concentration of 0.25 mg/ml (a l/4 dilution of the 1 mg/ml mixture used as standard). That at 0.1 mg/ml was made against virus assembled at 1 mg/ml, diluted l/10.

; 25 : \ s 0 1_

o-1

0.2

M NaCl FIG. 7. Effect of NaCl concentration on the assembly of PMV as measured by infectivity. PMV protein (1 mg/ml) and RNA (0.05 mg/ml) were incubated at 25” in 0.01 M Tris, pH 8.0, in the presence of various concentrations of NaCl added at zero time (U) and 2 min after the initiation of mmbly (O---O). After 30 min, the assembly mixtures were digested with RNase T1 and assayed for infectivity. Infectivities are expressed as percentages of those obtained with the samples to which no NaCl was added.

FIG. 8. Electron micrograph of “brushes.” PMV was assembled at 15” for 20 min at pH 8.0 (0.01 M Tris) and stopped by the addition of NaCl to 0.2 M. Bar represents 100 mn.

42

ERICKSON >t-

100

0

25 0

BANCROFT

b

5 75 F wo 50 2 P

AND

FL-I

5

l&-z PRoTElN/RNA

50

FIG. 9. Effect of protein to RNA ratio on PMV assembly at 25”. PMV protein at various concentrations was reacted with RNA maintained at a constant concentration (0.04 mg/ml) in 0.01 M Tris, pH 8.0, for 30 min before RNase TI digestion. Infectivities are expressed as percentages of that obtained at a protein to RNA ratio of 2O:l.

Protein Subassembly Products Attempts were made to relate the reconstitution results with the assembly behavior of the coat protein. The Effect ofpH The effect of pH on the behavior of coat protein is shown in Fig. 10. Coat protein at either pH 5.0 or 6.0 sedimented as a single species at 14 S. At pH 7.0, the polymers were heterogeneous, the principal components sedimenting at 13 and 36 S. From pH 7.5 to 9.0, the main species had sedimentation coefficients of, on the average, 25 S and were in equilibrium, as will be shown, with 14 S and slower sedimenting polymers. At pH 10.0 the 14 S species, which occurs in water, dissociated into two principal protein species sedimenting at 3 and 8 S. The rate at which protein was converted from the 14 S species in water at pH 5.5 to the predominately 25 S form upon addition of pH 8.0 Tris buffer to 0.01 M was measured by turbidity. The reaction was complete within 30 set at 25”. The Effect of Temperature and Concentration PMV protein at pH 6.0 sedimented at 14 S at l-2 mg/ml and at either 5 or 25’. In contrast, the protein existed in a dynamic equilibrium between the 14 and 25 S polymers, as well as some smaller aggregates, at pH 8.0. The extent of polymerization increased both with protein concentration and temperature (Fig. 11 and Table 1).

FIG. 10. Schlieren patterns of PMV protein at 3 mg/ml at various pH levels. A, 14 S species in 0.01 M acetate buffer, pH 5.0; B, upper, 14 S species in 0.01 M MES, pH 6.0; lower, 13 and 36 S species in 0.01 M MES, pH 7.0; C, upper, 12 and 24 S species in 0.01 M Tris, pH 7.5; lower, 12 and 27 S species in 0.01 M Tris, pH 8.0; D, upper, 15 and 27 S species in 0.01 M Tris, pH 8.5; lower, 13 and 24 S species in 0.01 M glycine, pH 9.0; E, 3 and 8 S species in 0.01 M glycine, pH 10.0. In all cases, buffers were added to the protein from the same large preparation 10 min before the cells were loaded. All runs were made at 22 or 23” at a speed of 52,640 rpm. Sedimentation is to the right.

The temperature-dependent conversion of the 14 S to 25 S species at 2 mg/ml was monitored by turbidity, which was maximal between 20 and 25”. The reaction was reversible between 5 and 25”, the association (heating) and dissociation (cooling) reactions both being complete within 1% min. The Effect of NaCl NaCl greatly affected the behavior of PMV protein at pH 8.0, with the equilib-

PMV

43

SELF-ASSEMBLY

ceeds best in stoichiometric mixtures at pH 8.0-8.5, requires moderate temperatures (25-30’) and low ionic strength. Assembly of PMV can be divided into two phases: TABLE

1

SEDIMENTATION COEFFICIENTS OF PMV PROTEIN AT DIFFERENT CONCENTRATIONS AND TEMPERATURES Temperature Protein COlXXntra-

tion (nip/

5

10

15

20

25

1.8 1.8, 12 3.5, 12 3.3, 12

1.8, 11 1.8, 12 12 13

1.8, 11 1.8, 13 13 13, 18

-1.8 1.8, 13 13 14, 21 14,26

-3.2 13 12, 186 14,27 14, 31 30

ml)”

0.10 0.25 0.50 1.0 2.0 8.0

a Protein in 0.01 M Tris, pH 8.0 b In some preparations, a 25 S rather species was observed.

than

an 18 S

FIG. 11. Schlieren patterns of PMV protein at 0.25, 0.50, 1.0, and 2.0 mg/mI (upper left, lower left, upper right, lower right, respectively) at A, 5’; B, lo’, C, 15”, D, 20”, and E, 25’ (where the inset corresponds to 0.10 mg/mI) in 0.01 M Tris, pH 8.0. The sedimentation coefficients are listed in Table 1. Ah runs were performed at 52,460 rpm. 30-mm cehs were used for protein concentrations at 0.5 mg/rnI or less. Sedimentation is to the right.

rium shifting from the 25 S to the 14 S polymer as the NaCl concentration increased (Fig. 12). These data were corroborated by turbidity studies on the NaClinduced disassembly (Fig. 13), which suggest a salting-in effect (Edsall and Wyman, 1958, pg. 304). The 25 S aggregate was dissociated to the 14 S polymer within 3 set upon the addition of NaCI. DISCUSSION

The in vitro reconstitution

of PMV

pro-

FIG. 12. Schheren patterns of PMV protein in 1 mg/ml in 0.01 M Tris, pH 8.0, containing: A, no NaCl; B, 0.025 M NaCl; C, 0.05 M NaCl; D, 0.10 M NaC1; E, 0.2 M NaCl. The sedimentation coefficients of the fastest sedimenting species from A to E are 27, 25, 20, 17, and 14 S, respectively. F is a direct comparison of PMV protein at 3 mg/ml in 0.01 M Tris, pH 8.0, in preparations containing no NaCl (upper) and 0.2 M NaCl (lower). All runs were performed at 52,460 rpm at 22 or 23’. Sedimentation is to the right.

44

ERICKSON

AND BANCROFT

mg/ml protein do not preclude the presence of a small quantity of the 14 S polymer (which appears to be a two-ring structure containing about 16 subunits (unpublished)). If, by analogy with TMV (see Butler and Durham, 1977) or cucumber green mottle virus (Ohno et al., 1972), the polyI 40 80 120 mer was necessary solely for initiation, it seconds would only have to represent about $45 by FIG. 13. Effect of NaCl concentration on the turweight of the protein inasmuch as the virus bidity of PMV protein at 25”. Protein at 2 mg/ml in is 540 nm long and the pitch is 3.6 nm water (pH 5.0) (A) was adjusted to pH 8.0 (0.01 M Tris) by the addition of 10 ~1 of 1.0 M Tris, pH 8.0 (Erickson et al., 1976) and would not be (arrow). The change in turbidity at 310 nm was mon- detected by Schlieren optics. itored in response to incremental additions of 2.0 M Elongation, which probably occurs on a NaCl to final concentrations of: B, 0.02 M; C, 0.04 M; concave surface and proceeds toward the 3’ D, 0.06 M; E, 0.08 M; F, 0.10 M; G, 0.20 M. The end of PMV-RNA (AbouHaidar and Banreference cell was as for G. The inset is a semilogacroft, 1978), is slow compared to initiation rithmic plot of turbidity uersus ionic strength (I) of (Erickson and Bancroft, 1978). The exNaCl. tended particles, which were produced by initiation and elongation. Initiation is tem- cooling growing rods at pH 8.0, and which perature-independent over the range O-25”, were also found at permissive temperatures whereas elongation requires elevated tem- after short times, need not necessarily be peratures. intermediates in the assembly reaction (ErInitiation is very rapid (Erickson and ickson and Bancroft, 1978). The temperaBancroft, 1978) and is probably mediated ture requirement for elongation probably by a high-affinity protein-nucleic acid rec- reflects the importance of protein-protein ognition step occurring near the 5’ end of interactions for virus formation and stabilthe RNA (AbouHaidar and Bancroft, 1978). ity. The formation of helical capsids of The average length of rods initiated in the PMV protein was found to require an elecold is about 50 nm, approximately %othe vated temperature (Erickson et al., 1976), as does the conversion of the 14 S to 25 S length of native PMV. It is unlikely that the RNA recognition site for the coat pro- aggregate at pH 8.0. It can be shown (untein amounts to 10% of the entire RNA. published) that the latter polymerization is Possibly there is a repeating, highly favorendothermic and entropy-driven, and helix able nucleotide sequence, such as the “dis- formation probably is as well if protein is location” sequence proposed by Zimmern first dialyzed overnight against 0.01 M, pH (1977) for TMV, in the first 600 nucleotides 4.0, citrate buffer at 4’. It is not unlikely on the initiating end of PMV-RNA which that the elongation phase of PMV assembly causes encapsidation even at low tempera- is similarly controlled, although a requireture, further assembly being stopped by an ment for a temperature-dependent conforunfavorable RNA sequence as postulated mational change in the nucleic acid or profor TMV (Stussi et al., 1969). Other expla- tein cannot yet be dismissed. Goodman nations for the 50-nm rodlet, such as elec- (1977) concluded from the effect of B-anilino-1-naphthalene sulfonate that hydrotrostatic inhibition of rod growth (Lauffer, 1975, pp. 92ff) or different protein polymer phobic regions of PVX protein may be inrequirements of the initiation and elonga- volved in virus assembly. tion reactions, cannot yet be ruled out. Inasmuch as PMV assembly takes place over a range of protein concentrations PMV protein exists in an apparently rapid, dynamic equilibrium among several where the 3, 14, and 25 S polymers may polymeric forms under reconstitution con- individually dominate the protein equilibrium, it is difficult to show experimentally ditions. The subassembly polymer required which polymer is essential to the elongation for initiation has not been unequivocally identified. The reconstitution results at 0.1 reaction. Whereas most of the encapsida-

PMV

SELF-ASSEMBLY

45

tion process on a weight basis involves elon- the presence of NaCl where assembly is gation, it is likely that no subassembly pol- inhibited. The reconstitution of PMV occurs speymer larger than a dimer or trimer (3 S) is required because that is all that has been cifically under reasonable conditions, exdetected at 0.1 mg/ml. Small protein aggre- cept for the low salt level required, which is troubling in view of what might be exgates may also have a role in the elongation environment. of TMV (Ohno et al., 1977), and PVX re- pected for an intracellular constitutes in the apparent absence of any However, not only PMV but all the flexuous large (14 S) polymer (Goodman et al., viruses so far examined (potato virus Y (McDonald and Bancroft, 1977); PMV 1976). However, polymers which at least initially are large can also be utilized by (Goodman, 1977); clover yellow mosaic virus (unpublished)) share the property of PMV and TMV but are probably not obliglow salt inhibition which is also found with atory. The formation of PMV nucleoproteins at certain rigid tube viruses (tobacco rattle pH 6.0 occurs at low temperature and takes virus (Morris and Semancik, 1973) and baronly seconds. The segmented or “kinked” ley stripe mosaic virus (Atabekov et al., appearance of particles made at lower pH 1970)). The reason for this behavior, which is not shared with TMV, has yet to be (up to 7.5) indicates that multiple initiations occur under these conditions. The determined. gaps between the segments may result from ACKNOWLEDGMENTS an intercalation problem, the helices being We thank Drs. M. AbouHaidar, M. Lauffer, and A. unable to anneal properly because they are out of phase. The overall relative affinity of C. H. Durham for helpful discussion on certain aspects the protein for the nucleic acid must be of this work, and Messrs. R. Johnston and D. Muirhead for technical assistance. This work was supported considerably higher at the lower pH, save in part by the National Research Council of Canada. for the recognition site, and the specificity of the reconstitution process is lost (ErickREFERENCES son et al., 1978). ABOUHAIDAR, M., and BANCROFT, J. B. (1978). The The presence of neither the 25 S, 14 S initiation of papaya mosaic virus assembly. Virology nor any minor protein species ensures that 90,54-59 reconstitution will occur at 25” if NaCl is ATABEKOV, J. G., NOVIKOV, V. K., VISCHNICHENKO, V. K., and KAFTANOVA, A. S. (1970). Some properpresent at a concentration of 0.025 M or ties of hybrid viruses reassembled in vitro. Virology greater at pH 8.0. The effect may be related 41,519-532. to the behavior of the protein in NaCl. BUTLER, P. J. G., and DURHAM, A. C. H. (1977). Protein helices, formed at pH 4.0 in the Tobacco mosaic virus protein aggregation and the absence of NaCl are very long, whereas virus assembly. A&. Protein Chem. 31, 187-251. they are relativeiy short at the same pH EDSALL, J. T., and WYMAN, J. (1958). Biophysical but in 0.2 M NaCl (Erickson et al., 1976). chemistry. In “Thermodynamics, Electrostatics, Similarly, the formation of the 25 S aggreand the Biological Significance of the Properties of gate at pH 8.0 is salt-sensitive, suggesting Matter,” Vol. I. Academic Press, New York. that NaCl destabilizes protein interactions ERICKSON, J. W., BANCROFT, J. B., and HORNE, R. W. and perhaps similar interactions in the vi(1976). The assembly of papaya mosaic virus protein. Virology 72,514-517. rus. It is unlikely that the salt-induced inhibition is the result of a chaotropic effect ERICKSON, J. W., and BANCROFT, J. B. (1978). The kinetics of papaya mosaic virus assembly. Virology as suggested for PVX (Goodman, 1977), 90,47-53. inasmuch as at the low concentrations emERICKSON, J. W., ABOUHAIDAR, M., and BANCROFT, ployed, the effect of NaCl on water strucJ. B. (1978). The specificity of papaya mosaic virus ture is minimal. Indeed, it is conceivable assembly. Virology 90, M-66. that the effect of salt (and temperature) FRAENKEL-CONRAT, H. (1957). Degradation of tocan be ascribed to RNA conformation, conbacco mosaic virus with acetic acid. Virology 4.1-4. sidering there would be less RNA second- GOODMAN, R. M. (1977). Reconstitution of potato ary structure under reconstitution condivirus X in vitro. III. Evidence for a role for hydrotions (25’, no NaCl) than in the cold or in phobic interactions. Virology 76, 72-78.

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AND

GOODMAN, R. M., MCDONALD, J. G., HORNE, R. W., and BANCROFT, J. B. (1976). Assembly of flexuous plant viruses and their proteins Philos. Trans. R. Sot. Land. [Biol. Sci.] 276, 173-179. HARRISON, B. D., FINCH, J. T., GIBBS, A. J., HOLLINGS, M., SHEPHERD, R. J., VALENTO, V., and WETTER, C. (1971). Sixteen groups of plant viruses. Virology 45,356-363. HIEBERT, E. (1970). Some properties of papaya mosaic virus and its isolated constituents. Phytopathology 60, 1295. LAUFFER, M. A. (1975). In “Entropy-Driven Processes in Biology: Polymerization of Tobacco Mosaic Virus Protein and Similar Reactions.” Molecular Biology, Biochemistry and Biophysics series, (A. KLEINZELLER, G. F. SPRINGER, and H. G. WITTMAN, eds.), Vol. 20, 264 pp. Springer-Verlag, New York. MCDONALD, J. G., and BANCROFT, J. B. (1977). Assembly studies on potato virus Y and its coat protein. J. Gen. Virol. 35,251-263. MORRIS, T. J., and SEMANCIK, J. S. (1973). In vitro protein polymerization and nucleoprotein reconstitution of tobacco rattle virus. Virology 53,215-224.

BANCROFT OHNO, T., INOUE, H., and OKADA, Y. (1972). Assembly of rod-shaped virus in vitro: reconstitution with cucumber green mottle mosaic virus protein and tobacco mosaic virus RNA. Proc. Nat. Acad. Sci. USA 69,3680-3683. OHNO, T., TAKAHASHI, M., and OKADA, Y. (1977). Assembly of tobacco mosaic virus in uitro: elongation of partially reconstituted RNA. Proc. Nat. Acad. Sci. USA 74, 552-555. PURCIFULL, D. E., and HIEBERT, E. (1971). Papaya mosaic virus. C.M.I./A.A.B. Descriptions of Plant Viruses, 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. STUSSI, Partial rology

C., LEBEURIER, reconstitution 38, 16-25.

ZIMMERN, D. (1977). origin for assembly Cell 11, 463-482.

G., and of tobacco

HIRTH, mosaic

L. (1969). virus. Vi-

The nucleotide sequence on tobacco mosaic virus

at the RNA.