日植 病 報 52: 422-427 (1986)

Ann. Phytopath. Soc. Japan 52: 422-427 (1986)

Properties of Virus Particles, Nucleic Acid and

Coat Protein of Cycas Necrotic Stunt Virus

Kaoru HANADA*, Manabu KUSUNOKI** and Mitsuro IWAKI***

Abstract

Cycas necrotic stunt virus (CNSV; considered to belong to nepovirus group) isolated

from cycad plants was characterized further. The sedimentation coefficients of the com

ponents M (middle) and B (bottom) were 85S and 112S, and buoyant densities in CsCl were

1.404 and 1.472g/cm3, respectively. When the infectivity of the components was tested, the

mixture of both components was five to twenty times more infectious than either particle

alone. M and B components contained single-stranded RNA species with molecular weight

(MW) of 1.5•~106 (RNA2) and 2.5•~106 (RNA1), respectively. Both RNA1 and RNA2 were

necessary for infection. M and B components contained a single major polypeptide with

identical MW of 65 K. These properties of virus particles, nucleic acid and coat protein

of CNSV confirmed affinities of CNSV to nepoviruses, particularly to tomato black ring vi

rus (TBRV). However, since no serological relationship between CNSV and TBRV or sero

logically TBRV-related viruses was detected in our previous work, CNSV would be a new

nepovirus having similarities to TBRV in biochemical properties.

(Received December 26, 1985)

Key words: nepovirus, cycas necrotic stunt virus, bipartite genome.

Introduction

A virus was isolated from Cycas revoluta in Chiba Prefecture in Japan. The virus

caused necrosis followed by stunting or top necrosis on infected cycad plants, and the

virus was called as cycas necrotic stunt virus (CNSV). CNSV consists of small spher

ical particles with ca. 28nm in diameter and had many properties similar to those of

nepoviruses, as described already6'. In this paper properties of virus particles, nucleic

acid and coat protein of CNSV will be described, confirming affinities of CNSV with

nepoviruses especially with those belonging to tomato black ring virus subgroup.

Materials and Methods

Virus purification. The virus isolate of CNSV used and the procedure for puri

fication was already described6). By sucrose density-gradient centrifugation (SDGC),

virus particles were separated into three components; top, middle and bottom com

-* National Agriculture Research Center, Tsukuba Science City, Ibaraki 305, Japan　 農 林 水 産 省

農 業 研 究 セ ン タ ー

** Forestry and Forest Products Research Institute, P.0. Box 16. Tsukuba Science City, Ibaraki

305, Japan　 農 林 水 産 省 林 業 試 験 場*** National Institute of Agro-Environmental Sciences

, Tsukuba Science City, Ibaraki 305, Japan

　農 林 水 産 省 農 業 環 境 技 術 研 究 所

Ann. Phytopath. Soc. Japan 52 (3). July, 1986 423

poments (hereafter called T, M and B components, respectively). Since T component

was usually present in small amount in virus samples and difficult to be separated

from host constituents, only M and B components were characterized after fractiona

tion by two cycles of SDGC. Purified virus samples and fractionated components were

immediately used or stored at -70C until use. Other viruses used for comparison or

marker were arabis mosaic virus (ArMV)5), cucumber mosaic virus (CMV-Y)12), tobac

co mosaic virus (TMV-OM)9) and tomato black ring virus (TBRV)4).

Analytical ultracentrifugation. Virus samples (0.5mg/ml) dissolved in 50mM

citrate buffer (CB), pH 6.8 were examined by M. S. E. centriscan 75 II, centrifuged at

30,000rpm. The sedimentation coefficients of virus components were calculated by

Markham's graphical method8). Buoyant densities in CsCl of virus components were

determined by the 'step' CsCl method described by Sehgal et al.11) using virus samples

dissolved in CB. Virus samples were centrifuged at 170,000•~g for 22 hr in a Hitachi

RPS 50-2 rotor and fractionated by an ISCO fractionator. Density of each fraction was

calculated from refractive index measured by an Abbe refractometer.

Preparation and identification of nucleic acid. The buffer (TNE) used for

nucleic acid preparation was reported already10). After addition of 1% sodium dodecyl

sulfate (SDS), nucleci acid was extracted by mixing virus samples with one volume

of TNE-saturated phenol containing 0.1% 8-hydroxyquinoline. After shaking and low

speed centrifugation, aqueous phase was re-extracted with half volume of TNE-phenol.

The nucleic acid was precipitated by adding 2.5 volumes of cold ethanol and stored at

-20C . Precipitated nucleic acid was collected by low-speed centrifugation, dissolved in

water or TNE and stored at -70C.

For nucleic acid identification, viral nucleic acid samples were treated with 10ƒÊg/ml

deoxyribonuclease (Sigma type I, RNase-free), with 10ƒÊg/ml ribonuclease A (Sigma

type I-A) either in 1•~SSC (0.15M NaCI and 0.015M sodium citrate, pH 7.0) or in

0.1•~SSC at 25C for 20min. After forty-fold dilution with TNE, the infectivity of un

treated control and nuclease treated samples was compared.

Electrophoresis. After disruption of virus particles by addition of 1% SDS and

heating at 60-65C for one min, viral nucleic acid was subjected to 1% agarose gel elec

trophoresis using threefold diluted Loening's buffer for running. After electrophoresis

gels were briefly stained with ethidium bromide then photographed under UV illumi

nation. Gel bands containing nucleic acid were cut out separately and ground with TNE,

then the homogenate was centrifuged at low-speed after phenol extraction. Nucleic acid

precipitated with ethanol was used for infectivity tests. Molecular weight (MW) of

nucleic acid was determined using RNA1 and RNA2 of TBRV as marker (2.5•~106 and

1.5•~108, respectively)3) under non-denaturing conditions.

Electrophoresis of viral protein was done as described by Laemmli7), then staining

and destaining was done by the method reported by Cleveland et al.1) MW of protein

was estimated as already described.2)

Infectivity assay. Fractionated virus components or nucleic acid was inoculated

either singly or in combination to Chenopodium murale plants which produced necrotic

local lesions in inoculated leaves with CNSV. Inoculated C. murale plants were incubat

ed in the growth chamber maintained at ca. 20C, then the local lesions produced were

424 日本植物病理学会報 第52巻 第3号 昭和61年7月

counted seven to ten days after inoculation.

Results

Properties of virus particles

When purified CNSV samples were subjected to analytical ultracentrifugation, sedi

mentation coefficients (S2, w) of M and B components were calculated to be 85S and

112S (not extraporated to infinite dilution), respectively. Both components were found

to be stable in CsCI, then at equilibrium M and B components sedimented as single

peaks corresponding to buoyant densities of 1.404 and 1.472g/cm3, respectively (mean of three determinations). Under similar conditions, CMV-Y sedimented as a single

peak with the density of 1.334g/cm3.

CNSV samples were separated into two major components (M and B) by two cycles

of SDGC. Fractionated M and B components were inoculated to C. murale plants singly

or after mixing in order to find whether both components are necessary for infection.

As results shown in Table 1, M component alone was infectious poorly and B component

alone was infections to some extent. The mixture of both components was highly infec

tious, indicating that both components are essential for infection.

Table 1. Infectivity of fractionated virus components of CNSV

a) In Expt. I, B component (0.05 A260 units/ml) and M component (0.035 A260 units/ml)

were used for inoculum. In Expt. II, two-fold diluted each component used in Expt.

I was inoculated.

b) Average numbers of local lesions produced in one leaf of Chenopodium murale (11-22

leaves were inoculated per inoculum).

Properties of nucleic acid

Nucleic acid samples extracted from CNSV particles were infectious when inoculated

to C. murale plants. After RNase treatments both in 1•~SSC and 0.1•~SSC, viral nu

cleic acid lost infectivity, while infectivity was not affected by DNase treatment. There

fore, nucleic acid of CNSV would be single-stranded RNA in positive sense. When

viral RNA samples were subjected to electrophoresis in 1% agarose gels, two distinct

bands were observed (Fig. 1). The more slowly migrating band comigrated with TBRV

RNA1 and ArMV-RNA1. The faster moving band comigrated with TBRV-RNA2 but

migrated more slowly than ArMV-RNA2. The larger and the smaller RNA of CNSV

were called RNA1 and RNA2, respectively. MW of CNSV-RNA1 was estimated under

non-denaturing conditions to be 2,5•~106 and that of CNSV-RNA2 was 1.5•~106, when

TBRV-RNA was used as MW marker. MW of RNA1 and RNA2 of CNSV was estimat

ed to be 2.9•~106 and 1.9•~106 when TMV-RNA and CMV-RNA were used as marker.

Ann. Phytopath. Soc. Japan 52 (3). July, 1986 425

Fig. 1. Electrophoretic patterns of CNSVRNA. Purified virus samples were

subjected to 1% agarose gel elect rophoresis after SDS-disruption.Electrophoresis was done at 80V for 2.5 hr. 1: CMV-Y, 2: TBRV 3: Middle component of CNSV, and 4: Bottom component of CNSV.

RNA1 and RNA2 of CNSV extracted from

agarose gels were inoculated singly or in

mixture. As results shown in Table 2, each

RNA species alone had no or very little

infectivity, while the mixture showed consi

derable infectivity. Both RNA1 and RNA2

were considered to be essential for infection

of CNSV.

When fractionated viral components were

subjected to electrophoresis after SDS-disr

uption, M component contained RNA2 almost

exclusively and B component contained RNA1.

These results confirmed that B component of

CNSV is homogeneous containing RNA1 alone,

like B component of TBRV.

Properties of protein

Polyacrylamide gel electrophoresis of pro

tein of CNSV particles after SDS treatment

revealed the presence of one major polypeptide

(Fig. 2). The major protein migrated slower than coat protein of TBRV. MW of the

protein of CNSV was estimated to be 65K

and that of TBRV was 60K. Two minor

proteins with estimated MW of 71K and 56K were also detected. Both M and B components

contained proteins with identical MW each

other. Coat protein of CNSV was seemed to

be unstable in comparison with that of TBRV.

When stored longer time at -70C, 56K pro

tein became the major protein indicating that

56K protein would be degradation product of 65K protein. Further analysis such as

peptide mapping must be necessary in order to clarify the relationship among major and minor proteins of CNSV.

Table 2. Infectivity of RNA species of CNSV

a) Total numbers of local lesions in six half-leaves of C, murale.

426 日本植物病理学会報 第52巻 第3号 昭和61年7月

Fig. 2. Electrophoretic patterns of coat protein of CNSV. Electrophoresis was done at 30 mA for 2 hr using 10% polyacrylamide slab gel. 1: CMV-Y, 2: TBRV, 3: Middle component of CNSV, and 4: Bottom component of CNSV.

Discussion

One of the most interesting features of CNSV is its characteristic severe symptoms

on cycad plants which belong to Gymnospermae. Although we have not tested yet

whether other nepovirus(es) can infect cycad, it is worth to be done. Virus diseases

of Gymnospermae plants have not been studied well. CNSV would be a useful tool for

study of such work. As described by Kusunoki et al.6), CNSV has many properties

which are commonly possessed by nepoviruses defined by Harrison and Murant3) although

tests for nematode transmission of CNSV have not been done.

The results described in this paper suggest that CNSV is a single-stranded RNA vi

rus having a bipartite genome of two functional RNA species with MW of 2.5•~106 and

1.5•~106 and a single major protein with MW of 65K. Both RNA species are separately

encapsidated to form M and B components of virus particle. All these evidences sug

gest that CNSV is most close to viruses belonging to TBRV-subgroup in the nepovirus

group. However, no serological relationship was detected between CNSV and members

of TBRV-subgroup (cocoa necrosis virus, grapevine chrome mosaic virus and TBRV) by

immunodiffusion tests6). Although more sensitive serological or hybridization tests

would be necessary to confirm uniqueness of CNSV, CNSV seems to be a new nepovirus.

Further analysis of viral RNA and protein including genome linked viral protein is in

progress.

We are grateful to Dr. H. Inoue for the great help to determine S value. We also thank to Dr.

H. Tochihara for encouraging this work.

Ann. Phytopath. Soc. Japan 52 (3). July, 1986 427

Literature cited

1. Cleveland, D. W., Fischer, S. G., Kirschner, M. W. and Laemmli, U. K. (1977). J. Biol. Chem. 252: 1120-1126.

2. Hanada, K. and Tochihara, H. (1982). Phytopathology 72: 761-764.3. Harrison, B. D. and Murant, A. F. (1977). Descriptions of plant viruses No. 185: Common W.

Mycol. Inst., Assoc. Applied Biologists, Kew, Surrey, England.4. Iwaki, M. and Komuro, Y. (1973). Ann. Phytopath. Soc. Japan 39: 279-287.5. Iwaki, M. and Komuro, Y. (1974). Ibid. 40: 344-353.6. Kusunoki, M., Hanada, K., Iwaki, M., Chang, M. U., Doi, Y. and Yora, K. (1986). Ibid. 52:

302-311.7. Laemmli, U. K. (1970). Nature 227: 680-685.8. Markham, R. (1962). Advances in Virus Research 9: 241-270.9. Nozu, Y. and Okada, Y. (1968). J. Mol. Biol. 35: 643-646.

10. Peden. K. W. C. and Symons R. H. (1973). Virology 53: 487-492.11. Sehgal, O. P., Jean, J. L., Bhalla, R. B., Soong, M. M. and Krause, G. F. (1970). Phytopaphology

60: 1778-1784.12. Tomaru, K. and Hidaka, Z. (1960). Bull. Hatano Tobacco Expt. Sta. 46: 143-149.

和 文 摘 要

花田 薫 ・楠木 学 ・岩 木満朗:ソ テツえそ萎 縮 ウイルスの ウイル ス粒子,核 酸及び外 被蛋 白質の諸性質

ソテツか ら分離 され たソテツえそ萎縮 ウイルス(CNSV;ネ ポ ウイルス群 に属す ると考 え られ る)の 諸性質

を さらに検討 した。CNSVのM成 分 とB成 分の沈降係数は それぞれ85Sと112Sで あった。 塩化セ シウム

中での浮遊密度 はM成 分 が1.4049/cm3, B成 分は1.4729/cm3で あ った。M成 分 とB成 分 は単 独では低い

感染性 しか な く,両 成分 を混 合す ると 感 染性 は5～20倍 高 くな ることか ら 感染に は両成分が必要 と考 え られ

た。両成分 は異 なる大 きさの1本 鎖RNA成 分 を別 々に含み, M成 分 に含 まれ るRNA2の 分子量 は1.5×

106, B成 分に含 まれ るRNA1の 分子量 は2.5×106で あ り, RNA1とRNA2の 両方が感染 に必要 であ っ

た。B成 分 とM成 分 に含 まれる外 被蛋白質は同 じ大 きさで あり,そ の主成 分の分 子量は65Kで あ った。これ

らの諸性質 はネポウィルスに 属す る トマ ト黒色 輪点 ウィル ス(TBRV)に 最 もよ く似て いるが, CNSVは

TBRV及 びTBRVと 血清 関係のあ るウイルス と血清学的類縁 関係が認め られなか った前報(1986)の 結果 か

ら考 えると, CNSVは 未報告の ネポウイルスであると結論 された。