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Formation of an infectious nucleoprotein from protein and nucleic acid isolated from a small spherical virus
VIROLOGY
32
(1967)
~1scussr0N
ASI) PRELIMTKBHY
REPORT
Spinco SW 39L swinging-bucket rotor, and the tjop three-fourt,hs of t’he supernatant was removed with a bent hypodermic a Small Spherical Virus’ needle. The E 260/280 of the protein was The reassembly of the rod-shaped t,o- 0.63, indicating little nucleic acid. An asbacco mosaic virus from its constituents, sumed Eit&, = 1.2 was used for tjhe profirst accomplished by Fraenkel-Conrat and tein and E$&,, = 25.0 for the nucleic Williams (I), serves as the only clear ex- acid. To assemble the nucleoprotein, 1 ample of the reconstitution of a virus, part of RNA was added to 3 parts of proalt’hough protein-nucleic acid complexes tein before dialysis for 2 hours at 4” against have been induced to form from the iso0.01 111 Tris, pH 7.4, 0.01 dl KCl, 5 X lated components of bacteriophages (6X174 low3 M MgC12 (TKbf) and 1 X 10-Z M (2) and fd (3). It has been suggested t,hat Cleland’s reagent. Protein and RNA to be the spherical cowpea chlorotic mottle virus added to density t’ubes separately were (CCMV) (4) would prove amenable t#o similarly dialyzed. Density-gradient caentrifrecon&ution studies because it can be ugat,ion was in a SW 397, rotor on gradients disassembled in such a way that its protein made t’he previous day of 1 ml each of 10, remains capable of forming 20, 30, and 40 mg sucrose per milliliter organized structures (5). Biologically active protein 0.1 M pH 5.0 sodium acetate buffer. The has also been prepared from brome mosaic cont,ent.sof the tubes were monitored by an and broad bean mottle viruses (,5; and ISCO densit’y-gradient analyzer (7). Localunpublished). We now wish t,o report the lesion assays of t,he different preparations all in TKRI were made with glass spatula formation of an infec%ious nucleoprotein, resembling CCMV, from prot,ein and RiYA on t,he primary leaves of soybean (GZ@ne ?UUZ (I,) Uerr. var. Lindarin) in nn inisolat’ed from CCMV. complete block design with 8 half-leaves CCMV was purified as before (5). RNA was prepared by two phenol extractions per t’reatment. Snake venom phosphodiesterase (Cal Biochem.) was used at a at room temperature, t.he first in the presence of bentonitc, followed by ether ex- final concentration of 20 u&s/ml TKM traction, removal of the ether wit,h piz, for 20 hours at 22”. and ethanol prcripitntion. The RNA was The sedimentat’ion distributions after dialyzed overnight in 0.01 M Tris, pH densit,y-gradient centrifugst,ion of RiYA, 7.4, 0.01 dl KCI, 5 X 10e4 M RIgClz before protein, reassembled nucleoprot~ein, and cont,rol virus are shown in Icig. 1. Neit,her use. Such preparations consisted of three the R?Sh nor the prot’ein by themselves sedimenting species with sedimentation coefficients of 23.1 S, 18.2 S, and 12.G S showed any absorption at 254 rnp at the depths reached by the nucleoproteins, nor (unpublished). To obt,ain protein, virus was could my infectivity be found at t)hat depth dialyzed overnight at 4” against a solution (Table 1). The reassembled nurleoprotjcin consisting of 1 M N&l, 0.02 111 Tris and reached t,he same depth as the c’ontjrol virus 1 X 10e3 d1 Cleland’s reagent. (fi), at. a ccntrifugeti in a sister tube, and bot’h nucleofknl pH of 7.4. The dialyzate was cbcntriproteins were infectious (Table 1). Sinchc fuged l;i hours at, 30,000 rpm in a little RN,4 remained at the meniscus of t,he tube containing the reassembled material, 1 Srlpported ill part by the National Science Foundation. Journd Paper No. 3013 of t,he Pldlre it is assumed t,hat most of the RNA was University Agricultural 1~2qIeriment, Sktlion. encapsulated by protein and that> the hetFormation
Protein
of
an
and
Infectious
Nucleic
Nucleoprotein
Acid
Isolated
from
from
354
DISCUSSION
0.4 c
2 (D
A
0.2
s: N l2
PRELIMINARY
A
0.3 -
AND
0.1 L!!!B
0.3 0.2 0.1 I
[q--Jy *
0.3
Q
0.2
D
0.1
3%
REPORT
fectivity of the RNA was completely abolished by snake venom phosphodiesterase whereas that, of the reassembled nucleoprotein and the virus were unaffected. Also, the virus and the reassembled material had about the same stability in TKM. The apparent instability of the nwleoprot,eins as compared to RNA probably- results from the 101~l\Ig++ level used at pH 7.4. The above data indiwte that WC have assembled an infectious spherical nucleoprotein similar in general properties to the virus from lvhi& the constituents of the rensscmbly produ& were derived. We have also found that, CC!\IV protein will form organized st,ruc+ures around KXA’s of widely divergent’ size and composition. A detailed description of this aspect of our
LJL 2
DEPTH
3
(cm)
FIG. 1. Tracings of the 254 mp absorbing distributions (from an ISCO scanner with a 2-mm light path and a range setting of 0.5) of (A) RNA, (B) protein, (C) reassembled nucleoproprotein, and (D) control virus after densitygradient cent,rifugation of the cowpea chlorotic mottle virrls or its constituents for IN hours at 36,000 rpm in a SW 39L rotor on a gradient of IO-40 mg of sucrose per milliliter of 0.1 ,V pH 5.0 acetate buffer. Sedimentation is toward t,he right.
erogeneity of the assembled material (Fig. 1, C) reflects the different sizes of RNA in the original RNA preparation. Electron microscopic observat’ions, kindly made by Dr. Charles Bracker, showed no viruslike particles in the RKA and protein preparations [although if the protein lvas backdialyzed against 0.05 M pH 7.3 phosphat)e buffer, tubular and other strwkures (5) were seen] whereas large numbers of sphcrical particles of the general appearance of virus mere seen in the material that had been reassembled.The reassembledmaterial react’cd against,antiserum made t)o t,he virus, had a similar E 260/280 (1.67) and behaved generally like virus when centrifuged to equilibrium in C&l. The infectivities of virus and reassembled nucleoprotein removed from the densitygradient tubes and RNA which had not been centrifuged are given in Table 1. The in-
TBBLE
1
IKFECTIVITIES OF PREP.LR.\TIONS USED IN FIG. 1
Material
assayed
Days after preparation
Treatment 1
Virus-DCp Virus-DC: Reassembled-DG Reassembled-DG RNA RNA RNA-DC; Protein IX
Enzyme*
~
2
~ I
5
149V
~-
263
1266
365
Enzyme
1114 1109
--301
142 248
Enzyme
0 2000
' 1160
0 1 1300 0~
--
404
-
oi-
n The designation T)G means that the sources of the assayed preparations were t,he UT--absorbing materials found a little more than half way down the density-gradient, tubes after centrifugation. RNB-DG and protein-DC were removed from the same level as reached by the virlls. RNA refers to the starting nucleic acid before densitygradient centrifugation. b EnzJ-me refers to a final concentration of 20 units of snake venom phosphodiesterase per milliliter of material in TK&I. Incubat,ion for enzyme-treated and control material was for 20 hours at 2”“. I r Number of local lesions on 8 half-leaves of soybean at. fi.‘GO = 0.100 with the except,ion of RNA-DC alld proteiil-DG, which shoned no absorption at the level reached by the nucleoproteins. All mat,crial was incubated and assayed in TK&I, and preparations were stored at 4” bet,ween treatments reqlliring storage at 22”.
356
DISCUSSION
AND
PRELIMINARY
work and on the in vifro formation of hybrid spherical viruses will be presentsed later.
REPORT
5. BANCROFT, J. B., HILLS, G., and &I.~RKHAM, R., Virology 31, 354-379. 6. CLELAXD, W. W., Biochemistry 3,480-482 (19&I). 7. BRAKKE, RI. Ii., Anul. Kiochem. 5, 271-283 (1963). J. B. B.4NCIWFT ERNEST HIEBERT Deparlment of Botany and Plant Pathology Lilly Hall of 1,ije Science.s Purdue University Lafayette, Indiana 4790Y
REFERENCES 1. FRAENKEL-CONRAT, H., ad WILLIAMS, R. C., hoc. ,JTatl. Acad. Sci. U.S. 41,69@698 (1955). 2. TAKAI, M., Biochim. Riophys. Actu 119, 20-28 (1966). 3. KNIPPERS, IL, and HOFFMANN~EELING, H., J. Mol. Biol. 21, 293-304 (196G). 4. KUHN, C. W., Phytopalhology 54,853%857 (196-i).
Accepted
April
4,
1967
Errata Vol. 31, No. 4 (1967), in the article “Restriction of NonglucosyMed T-even Bacteriophage: Propert,ies of Permissive Mut’ants of Escherichia coli B and IZ12,” by Helen R. Revel, pp. ASS%701: Page 696, Table 6: The endonuclease units/mg protein, not 100.
I activity
Page 69S, Table 25,column reading “Growth reference following “Ratio” should be d.
of st,rain Sh should read 1000
of 7r on spheropla&s,”
bhe footnote
32
(1967)
~1scussr0N
ASI) PRELIMTKBHY
REPORT
Spinco SW 39L swinging-bucket rotor, and the tjop three-fourt,hs of t’he supernatant was removed with a bent hypodermic a Small Spherical Virus’ needle. The E 260/280 of the protein was The reassembly of the rod-shaped t,o- 0.63, indicating little nucleic acid. An asbacco mosaic virus from its constituents, sumed Eit&, = 1.2 was used for tjhe profirst accomplished by Fraenkel-Conrat and tein and E$&,, = 25.0 for the nucleic Williams (I), serves as the only clear ex- acid. To assemble the nucleoprotein, 1 ample of the reconstitution of a virus, part of RNA was added to 3 parts of proalt’hough protein-nucleic acid complexes tein before dialysis for 2 hours at 4” against have been induced to form from the iso0.01 111 Tris, pH 7.4, 0.01 dl KCl, 5 X lated components of bacteriophages (6X174 low3 M MgC12 (TKbf) and 1 X 10-Z M (2) and fd (3). It has been suggested t,hat Cleland’s reagent. Protein and RNA to be the spherical cowpea chlorotic mottle virus added to density t’ubes separately were (CCMV) (4) would prove amenable t#o similarly dialyzed. Density-gradient caentrifrecon&ution studies because it can be ugat,ion was in a SW 397, rotor on gradients disassembled in such a way that its protein made t’he previous day of 1 ml each of 10, remains capable of forming 20, 30, and 40 mg sucrose per milliliter organized structures (5). Biologically active protein 0.1 M pH 5.0 sodium acetate buffer. The has also been prepared from brome mosaic cont,ent.sof the tubes were monitored by an and broad bean mottle viruses (,5; and ISCO densit’y-gradient analyzer (7). Localunpublished). We now wish t,o report the lesion assays of t,he different preparations all in TKRI were made with glass spatula formation of an infec%ious nucleoprotein, resembling CCMV, from prot,ein and RiYA on t,he primary leaves of soybean (GZ@ne ?UUZ (I,) Uerr. var. Lindarin) in nn inisolat’ed from CCMV. complete block design with 8 half-leaves CCMV was purified as before (5). RNA was prepared by two phenol extractions per t’reatment. Snake venom phosphodiesterase (Cal Biochem.) was used at a at room temperature, t.he first in the presence of bentonitc, followed by ether ex- final concentration of 20 u&s/ml TKM traction, removal of the ether wit,h piz, for 20 hours at 22”. and ethanol prcripitntion. The RNA was The sedimentat’ion distributions after dialyzed overnight in 0.01 M Tris, pH densit,y-gradient centrifugst,ion of RiYA, 7.4, 0.01 dl KCI, 5 X 10e4 M RIgClz before protein, reassembled nucleoprot~ein, and cont,rol virus are shown in Icig. 1. Neit,her use. Such preparations consisted of three the R?Sh nor the prot’ein by themselves sedimenting species with sedimentation coefficients of 23.1 S, 18.2 S, and 12.G S showed any absorption at 254 rnp at the depths reached by the nucleoproteins, nor (unpublished). To obt,ain protein, virus was could my infectivity be found at t)hat depth dialyzed overnight at 4” against a solution (Table 1). The reassembled nurleoprotjcin consisting of 1 M N&l, 0.02 111 Tris and reached t,he same depth as the c’ontjrol virus 1 X 10e3 d1 Cleland’s reagent. (fi), at. a ccntrifugeti in a sister tube, and bot’h nucleofknl pH of 7.4. The dialyzate was cbcntriproteins were infectious (Table 1). Sinchc fuged l;i hours at, 30,000 rpm in a little RN,4 remained at the meniscus of t,he tube containing the reassembled material, 1 Srlpported ill part by the National Science Foundation. Journd Paper No. 3013 of t,he Pldlre it is assumed t,hat most of the RNA was University Agricultural 1~2qIeriment, Sktlion. encapsulated by protein and that> the hetFormation
Protein
of
an
and
Infectious
Nucleic
Nucleoprotein
Acid
Isolated
from
from
354
DISCUSSION
0.4 c
2 (D
A
0.2
s: N l2
PRELIMINARY
A
0.3 -
AND
0.1 L!!!B
0.3 0.2 0.1 I
[q--Jy *
0.3
Q
0.2
D
0.1
3%
REPORT
fectivity of the RNA was completely abolished by snake venom phosphodiesterase whereas that, of the reassembled nucleoprotein and the virus were unaffected. Also, the virus and the reassembled material had about the same stability in TKM. The apparent instability of the nwleoprot,eins as compared to RNA probably- results from the 101~l\Ig++ level used at pH 7.4. The above data indiwte that WC have assembled an infectious spherical nucleoprotein similar in general properties to the virus from lvhi& the constituents of the rensscmbly produ& were derived. We have also found that, CC!\IV protein will form organized st,ruc+ures around KXA’s of widely divergent’ size and composition. A detailed description of this aspect of our
LJL 2
DEPTH
3
(cm)
FIG. 1. Tracings of the 254 mp absorbing distributions (from an ISCO scanner with a 2-mm light path and a range setting of 0.5) of (A) RNA, (B) protein, (C) reassembled nucleoproprotein, and (D) control virus after densitygradient cent,rifugation of the cowpea chlorotic mottle virrls or its constituents for IN hours at 36,000 rpm in a SW 39L rotor on a gradient of IO-40 mg of sucrose per milliliter of 0.1 ,V pH 5.0 acetate buffer. Sedimentation is toward t,he right.
erogeneity of the assembled material (Fig. 1, C) reflects the different sizes of RNA in the original RNA preparation. Electron microscopic observat’ions, kindly made by Dr. Charles Bracker, showed no viruslike particles in the RKA and protein preparations [although if the protein lvas backdialyzed against 0.05 M pH 7.3 phosphat)e buffer, tubular and other strwkures (5) were seen] whereas large numbers of sphcrical particles of the general appearance of virus mere seen in the material that had been reassembled.The reassembledmaterial react’cd against,antiserum made t)o t,he virus, had a similar E 260/280 (1.67) and behaved generally like virus when centrifuged to equilibrium in C&l. The infectivities of virus and reassembled nucleoprotein removed from the densitygradient tubes and RNA which had not been centrifuged are given in Table 1. The in-
TBBLE
1
IKFECTIVITIES OF PREP.LR.\TIONS USED IN FIG. 1
Material
assayed
Days after preparation
Treatment 1
Virus-DCp Virus-DC: Reassembled-DG Reassembled-DG RNA RNA RNA-DC; Protein IX
Enzyme*
~
2
~ I
5
149V
~-
263
1266
365
Enzyme
1114 1109
--301
142 248
Enzyme
0 2000
' 1160
0 1 1300 0~
--
404
-
oi-
n The designation T)G means that the sources of the assayed preparations were t,he UT--absorbing materials found a little more than half way down the density-gradient, tubes after centrifugation. RNB-DG and protein-DC were removed from the same level as reached by the virlls. RNA refers to the starting nucleic acid before densitygradient centrifugation. b EnzJ-me refers to a final concentration of 20 units of snake venom phosphodiesterase per milliliter of material in TK&I. Incubat,ion for enzyme-treated and control material was for 20 hours at 2”“. I r Number of local lesions on 8 half-leaves of soybean at. fi.‘GO = 0.100 with the except,ion of RNA-DC alld proteiil-DG, which shoned no absorption at the level reached by the nucleoproteins. All mat,crial was incubated and assayed in TK&I, and preparations were stored at 4” bet,ween treatments reqlliring storage at 22”.
356
DISCUSSION
AND
PRELIMINARY
work and on the in vifro formation of hybrid spherical viruses will be presentsed later.
REPORT
5. BANCROFT, J. B., HILLS, G., and &I.~RKHAM, R., Virology 31, 354-379. 6. CLELAXD, W. W., Biochemistry 3,480-482 (19&I). 7. BRAKKE, RI. Ii., Anul. Kiochem. 5, 271-283 (1963). J. B. B.4NCIWFT ERNEST HIEBERT Deparlment of Botany and Plant Pathology Lilly Hall of 1,ije Science.s Purdue University Lafayette, Indiana 4790Y
REFERENCES 1. FRAENKEL-CONRAT, H., ad WILLIAMS, R. C., hoc. ,JTatl. Acad. Sci. U.S. 41,69@698 (1955). 2. TAKAI, M., Biochim. Riophys. Actu 119, 20-28 (1966). 3. KNIPPERS, IL, and HOFFMANN~EELING, H., J. Mol. Biol. 21, 293-304 (196G). 4. KUHN, C. W., Phytopalhology 54,853%857 (196-i).
Accepted
April
4,
1967
Errata Vol. 31, No. 4 (1967), in the article “Restriction of NonglucosyMed T-even Bacteriophage: Propert,ies of Permissive Mut’ants of Escherichia coli B and IZ12,” by Helen R. Revel, pp. ASS%701: Page 696, Table 6: The endonuclease units/mg protein, not 100.
I activity
Page 69S, Table 25,column reading “Growth reference following “Ratio” should be d.
of st,rain Sh should read 1000
of 7r on spheropla&s,”
bhe footnote