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Diffraction studies of papaya mosaic virus
VIROLOGY
98,
108-115
(Wi’g)
Diffraction
Studies
of Papaya
Mosaic
Virus
P. TOLLIN,’ J. B. BANCROFT,* J. F. RICHARDSON,3 N. C. PAYNE,3 AND T. J. BEVERIDGE4 Departments of Plant Sciences, Chemistry, and Microbiology, The University of Western Ontario, London, Ontario N6A 5B7, Canada Accepted June 7, 1979 X-ray and optical diffraction studies of the flexuous papaya mosaic virus are described. The virus is constructed so that there are 35 coat protein subunits in 4 turns of the helix. The virus contains about 1410 protein subunits and 6300 nucleotides and has a molecular weight of about 33 x 106. The structure of tubes assembled in vitro from coat protein both in the presence and absence of nucleic acid resembles that of the native virus.
X-ray diffraction. Specimens for X-ray diffraction experiments were prepared by drying a solution of virus, at an original concentration of about 30 mg/ml in 0.01 M borate buffer, pH 9.0, in the manner described by Tollin et al. (1968) adapted from Bernal and Fankuchen (1941). Some of the X-ray diffraction photographs were recorded in Dundee, Scotland using a camera with two 100~pm pinholes and a 6-cm specimen to film distance. Hydrogen gas was allowed to flow continuously through this camera, the humidity being controlled by passage of the gas through suitable salt solutions. Other photographs were taken in London! Ontario, on a Norelco microcamera fitted with a 50-pm collimator. Cu radiation was used from a Rigaku Rotaflex high brilliance rotating anode X-ray generator employing a 1 X O.l-mm* focal spot. Preliminary studies and alignments were carried out with a 1.5cm specimen to film distance. Regions of the gel showing a high degree of order were then photographed using a locally constructed camera extension, corresponding to a 6-cm specimen to film distance, and an atmosphere of helium with controlled humidity. Optical di,ffraction. The optical diffractometer is similar to that developed by Taylor and Lipson (1964) with lenses mounted on 1-in.-thick cast steel plate. Straight regions of virus particles were
INTRODUCTION
Papaya mosaic virus (PMV) is a flexuous virus in the potato virus X family which has been used in self-assembly studies (Erickson and Bancroft, 1978a,b; AbouHaidar and Bancroft, 1978; Erickson et al., 1978). We wished to know the structure of PMV and now describe its gross architecture and provide a molecular weight. We also present evidence that various products assembled in vitro are similar in structure to the virus from which their constituents were obtained. MATERIALS
AND METHODS
Specimen preparation. PMV was grown and purified as described (Erickson and Bancroft, 1978a). Reconstitution of virus and protein rods was done as before (Erickson and Bancroft, 1978a, Erickson et al., 1976). * Visiting’ scientist at University of Western Ontario (U. W.O.). Permanent address: Carnegie Laboratory of Physics, University of Dundee, Dundee, Scotland. 2 Department of Plant Sciences, U.W.O., to which reprint request should be sent. B Department of Chemistry, U. W.O. 4 Department of Microbiology, U.W.O. Present address: Department of Microbiology, University of Guelph, Guelph, Ontario, Canada. 0042~6822/79/130108-08$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
108
PMV DIFFRACTION
masked off on original micrographs at a magnification of ~65,000 calibrated with catalase and used as diffraction objects (Klug and Berger, 1964). The diffracting lens system was arranged to give a diffraction pattern about 5 cm across. Routine screening of diffraction patterns was done on a television monitor. When suitable patterns were obtained, masks for reconstruction could be easily made from patterns recorded on Polaroid film because of the scale. Electron micrographs of virus particles stained with uranyl acetate were usually obtained on a Philips EM200. RESULTS
X-Ray
Diffraction
X-ray diffraction patterns obtained with the Norelco and double pinhole cameras (Figs. 1A and B) of the same dry specimen of PMV show the reflections expected from a helical particle with a pitch of 3.36 f 0.03 nm. However, there are layer lines, which are particularly clear in the picture from the Norelco camera, which do not correspond to spacings of 0.297 nm-‘, indicating that the particle does not repeat in one turn. The values of 1 which give the
109
best agreement and the smallest standard deviation for the true repeat are listed in Table 1. These measurements suggest that the true repeat centers around four turns of the helix (Table 1). The general structure of the virus remains the same in the presence of phosphorus pentoxide but the pitch of the helix is reduced to 3.28 ? 0.05 nm (Table 2). The equatorial X-ray diffraction pattern appears to be that from a hexagonal closepacked array (see Fig. 2 for electron micrograph of a cross section of a dried gel) having an interparticle spacing of approximately 11 nm. So, considering the diameter of single particles to be described, there is significant interpenetration of the particles similar to that observed in narcissus mosaic virus (Tollin et al., 1968). The intensities are consistent with their being due to the crystalline sampling of aJ, Bessel function caused by a concentration of scattering matter at a radius of close to 3.5 nm. This is consistent with results obtained from NMV (Tollin et al., 1968; Wilson et al., 1973), and a full discussion of the interpretation of such equatorial patterns will appear in a subsequent publication (Tollin et al., unpublished) concerned with a re-
FIG. 1. X-ray diffraction patterns of an oriented gel of PMV taken with the Norelco (A) and double pinhole (B) cameras.
110
TOLLIN TABLE
MEASUREMENTS PMV Distance on film (mm) 5.55 11.15 15.35 16.85 20.75 22.35 26.80 29.00 32.35 34.35 38.45 40.70 44.80
1
OF X-RAY REFLECTIONS AT ROOM HUMIDITY d
FROM
Spacing
Layer line
(nm)
1
True repeat (nm)
m
3.34 1.67 1.21 1.11 0.90 0.84 0.71
W
0.66
W
0.59 0.56 0.50 0.48 0.44
4 8 11 12 15 16 19 20 23 24 27 28 31
13.4 13.4 13.3 13.3 13.5 13.4 13.5 13.2 13.6 13.4 13.5 13.4 13.6
lntensity” S
s S S S S
W
m W m
Mean
13.4 2 0.12
” s, m, and w designate strong, intensity reflections, respectively.
medium,
and weak
lated virus, clover yellow mosaic virus. This is likely to indicate the position of the RNA of the virus. We were unable to make satisfactory oriented specimens of protein polymerized in vitro. TABLE
2
MEASUREMENTS OF X-RAY REFLECTIONS FROM PMV WITH PHOSPHORUS PENTOXIDE IN THE CAMERA Distance on film
d Spacing
Layer line
(mm)
(nm)
1
(nm)
5.6 11.1 15.2 16.7 20.5 21.7 26.0 31.6 38.2 43.4
3.14 1.58 1.16 1.05 0.87 0.81 0.69 0.57 0.48 0.43
4 8 11 12 15 16 19 23 27 31
12.7 12.8 12.9 12.8 13.2 13.3 13.3 13.3 13.1 13.4
Mean
ET AL.
True repeat
13.1 2 0.2
FIG. 2. Electron micrograph of an oriented gel. The bar is 600 nm.
Optical
a cross
section
of
Diffraction
Optical diffraction patterns from about 60 individual particles were examined. Some of these only showed diffraction on a layer line corresponding to a helix pitch with a spacing of 3.4 nm. About 20 patterns also showed maxima on a layer line having a spacing about one-quarter that of the pitch. This suggests, as do the X-ray data, that the primary helix repeats in about four turns or 13.6 nm as measured from the micrographs. The determination of the number of subunits per turn of the helix requires that many diffraction patterns with spots at the first and, with PMV, the fourth layer lines be examined. Some patterns showed maxima equally spaced about the meridian (Fig. 3A) suggesting that they arose from two-sided undistorted images. Others, such as in Fig. 3B, were not equally spaced about the meridian perhaps reflecting a particle in which one side is undistorted while the other has suffered some flattening, although not to such a degree as that in polysheaths (Moody, 1967). Alternatively, such patterns could mean that the negative stain penetrated to different depths on the two sides of the particles. Other patterns corresponded to a one-sided image only and Fig. 3C shows one on which the maximum on the appropriate fifth layer line also occurs. While the optically fil-
PMV DIFFRACTION
111
information on the fifth layer line to appear, seems to show some evidence for the shape of the subunit as visualized in the optical reconstruction in which the first, fourth, and fifth layer line spots were used. The information from analyses of diffraction patterns from a number of virus particles in Table 3 suggest, as will be discussed, there there are probably 35 subunits per 4 turns of the helix. This information coupled with the shape from Fig. 3C gives rise to the model in Fig. 4. The optical diffraction patterns of particles assembled in vitro have also been examined. Protein assembled into tubes of various lengths at pH 4, 5, and 6 gave patTABLE
3 PMV
MEASCREMENTSFROMOPTICALTRANSFORMSOF AND ITSASSEMBLEDPROTEIN
PMV Pattern type”
Assembled protein
R
R
(nm ’ X 10:‘) P&r”
(nm ’ X l(Y) ZnRv
FIG. 3. Optical diffraction patterns of PMV. (A) no distortion showing opposite spots on the first and fourth layer lines; (B) some distortion; (C) some distortion and showing the fifth layer line spot. Note that the first, fourth, and fifth layer line maxima are in the same quadrant indicating q - 1. The particle to the left is an unfiltered image and that on the right, a filtered one of the same particle.
Even Smgle Even Uneven Smgle Single Single Single Uneven Even Single Single Smgle Even Even Even Smgle Smgle Smgle Single Uneven
tered one-sided image of a particle such as the one in Fig. 3A would contain only information about the position of the subunits, the distortion to the particle in Fig. 3C which has allowed the higher resohtion _.
” Even means that maxima from the upper and lovver surface of the sample were evenly spaced; unev.en means that they were not; single designates maxima from one surface. ’ R is the position of the maximum on the layer lme and T is the radius of the particle.
23.4 24.0
2‘4.1 23.8 22.4
10.7 11.0 ll.u lU.9 10.7
25.0
11.4
25.0 25.0 25.4
11.4 11.4 I l.‘I 11.6
24.7
11.3
26.ti
12.2
25.6
11.7
25.0 23.5 22.6 22.3 22.9 20.8 28.9 25.7 25.7 20.6 25.9 23.8
23.7
10.7 10.3
10.2 10.5 9.5 13.2 11.6 Il.8 9.4 11.9 10.9 10.8
112
TOLLIN
ET AL.
FIG. 4. Computer model of PMV combining all features derived from diffraction patterns and a diffraction pattern of the computer model.
terns similar to those obtained from natural virus. Symmetrical patterns (Fig. 5A) as well as those showing distortion (Fig. 5B) were observed. Analysis again indicated a repeat in 4 turns containing 35 subunits (Table 3). Figure 5C shows a slightly distorted two-sided image of a reconstituted virus particle like that of the natural virus in Fig. 3B. Thus, there is no reason to believe that particles assembled outside the host in the absence or presence of homologous RNA have a gross structure different from that of the virus. Particle Diameter
Since the particle diameter is critical in assigning the correct Bessel function from the optical diffraction patterns, measurements of the diameter of about 140 single negatively stained particles of PMV were made. These showed considerable variation but the average diameter was 14.2 nm with a standard deviation of 0.9 nm. The corresponding figure for the protein rods from 30 observations was 13.6 nm again with a standard deviation of 0.9 nm. DISCUSSION
The scattering from a helical particle is concentrated on discrete layer lines. The theory of helical diffraction predicts that if there is scattering matter at one particular radius then the scattering along the layer line will be continuous and of the form of a superposition of Bessel function J,(2z-Rr), where R is the position
FIG. 5. Diffraction patterns of PMV protein assembled into tubes in vitro. (A) No distortion; (B) distortion; (C) assembled with PMV-RNA, shoq ring some distortion.
PMV DIFFRACTION
FIG. 6. An n, 1 plot showing permitted functions and their positions for PMV.
Bessel
on the layer line and r is the radial position of the scattering matter. Only certain Bessel functions may occur on a particular layer line, and are given by the selection rule 1 = tn + urn where t is the number of turns in the true repeat, u is the number of subunits per true repeat, 1 is the layer line number, n is the order of the Bessel function, and m is an integer. An (n,Z) plot shows the allowed Bessel functions on each layer line and their relative positions. Figure 6 shows an (n, 1) plot for a PMV particle having 35 subunits in 4 turns. The comments to be made would, however, apply in other cases. The concept of a true repeat is a useful one although it may be artificial. In principle, and at high enough resolution, there is no reason why a helical particle should repeat at all. For example, if the ratio of the distance from the equator of the closer layer line in the PMV optical diffraction pattern to that further from the equator is exactly 4, then the particle repeats in 4 turns and the selection rule for the Bessel function on the inner layer line is 1 = 4n + 35m, and there are 8.75 subunits/turn. However, for an optical diffraction pattern for which this ratio is 4.4 the true repeat would be 22 turns. The inner layer line would now have I = 5, the selection rule would be 5 = 22n + 193m and there would be 8.76 subunits per turn. Thus the parameter which more clearly describes the architecture of the virus may be
113
the number of subunits per turn. The substantial variation in this ratio for the optical diffraction pattern thus corresponds to very slight local twisting and untwisting of the helix, likely to occur in tivo anyway, and does not imply any serious distortion of this type. The number of subunits in the true repeat of a virus which repeats in four turns is either 4q + 1 or 4q - 1 where q is an integer. Considering the (n, 1) plot in Fig. 6 for the (4q - 1) case the intensities close to the meridian on higher layer lines occur on layer lines which are just below the layer lines corresponding to multiples of the reciprocal of the pitch. The better fit to the spacings obtained from the X-ray diffraction pattern is obtained by assigning the maxima near the meridian at higher angles to just such layer lines. Were the virus to repeat in four turns with 4q -t- 1 subunits then the higher layer lines would occur closer to the meridian just above those corresponding to the pitch. The fit to the X-ray diffraction pattern on this assumption is less satisfactory. Optical diffraction patterns obtained from one-sided particles with 4q - 1 subunits in the true repeat would have a maximum on the first layer line and a maximum on the fourth layer line on the same side of the meridian. The Bessel functions marked on the (n,E) plot of Fig. 6 show this. In the (4q + 1) case these maxima would be on opposite sides of the meridian. All the single-sided diffraction patterns (including that in Fig. 3C) show the former case. Further inspection of the (n, 1) plot shows that if a maximum occurs on the fifth layer line it should be on the same side of the meridian as the previous two if the number is (4q - 1) and Fig. 3C shows this. This arises because the maxima due to one side of the particle must all lie on the line from one higher origin, in this case the upper origin in Fig. 6, corresponding to m = +1 in the selection rule. Single-sided protein rods give the same result. The determination of the integer q is a most difficult task in the elucidation of the viral architecture. Were it possible to determine the first truly meridional reflection
114
TOLLIN
in the X-ray pattern, q would be defined. However, the degree of disorientation in the specimens makes this impossible. Alternatively, if the value of n for the Bessel function on the first layer line of spacing 0.074 -l in the optical diffraction pattern can Er found, q can be deduced. From the values of R the reciprocal space distance from the meridian given in Table 3, values of Z?rRr can be computed, where r is the presumed radius of the scattering matter. The assignment of a value of r is the problem which leads to some difficulty in determining q precisely. We have chosen to assume that there is stain at the surface of the particle. Stain further in would then merely extend the maximum further from the equator but would not affect its nearest approach to the meridian. Biological specimens are subject to distortion by negative stain. By concentrating on the symmetrical particles we assume that we are considering undistorted helical particles. Five of these particles give a value of 27rRr of 11.2 and one a value of 13.2. The two uneven two-sided particles gave a value of 10.8 for the half-helical side of the pattern. Such values for 27rRr are entirely consistent with the J, Bessel function’s first maximum at 2nRr of 10.7 for a circular, or 10.87 for a half-circular helix. (The corresponding values for Js and Jlo are 9.65, 9.78, 11.77, 11.91). Treating the various distortions in the other particles as random variations from best value, the mean of all the values obtained is 11.0. Corresponding values for the PMV protein are 11.0 for undistorted particles and 10.5 for semi-circular helices. If the Bessel function on the first layer line is Jg then q is 9 and there are about 35 subunits in 4 turns or the number of subunits per turn is 8.75. While the evidence for Jg is not conclusive because of the assumptions made, other evidence helps to confirm it. PMV has a length of 540 nm and since there are about 35 subunits in 4 turns the true repeat is 13.4 nm and thus there are about 1410 subunits in the structure. The molecular weight of the RNA in the virus is 2.2 x lo6 (Purcifull and Hiebert, 1971 and verified by us) and the molecular
ET AL.
weight of an average nucleotide, estimated from the known base ratio for this virus (AbouHaidar and Bancroft, 1978), is 322. There are therefore about 6800 nucleotides or 5 nucleotides per subunit. The fact that the number of nucleotides per subunit is not the same or a multiple of the degree of overlap of the protein subunits in the helix need not present a problem. The molecular weight of the protein subunit calculated from amino acid analyses is close to 22,000 (Rees, personal communication) and hence the total molecular weight of the virus particle is about 33 x lo6 and the percentage RNA 6.7% in good agreement with the measured value of 7% (Purcifull and Heibert, 1971). The X-ray diffraction results suggest a position for the nucleic acid at a radius of about 3.5 nm. Assuming that it is the most electron dense part of the nucleic acid, that is the phosphate backbone, these positions would give a phosphorus to phosphorus distance along the chain of 0.5 nm. This shows that a RNA of such a molecular weight can be accommodated in the structure. Thus the X-ray and optical diffraction results for PMV are consistent with a particle having 35 subunits in four turns of its helix and the computer drawn illustration (ORTEP (Johnson, 1965)) presented in Fig. 4 is an accurate representation of the gross structure of the virus. Descriptions of other members of the large and important group of viruses to which PMV belongs are underway and are so far consistent with a value of J9 but with different repeats of the primary helix. ACKNOWLEDGMENTS This work was supported in part by grants from the Academic Development Fund of the University of Western Ontario and the National Research Council of Canada. The authors would like to thank Professors W. P. Alford and P. W. Whippey of the Department of Physics of the University of Western Ontario, who were largely responsible for the design and construction of the optical diffractometer. REFERENCES EA., and BANCROFT, J. B. (1978). The initiation of papaya mosaic virus assembly. Vivology 90, 54-5s.
AB~UHAIDAR
PMV DIFFRACTION BERNAL, J. D., and FANKUCHEN, I. (1941). X-ray and crystallographic studies of plant virus preparations. J. Gen. Physiol. 25, 111-165. 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. (197813). 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. JOHNSON, C. K. (1965). Report ORNL-3794. Oak Ridge National Laboratory, Oak Ridge, Tenn. KLUG, A., and BERGER, 3. E. (1964). An optical method for the analysis of periodicities in electron-
115
micrographs and some observations on the mechanism of negative staining. J. Mol. Biol. 10, 565-569. MOODY, M. F. (1967). Structure of the sheath of bacteriophage T4. 1. Structure of the contracted sheath and polysheath. J. Mol. Biol. 25, 167-200. PURCIFULL, D. E., and HIEBERT, E. (1971). “Papaya Mosaic Virus.” C.M.1.IA.A.B. Descriptions of Plant Viruses, No. 56. TAYLOR, C. A., and LIPSON, H. (1964)” Optical Transforms: Their Preparation and Application to X-Ray Diffraction Problems: Bell, London. TOLLIN, P., WILSON, H. R., and YOUNG, D. W. (1968). X-ray diffraction evidence of the helical structure of narcissus mosaic virus. J. Mol. Biol. 34, 189-192. WILSON, H. R., TOLLIN, P., and RAHMAN, A. (1973). The structure of narcissus mosaic virus. J. Gen. Viral. 18, 181.
98,
108-115
(Wi’g)
Diffraction
Studies
of Papaya
Mosaic
Virus
P. TOLLIN,’ J. B. BANCROFT,* J. F. RICHARDSON,3 N. C. PAYNE,3 AND T. J. BEVERIDGE4 Departments of Plant Sciences, Chemistry, and Microbiology, The University of Western Ontario, London, Ontario N6A 5B7, Canada Accepted June 7, 1979 X-ray and optical diffraction studies of the flexuous papaya mosaic virus are described. The virus is constructed so that there are 35 coat protein subunits in 4 turns of the helix. The virus contains about 1410 protein subunits and 6300 nucleotides and has a molecular weight of about 33 x 106. The structure of tubes assembled in vitro from coat protein both in the presence and absence of nucleic acid resembles that of the native virus.
X-ray diffraction. Specimens for X-ray diffraction experiments were prepared by drying a solution of virus, at an original concentration of about 30 mg/ml in 0.01 M borate buffer, pH 9.0, in the manner described by Tollin et al. (1968) adapted from Bernal and Fankuchen (1941). Some of the X-ray diffraction photographs were recorded in Dundee, Scotland using a camera with two 100~pm pinholes and a 6-cm specimen to film distance. Hydrogen gas was allowed to flow continuously through this camera, the humidity being controlled by passage of the gas through suitable salt solutions. Other photographs were taken in London! Ontario, on a Norelco microcamera fitted with a 50-pm collimator. Cu radiation was used from a Rigaku Rotaflex high brilliance rotating anode X-ray generator employing a 1 X O.l-mm* focal spot. Preliminary studies and alignments were carried out with a 1.5cm specimen to film distance. Regions of the gel showing a high degree of order were then photographed using a locally constructed camera extension, corresponding to a 6-cm specimen to film distance, and an atmosphere of helium with controlled humidity. Optical di,ffraction. The optical diffractometer is similar to that developed by Taylor and Lipson (1964) with lenses mounted on 1-in.-thick cast steel plate. Straight regions of virus particles were
INTRODUCTION
Papaya mosaic virus (PMV) is a flexuous virus in the potato virus X family which has been used in self-assembly studies (Erickson and Bancroft, 1978a,b; AbouHaidar and Bancroft, 1978; Erickson et al., 1978). We wished to know the structure of PMV and now describe its gross architecture and provide a molecular weight. We also present evidence that various products assembled in vitro are similar in structure to the virus from which their constituents were obtained. MATERIALS
AND METHODS
Specimen preparation. PMV was grown and purified as described (Erickson and Bancroft, 1978a). Reconstitution of virus and protein rods was done as before (Erickson and Bancroft, 1978a, Erickson et al., 1976). * Visiting’ scientist at University of Western Ontario (U. W.O.). Permanent address: Carnegie Laboratory of Physics, University of Dundee, Dundee, Scotland. 2 Department of Plant Sciences, U.W.O., to which reprint request should be sent. B Department of Chemistry, U. W.O. 4 Department of Microbiology, U.W.O. Present address: Department of Microbiology, University of Guelph, Guelph, Ontario, Canada. 0042~6822/79/130108-08$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
108
PMV DIFFRACTION
masked off on original micrographs at a magnification of ~65,000 calibrated with catalase and used as diffraction objects (Klug and Berger, 1964). The diffracting lens system was arranged to give a diffraction pattern about 5 cm across. Routine screening of diffraction patterns was done on a television monitor. When suitable patterns were obtained, masks for reconstruction could be easily made from patterns recorded on Polaroid film because of the scale. Electron micrographs of virus particles stained with uranyl acetate were usually obtained on a Philips EM200. RESULTS
X-Ray
Diffraction
X-ray diffraction patterns obtained with the Norelco and double pinhole cameras (Figs. 1A and B) of the same dry specimen of PMV show the reflections expected from a helical particle with a pitch of 3.36 f 0.03 nm. However, there are layer lines, which are particularly clear in the picture from the Norelco camera, which do not correspond to spacings of 0.297 nm-‘, indicating that the particle does not repeat in one turn. The values of 1 which give the
109
best agreement and the smallest standard deviation for the true repeat are listed in Table 1. These measurements suggest that the true repeat centers around four turns of the helix (Table 1). The general structure of the virus remains the same in the presence of phosphorus pentoxide but the pitch of the helix is reduced to 3.28 ? 0.05 nm (Table 2). The equatorial X-ray diffraction pattern appears to be that from a hexagonal closepacked array (see Fig. 2 for electron micrograph of a cross section of a dried gel) having an interparticle spacing of approximately 11 nm. So, considering the diameter of single particles to be described, there is significant interpenetration of the particles similar to that observed in narcissus mosaic virus (Tollin et al., 1968). The intensities are consistent with their being due to the crystalline sampling of aJ, Bessel function caused by a concentration of scattering matter at a radius of close to 3.5 nm. This is consistent with results obtained from NMV (Tollin et al., 1968; Wilson et al., 1973), and a full discussion of the interpretation of such equatorial patterns will appear in a subsequent publication (Tollin et al., unpublished) concerned with a re-
FIG. 1. X-ray diffraction patterns of an oriented gel of PMV taken with the Norelco (A) and double pinhole (B) cameras.
110
TOLLIN TABLE
MEASUREMENTS PMV Distance on film (mm) 5.55 11.15 15.35 16.85 20.75 22.35 26.80 29.00 32.35 34.35 38.45 40.70 44.80
1
OF X-RAY REFLECTIONS AT ROOM HUMIDITY d
FROM
Spacing
Layer line
(nm)
1
True repeat (nm)
m
3.34 1.67 1.21 1.11 0.90 0.84 0.71
W
0.66
W
0.59 0.56 0.50 0.48 0.44
4 8 11 12 15 16 19 20 23 24 27 28 31
13.4 13.4 13.3 13.3 13.5 13.4 13.5 13.2 13.6 13.4 13.5 13.4 13.6
lntensity” S
s S S S S
W
m W m
Mean
13.4 2 0.12
” s, m, and w designate strong, intensity reflections, respectively.
medium,
and weak
lated virus, clover yellow mosaic virus. This is likely to indicate the position of the RNA of the virus. We were unable to make satisfactory oriented specimens of protein polymerized in vitro. TABLE
2
MEASUREMENTS OF X-RAY REFLECTIONS FROM PMV WITH PHOSPHORUS PENTOXIDE IN THE CAMERA Distance on film
d Spacing
Layer line
(mm)
(nm)
1
(nm)
5.6 11.1 15.2 16.7 20.5 21.7 26.0 31.6 38.2 43.4
3.14 1.58 1.16 1.05 0.87 0.81 0.69 0.57 0.48 0.43
4 8 11 12 15 16 19 23 27 31
12.7 12.8 12.9 12.8 13.2 13.3 13.3 13.3 13.1 13.4
Mean
ET AL.
True repeat
13.1 2 0.2
FIG. 2. Electron micrograph of an oriented gel. The bar is 600 nm.
Optical
a cross
section
of
Diffraction
Optical diffraction patterns from about 60 individual particles were examined. Some of these only showed diffraction on a layer line corresponding to a helix pitch with a spacing of 3.4 nm. About 20 patterns also showed maxima on a layer line having a spacing about one-quarter that of the pitch. This suggests, as do the X-ray data, that the primary helix repeats in about four turns or 13.6 nm as measured from the micrographs. The determination of the number of subunits per turn of the helix requires that many diffraction patterns with spots at the first and, with PMV, the fourth layer lines be examined. Some patterns showed maxima equally spaced about the meridian (Fig. 3A) suggesting that they arose from two-sided undistorted images. Others, such as in Fig. 3B, were not equally spaced about the meridian perhaps reflecting a particle in which one side is undistorted while the other has suffered some flattening, although not to such a degree as that in polysheaths (Moody, 1967). Alternatively, such patterns could mean that the negative stain penetrated to different depths on the two sides of the particles. Other patterns corresponded to a one-sided image only and Fig. 3C shows one on which the maximum on the appropriate fifth layer line also occurs. While the optically fil-
PMV DIFFRACTION
111
information on the fifth layer line to appear, seems to show some evidence for the shape of the subunit as visualized in the optical reconstruction in which the first, fourth, and fifth layer line spots were used. The information from analyses of diffraction patterns from a number of virus particles in Table 3 suggest, as will be discussed, there there are probably 35 subunits per 4 turns of the helix. This information coupled with the shape from Fig. 3C gives rise to the model in Fig. 4. The optical diffraction patterns of particles assembled in vitro have also been examined. Protein assembled into tubes of various lengths at pH 4, 5, and 6 gave patTABLE
3 PMV
MEASCREMENTSFROMOPTICALTRANSFORMSOF AND ITSASSEMBLEDPROTEIN
PMV Pattern type”
Assembled protein
R
R
(nm ’ X 10:‘) P&r”
(nm ’ X l(Y) ZnRv
FIG. 3. Optical diffraction patterns of PMV. (A) no distortion showing opposite spots on the first and fourth layer lines; (B) some distortion; (C) some distortion and showing the fifth layer line spot. Note that the first, fourth, and fifth layer line maxima are in the same quadrant indicating q - 1. The particle to the left is an unfiltered image and that on the right, a filtered one of the same particle.
Even Smgle Even Uneven Smgle Single Single Single Uneven Even Single Single Smgle Even Even Even Smgle Smgle Smgle Single Uneven
tered one-sided image of a particle such as the one in Fig. 3A would contain only information about the position of the subunits, the distortion to the particle in Fig. 3C which has allowed the higher resohtion _.
” Even means that maxima from the upper and lovver surface of the sample were evenly spaced; unev.en means that they were not; single designates maxima from one surface. ’ R is the position of the maximum on the layer lme and T is the radius of the particle.
23.4 24.0
2‘4.1 23.8 22.4
10.7 11.0 ll.u lU.9 10.7
25.0
11.4
25.0 25.0 25.4
11.4 11.4 I l.‘I 11.6
24.7
11.3
26.ti
12.2
25.6
11.7
25.0 23.5 22.6 22.3 22.9 20.8 28.9 25.7 25.7 20.6 25.9 23.8
23.7
10.7 10.3
10.2 10.5 9.5 13.2 11.6 Il.8 9.4 11.9 10.9 10.8
112
TOLLIN
ET AL.
FIG. 4. Computer model of PMV combining all features derived from diffraction patterns and a diffraction pattern of the computer model.
terns similar to those obtained from natural virus. Symmetrical patterns (Fig. 5A) as well as those showing distortion (Fig. 5B) were observed. Analysis again indicated a repeat in 4 turns containing 35 subunits (Table 3). Figure 5C shows a slightly distorted two-sided image of a reconstituted virus particle like that of the natural virus in Fig. 3B. Thus, there is no reason to believe that particles assembled outside the host in the absence or presence of homologous RNA have a gross structure different from that of the virus. Particle Diameter
Since the particle diameter is critical in assigning the correct Bessel function from the optical diffraction patterns, measurements of the diameter of about 140 single negatively stained particles of PMV were made. These showed considerable variation but the average diameter was 14.2 nm with a standard deviation of 0.9 nm. The corresponding figure for the protein rods from 30 observations was 13.6 nm again with a standard deviation of 0.9 nm. DISCUSSION
The scattering from a helical particle is concentrated on discrete layer lines. The theory of helical diffraction predicts that if there is scattering matter at one particular radius then the scattering along the layer line will be continuous and of the form of a superposition of Bessel function J,(2z-Rr), where R is the position
FIG. 5. Diffraction patterns of PMV protein assembled into tubes in vitro. (A) No distortion; (B) distortion; (C) assembled with PMV-RNA, shoq ring some distortion.
PMV DIFFRACTION
FIG. 6. An n, 1 plot showing permitted functions and their positions for PMV.
Bessel
on the layer line and r is the radial position of the scattering matter. Only certain Bessel functions may occur on a particular layer line, and are given by the selection rule 1 = tn + urn where t is the number of turns in the true repeat, u is the number of subunits per true repeat, 1 is the layer line number, n is the order of the Bessel function, and m is an integer. An (n,Z) plot shows the allowed Bessel functions on each layer line and their relative positions. Figure 6 shows an (n, 1) plot for a PMV particle having 35 subunits in 4 turns. The comments to be made would, however, apply in other cases. The concept of a true repeat is a useful one although it may be artificial. In principle, and at high enough resolution, there is no reason why a helical particle should repeat at all. For example, if the ratio of the distance from the equator of the closer layer line in the PMV optical diffraction pattern to that further from the equator is exactly 4, then the particle repeats in 4 turns and the selection rule for the Bessel function on the inner layer line is 1 = 4n + 35m, and there are 8.75 subunits/turn. However, for an optical diffraction pattern for which this ratio is 4.4 the true repeat would be 22 turns. The inner layer line would now have I = 5, the selection rule would be 5 = 22n + 193m and there would be 8.76 subunits per turn. Thus the parameter which more clearly describes the architecture of the virus may be
113
the number of subunits per turn. The substantial variation in this ratio for the optical diffraction pattern thus corresponds to very slight local twisting and untwisting of the helix, likely to occur in tivo anyway, and does not imply any serious distortion of this type. The number of subunits in the true repeat of a virus which repeats in four turns is either 4q + 1 or 4q - 1 where q is an integer. Considering the (n, 1) plot in Fig. 6 for the (4q - 1) case the intensities close to the meridian on higher layer lines occur on layer lines which are just below the layer lines corresponding to multiples of the reciprocal of the pitch. The better fit to the spacings obtained from the X-ray diffraction pattern is obtained by assigning the maxima near the meridian at higher angles to just such layer lines. Were the virus to repeat in four turns with 4q -t- 1 subunits then the higher layer lines would occur closer to the meridian just above those corresponding to the pitch. The fit to the X-ray diffraction pattern on this assumption is less satisfactory. Optical diffraction patterns obtained from one-sided particles with 4q - 1 subunits in the true repeat would have a maximum on the first layer line and a maximum on the fourth layer line on the same side of the meridian. The Bessel functions marked on the (n,E) plot of Fig. 6 show this. In the (4q + 1) case these maxima would be on opposite sides of the meridian. All the single-sided diffraction patterns (including that in Fig. 3C) show the former case. Further inspection of the (n, 1) plot shows that if a maximum occurs on the fifth layer line it should be on the same side of the meridian as the previous two if the number is (4q - 1) and Fig. 3C shows this. This arises because the maxima due to one side of the particle must all lie on the line from one higher origin, in this case the upper origin in Fig. 6, corresponding to m = +1 in the selection rule. Single-sided protein rods give the same result. The determination of the integer q is a most difficult task in the elucidation of the viral architecture. Were it possible to determine the first truly meridional reflection
114
TOLLIN
in the X-ray pattern, q would be defined. However, the degree of disorientation in the specimens makes this impossible. Alternatively, if the value of n for the Bessel function on the first layer line of spacing 0.074 -l in the optical diffraction pattern can Er found, q can be deduced. From the values of R the reciprocal space distance from the meridian given in Table 3, values of Z?rRr can be computed, where r is the presumed radius of the scattering matter. The assignment of a value of r is the problem which leads to some difficulty in determining q precisely. We have chosen to assume that there is stain at the surface of the particle. Stain further in would then merely extend the maximum further from the equator but would not affect its nearest approach to the meridian. Biological specimens are subject to distortion by negative stain. By concentrating on the symmetrical particles we assume that we are considering undistorted helical particles. Five of these particles give a value of 27rRr of 11.2 and one a value of 13.2. The two uneven two-sided particles gave a value of 10.8 for the half-helical side of the pattern. Such values for 27rRr are entirely consistent with the J, Bessel function’s first maximum at 2nRr of 10.7 for a circular, or 10.87 for a half-circular helix. (The corresponding values for Js and Jlo are 9.65, 9.78, 11.77, 11.91). Treating the various distortions in the other particles as random variations from best value, the mean of all the values obtained is 11.0. Corresponding values for the PMV protein are 11.0 for undistorted particles and 10.5 for semi-circular helices. If the Bessel function on the first layer line is Jg then q is 9 and there are about 35 subunits in 4 turns or the number of subunits per turn is 8.75. While the evidence for Jg is not conclusive because of the assumptions made, other evidence helps to confirm it. PMV has a length of 540 nm and since there are about 35 subunits in 4 turns the true repeat is 13.4 nm and thus there are about 1410 subunits in the structure. The molecular weight of the RNA in the virus is 2.2 x lo6 (Purcifull and Hiebert, 1971 and verified by us) and the molecular
ET AL.
weight of an average nucleotide, estimated from the known base ratio for this virus (AbouHaidar and Bancroft, 1978), is 322. There are therefore about 6800 nucleotides or 5 nucleotides per subunit. The fact that the number of nucleotides per subunit is not the same or a multiple of the degree of overlap of the protein subunits in the helix need not present a problem. The molecular weight of the protein subunit calculated from amino acid analyses is close to 22,000 (Rees, personal communication) and hence the total molecular weight of the virus particle is about 33 x lo6 and the percentage RNA 6.7% in good agreement with the measured value of 7% (Purcifull and Heibert, 1971). The X-ray diffraction results suggest a position for the nucleic acid at a radius of about 3.5 nm. Assuming that it is the most electron dense part of the nucleic acid, that is the phosphate backbone, these positions would give a phosphorus to phosphorus distance along the chain of 0.5 nm. This shows that a RNA of such a molecular weight can be accommodated in the structure. Thus the X-ray and optical diffraction results for PMV are consistent with a particle having 35 subunits in four turns of its helix and the computer drawn illustration (ORTEP (Johnson, 1965)) presented in Fig. 4 is an accurate representation of the gross structure of the virus. Descriptions of other members of the large and important group of viruses to which PMV belongs are underway and are so far consistent with a value of J9 but with different repeats of the primary helix. ACKNOWLEDGMENTS This work was supported in part by grants from the Academic Development Fund of the University of Western Ontario and the National Research Council of Canada. The authors would like to thank Professors W. P. Alford and P. W. Whippey of the Department of Physics of the University of Western Ontario, who were largely responsible for the design and construction of the optical diffractometer. REFERENCES EA., and BANCROFT, J. B. (1978). The initiation of papaya mosaic virus assembly. Vivology 90, 54-5s.
AB~UHAIDAR
PMV DIFFRACTION BERNAL, J. D., and FANKUCHEN, I. (1941). X-ray and crystallographic studies of plant virus preparations. J. Gen. Physiol. 25, 111-165. 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. (197813). 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. JOHNSON, C. K. (1965). Report ORNL-3794. Oak Ridge National Laboratory, Oak Ridge, Tenn. KLUG, A., and BERGER, 3. E. (1964). An optical method for the analysis of periodicities in electron-
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micrographs and some observations on the mechanism of negative staining. J. Mol. Biol. 10, 565-569. MOODY, M. F. (1967). Structure of the sheath of bacteriophage T4. 1. Structure of the contracted sheath and polysheath. J. Mol. Biol. 25, 167-200. PURCIFULL, D. E., and HIEBERT, E. (1971). “Papaya Mosaic Virus.” C.M.1.IA.A.B. Descriptions of Plant Viruses, No. 56. TAYLOR, C. A., and LIPSON, H. (1964)” Optical Transforms: Their Preparation and Application to X-Ray Diffraction Problems: Bell, London. TOLLIN, P., WILSON, H. R., and YOUNG, D. W. (1968). X-ray diffraction evidence of the helical structure of narcissus mosaic virus. J. Mol. Biol. 34, 189-192. WILSON, H. R., TOLLIN, P., and RAHMAN, A. (1973). The structure of narcissus mosaic virus. J. Gen. Viral. 18, 181.