Inhibition of the multiplication of a plant virus by actinomycin D

504

SHORT

COMMUNICATIONS

REFER.ENCES 1. HOLME:S, I. H., and WARBURTON, M. F., Lancet II, 1233-1236 (1967). 2. CARVER, D. H., and MARCUS, P. I., Boxteriol. Proc. 681h Ann. Meeting Am. Sot. Microbial., p. 181 (1968). S. CASALS, J., Trans. N.Y. Acad. Sci., Ser. II, 19, 219-235 (1957). 4. Scientific Group, World Health Organ. Tech. Rept. Ser. 369, (1967). 5. STEWART, G. L., PARKMAN, P. D., HOPPS, H. E., DOUGL.~S, It. D., HAMILTON, J. P., and MEYER, H. M., JR., !Vew Engl. J. Med. 276, 554-557 (1967). 6. CLARKE, D. H., and CAS~LS, J., Am. J. Trop. Med. Hyg. 7,561-573 (1958). Y. C~SALS, J., Proc. Symp. 9th Intern. Congr. Microbial., Moscow, pp. 441-452 (1966). NORMA E. METTLER RICHARD L. PETRELLI Department of Epidemiology and Public Health Yale University School of Medicine New Haven, Connecticut 06510 JORDI

CASALS

Yale Arbovirus Research Unit Department of Epidemiology and Public Yale University School of Medicine New Haven, Connecticut 06510 Accepted August 25, 1968

Inhibition

of the Multiplication

Plant Virus by Actinomycin

Health,

of a D

Actinomycin D at certain concentrations and under certain conditions has been reported both to inhibit (l-4) and to be without effect on (2-S) the multiplication of several animal and bacterial RNA viruses. The multiplication of two plant viruses, tobacco mosaic virus in detached tobacco leaves (7) and bean pod mottle virus in excised soybean hypocotyls (8) has been reported to be unaffected by levels of the antibiotic that inhibited 77-95% of host RNA synthesis. In both cases the antibiotic had been applied 24 and 72 hours, respectively, after inoculation. The studies reported here were undertaken to determine the effect of actinomycin D on the multiplication of another single-stranded RNA plant virus, cowpea yellow mosaic virus, when the drug was applied at different times after inoculation.

Etiolated hypocotyls of cowpea (T’igna unguiculata cvar. ‘Early Ramshorn’) were inoculated 45 hours after germination with a suspension of purified virus (0.1 mg/ml) and Celite in 0.01 $1 phosphate buffer, pH 7.0. At various intervals after inoculation, hypocotyls were cut into 2.0-cm segments, were randomized, and 10-g samples were incubated in shake culture in 10 ml of a buffered sucrose solution (1% sucrose in 0.005 M KH,POk , brought to pH 6.0 with KHdOH, containing 25 mg/ml 2,4-D and 20 mg/ml streptomycin) at 30” in the dark. Actinomycin D was added to a final concentration of 10 pg/ml. At this level the antibiotic inhibits the incorporation of 32P into host RNA by 75-85%. After 3640 hours the tissue was washed, blotted dry, and ground with sand in a chilled mortar in 0.01 M phosphate, pH 7.0 (1: 2 w/v). The extract was centrifuged at 8000 g for 15 min and the supernatant was diluted and assayed for host, on a local-lesion virus content Phase&u vulgar& var. ‘Pinto.’ The results as indicated in Fig. 1 (averaged data from five similar experiments), show that actinomycin D reduced the yield of virus when applied shortly after inoculation, but that this apparent inhibitory effect was progressively reduced with time after inoculation. Percentage inhibition of infectivity was determined by comparison of lesion numbers from infected hypocotyl tissue excised at the same time after inoculation and incubated

GO-

s

‘IO-

/

20-

- 300 z w > F

OO TIME

16

24

(HOURS

AFTER

32 40 INOCULATION)

48

O

FIG. 1. Growth curve of virus in excised hypocotyl tissue (m--q); total number of local lesions on 8 half-leaves corrected for dilution factor. Percentage reduction in infectivity from tissue incubated in 10 pg/ml actinomycin D at 2, 4, 6, 8, 10,12,15, and 20 hours after inoculation and maintained for 3640 hours (O---O).

SHORT

CORIMUNICATIO~S

for t’he same number of hours with or nithout’ the addit,ion of actinomycin D. The ea.rly inhibitory effect, not,ed above could also be obkned at a concentration of 5 pg/ml, but llot at 1.0 pg/ml or 0.1 pg/ml. The effect could also be obtained by exposure of inoculated &sue to the drug for only 5 hours, after which the segments were floated in nctinomycin D-free medium for t’he remaiw ing :
1

RESPIR.~TION I~ATES OF EXCISED HYPOCOTYL SEGMI~WS INCUHA-IY:D IN THF, PRCSENCE MXD ABSIGNCE OF 10 &ML ACTINOMYCIN II AT 30”

.-lo.5

replicative form (RF) of the virus. A replicative form of CYKV has not, been isolated and characterized, but assuming that’ t’his viws replicates by the mechanism presentl\* post ulated for single-stranded RSA vinlses, all effect of :&nom\-tin 1) on the struct,ur;~l integrity or biologica! activity of the RI* cannot be ruled o:lt The role of t,hc host genome in the mult#iplication of ItSA virtises has not, been ascertained, and there have been suggestions (2, 3, 8, 10) that a host’ DSA-dependent, function may possibly be involved in tbn early stages of replication of some of t-how viruses. Our findings appear to be consistent wit,h the previous reports (‘7, 8) which indicate that once infection has been ctitablishetl, a DSA template is no longer involvctl in thcl production of progeny viral RSIZ. There is considerable asynchrolly of infcct#ion in t)hc system described hcrc, as it is estimat’ed from microscopic examination of hypocotyl cross sections that a maximum of 0111~ S--l0 72 of t’hc tot al number of ~11s can be inoculated initially. If the inhibition levels obtained bear an inverse rel:ttiolwhil) to the percentage of cells infected, then this may indicate ~1 inhibit’ion by actinomycin 1) of early intracellular events itt t hc cstahlishment of virus infection. X decreased inhibition of infectivity with time after inoculation with tobacco mosaic virus has been reported for blasticidin S (1 I), a11 inhibitor of protein synthesis, alt,hough t,he effect, in t)his case \vas ascribed to an inhibition of TAl\r IZSA synthesis. A similar situat)ion has been reported for Ke\vrastle disease virus IZSX (12). The rcplk6on of this virus is inhibited by profl:rvinc~~-:t revertiible inhibitor of RSX synt,hesis ~~at, early stages of infect’ion. The possibilit#y, IWRXW~I., cannot~ he discarded that the effect of actinomycin I) and other antibiotics ma!’ be mediate6 not through inhibition of a virusspecific functjion, but by interference with a constitutive host fwction involwtl in early event,? 01‘ virus multiplication.

02 uptake &l/mg drv wt/hr) Hours of incubaiion

--

18 -15 40

24 42 40

40 25 25

1. PoNS, &I., I’iroloyy 33, 150~15-1 (1907). 2. BIKIF, R. U., IVKS, 1.). R., arid CRTJIVKSH {SIC, J. C; ., AValure 194, 1139-1140 (19W). 3. GR\DO~ C., FISC-HEIZ S. awl
506

SHORT

COMMUNICATIONS

4. BADER, J. P., Virology 26, 253%2Gl (1965). 5. HAYIVOOD, A. M., and HARRIS, J. M., J. Mol. Biol. IO, 448-463 (1966). 6. POLATNICK, J., and ARLINGH.~US, R. B.. J. Viral. 1, 1130-1134 (1967). 7. S;~NGER, H. L., and KNIGHT, C. A., Biochem. Biophys. Res. Commun. 13, 455-461 (1963). 8. BANCROFT, J. B., and KEY, J. L., hiature 202, 729-730 (1964). 9. MUKHERJEE, A. K., SOANS, L. C., and CHCSSIN, M., fVature 216, 1344-1345 (1967). IO. BORLAND, R., and MAHY, B. W. J., Virol. 2, 33-39 (1968). 11. HIRAI, T., HIRASHIMA, A., ITOH, T., TamHASHI, T., SHIMOMURA, T., and HAYASHI, Y., Phytopathology 56, 1236-1240 (1966). 11. STAKHANOVA, V. M., and SCHOLTISSEK, C., J. Gen. Viral. I, 571-573 (1967). B. E. L. LOCKHART J. S. SIZMANCIK Department of Plant Pnthology Unioersity of California Riverside, California. Accepted August 26, 1.968

Pea Enation Mosaic Virus: Differential Suspension of Virus Components in Buffer and Sucrose A constant problem in the study of the components of small spherical viruses is the purification of significant quantities of component fractions. These components are generally recognized only by difference in their sedimentation velocities or density. They are separated by centrifugation in density gradients, and there have been few reports of their purification by other means (1, 2). When their sedimentation velocities are close as in the case of pea enation mosaic virus (PEXIV), which contains an upper component of 94 S and a bottom component of 113 S, supplementary methods for obtaining one or the other component in pure form would be useful. This communication describes how weak phosphate buffer and sucrose solutions were used in the purification of bottom component free of upper component and samples enriched with upper component. The buffer used was 0.05 M potassium phosphate pH 6 and the sucrose solutions used for suspending virus pellets contained

5 % sucrose in this buffer. High speed centrifugations to pellet the virus were made iI1 a Spinco Model L ultracentrifuge at 66,000 g for 1.5 hours and low speed centrifugations were made in a refrigerated Sorvall centrifuge at 4500 g for 10 min. Sucrose density gradients (1.037-l. 113 g/cm3, linear) were prepared in buffer as previously described (3). Rate zonal sucrose density gradient centrifugations were made at 63,000 g for 4 hours using the SW 25.1 head, and the gradients were fractionated by puncturing the bottom of the centrifuge tubes and passing the sample through an ultraviolet monitor (3). Partially purified virus was prepared by grinding PEMV-infected pea tissue in a Waring Blendor together with an equal weight of buffer and chloroform, clarification by low speed centrifugation, dialysis overnight against buffer, clarification again by low speed centrifugation, and pelleting the virus by high speed centrifugation. The virus pellets were suspended in sucrose or buffer solution and clarified by low speed centrifugation. Then the’ remaining low speed pellets were suspended in 5 % sucrose solution. To determine the amount of virus components present, an aliquot of each extraction was further purified by rate zonal centrifugation in sucrose density gradient. The virus components banded as expected from previous experiments (5) when it was shown that healthy pea tissue produces no band in the same region of density gradient columns. The gradients were fractionated, and the amount of components estimated from the optical density of the virus peak. In the first suspension of the virus pellet, the sucrose solution extracted about 4 times as much virus as the buffer solution (Table 1). Resuspension of both pellets with sucrose solution produced an additional 17% from the pellet previously extracted with sucrose, but 3 times more virus components from the pellet previously extracted with buffer. Successive experiments varied in the exact quantity of virus components extracted but the first suspension with sucrose solution consistently extracted at least 80 % of the virus and frequently no additional virus was obtained in the second suspension. Buffer, however,