The nucleic acid of human cytomegalovirus

DISCUSSION AND PRELIMINARY REPORTS

trated and not penetrated by phosphotungstate.

105

suspension obtained by suspending infected cultures in 10 ml of growth medium. Five to seven days later the cultures contained REFERENCES many of the large rounded refractile cells 1. KLI~KOWSKI, M., ~nd KRETSTZBERG,G. (1958). characteristic of cytomegalovirus infection. Phytopathol. Z. 32, 1-24. Extracts of these cultures examined in the 2. MULLmAN, T. F. Ann. Appl. Biol. 48, 575-579. electron microscope were found to contain (1960). particles of the size and morphology de3. MULLIGAN,T. F. Rept. Rothamsted Exptl. Sta. scribed for cytomegalovirus (2). Infected 1957, 110-111. (1958). cultures were harvested by scraping the cells ~. OHMA~N-KREUTZBERG, G., PAWLITSCItEK, W., into the medium and sedimenting the cells and SCttMIDT, H. B. (1960). Phytopathol. Z. and cell debris (5000 g for 10 minutes). The 38, 13-17. 5. OHMANN-KREUTZBERG, G. (1963). Phytopathol. virus was then sedimented (40,000 g for 60 minutes) and subjected to a cycle of differZ. 47, 1-17. ~. SCttMIDT, I-I. B., ERITZSCttE, R., and LEtIMANI~, ential centrifugation (5000 g for 10 minutes followed by 40,000 g for 60 minutes). W. (1963). Naturwissenseha]ten S0, 386. 7. TAh*IG~JCHI,T. (1962). Nature 194, 708. The DNA was extracted from the in8. HAaRISO~, B. D., and NlXON, H. L. (1960). Vi- fected cells and from the virus using the rology 12, 104-117. techniques developed for herpes simplex 9. NlxoN, H. L., and GIBBS, A. J. (1960). J. Mol. virus (1, 3). The infected cell suspension Biol. 2, 197-200. was treated with sodium dodecyl sulphate E. PROLL I-I. B. SCI-IMIDT (1%) for 2 minutes at 25 ° and then made 1 N with sodium perchlorate and deproInstitute o] Phytopathology Aschersleben teinized by shaking with chloroform:isoGerman Academy o] Agriculture Sciences amyl alcohol (24:1) as described by MarBerlin, German Democratic Republic tour (4). The DNA was then deposited on Accepted January 11, 196~ a glass rod and redissolved in standard saline citrate (4). The virus pellet obtained as described above was suspended in 0.8 ml The Nucleic Acid of Human Cytomegalovirus of phosphate buffer (0.1 M, pH 7), and the In a recent study of the nucleic acids of DNA was extracted as follows. Sodium herpes related viruses (1) it was not pos- dodecyl sulphate (0.2 ml, 0.5%) and watersible to include human cytomegalovirus saturated phenol (0.8 ml) were added and owing to difficulties in virus production. the suspension was mixed gently for 5 minSufficient virus has now been obtained and utes at 25 °. After cooling to 0 °, the phases the approximate size and base composition were separated (5000 g for 10 minutes) of the virus nucleic acid have been de- and the aqueous phase was collected. Retermined. sidual phenol was removed by dialysis for The strain of human cytomegalovirus 24 hours against phosphate buffer (0.01 M, used was obtained from Dr. H. Stern, St. pH 8) containing Versene (0.01 M). George's Hospital, London. After being Equilibrium density gradient centrifugapassed several times in human adult skin tion (5) was used to estimate the base comfibroblasts by Dr. W. C. Russell, it was position of the DNA and its molecular transferred to human embryo skin fibro- weight. The DNA, either from infected cells blasts, where the virus yield was improved. or from virus, was suspended in CsC1 (1.71 The cells were grown at 37 ° in Pyrex bot- g/ml) containing Tris buffer (0.05 M, pH tles with Eagle's medium containing twice 8.5) and centrifuged at 44,770 rpm for 24 the standard concentration of amino acids hours at 25 ° in a Spinco model E centrifuge. and vitamins plus 10% tryptose phosphate DNA from Clostridium welchii was added broth and 10% unheated calf serum. Virus- to the virus DNA to provide a reference infected cultures were propagated in the of known density and base composition. same medium but with 2% calf serum. Ultraviolet absorption photographs were Cultures were infected with 5 ml of a celt taken and scanned with a microdensitom-

106

DISCUSSION

AND

PRELIMINARY

eter. The base composition of the virus DNA was then calculated from the relative positions of the absorbing bands (6). For band-width molecular weight determination, the gradients were centrifuged at 44,770 rpm for I day then at 31,410 rpm for 2 days. The band profiles at the lower speed showed no detectable change during the last 12 hours of centrifugation. Human cytomegalovirus DNA formed a sharp band in the equilibrium density gradient, the position of the band corresponding to a base composition of 58% guanine plus cytosine (% GC) if the DNA is assumed to be double stranded (Fig. 1). The DNA extracted from infected cells showed a small peak in the position corresponding to 58% GC, in addition to the main peak of cellular DNA. The band profile of human tyromegalovirus DNA (Fig. 2) corresponded to a band-width molecular weight of 32 million for the sodium salt of the DNA. This is a minimmn estimate for the toolecular weight since band-width molecu-

REPORTS [

,

,

)

0.8

Q6

× 0.4

i

o~ Q3

0

0.2

0

0

2

4

8 O.

i

I

60

6,5

Z0

Radius (cm,) FIO. 1. M i c r o d e n s i t o m e t e r

tracing of the ultra-

violet absorption photograph of an equilibrium density gradient containing h u m a n cytomegalovirus D N A and C l o s t r i d i u m welchii D N A marker in CsC1 (density 1.71 g/ml). Photographs were t a k e n after 24 hours' centrifugation at 44,770 rpm and 25 ° . The meniscus is at the left of the figure and density increases towards the right. The sharp band of h u m a n cytomegalovirus D N A appears at a higher density t h a n the marker DNA.

6' 8' w2. iO s 8/9

I0 '

I'Z

I'4

FIG. 2. Band-width molecular weight determination of h u m a n cytomegalovirus DNA. The

virus DNA was centrifuged in a CsC1 (density 1.71 g/ml) density gradient for 1 day at 44,770 rpm followed by 2 days at 31,410 rpm. Ultraviolet absorption photographs were taken and scanned with a microdensitometer. The method of presentation of the band profile is taken from Thomas and Berns (9). The microdensitometertracing is inset on the plot of band width squared (W~) against relative DNA concentration (C/Cm,x) : fl =

o

i Imm.

RI' p(ro) (dp/dr)roo~ro

lar weights are in general lower than those determined by other methods. Human cytomegalovirus has been placed in the same group as herpes simplex virus (7), the two viruses being similar in many respects. Indirect evidence for the presence of DNA in cytomegalovirus has been obtained by electron microscopy (2) and by the use of 5-ftuorodeoxyuridine (8) and is supported by the data presented in this paper. The molecular weight of the virus DNA appears to be high and similar to that of herpes simplex virus DNA (1). The composition of cytomegalovirus DNA, however, appears to be markedly different from that of herpes simplex virus, 58% GC as compared to 68% GC. This relatively low

DISCUSSION AND PRELIMINARY REPORTS GC content is more similar to t h a t of the equine herpes viruses, L K virus (56% GC) and equine abortion virus (55% GC)

(1). ACKNOWLEDGMENTS We should like to thank Dr. It. Stern for proriding the strain of human cytomegalovirus used and Dr. D. H. Watson for examination of virus preparations in the electron microscope. The electron microscope and analytical centrifuge were provided by the Wellcome Foundation. We are also indebted to Miss M. Ballantyne for her excellent technical assistance. REFERENCES 1. RUSSELL,W. C., and CRAWFOm),L. V., Virology, 22,288-292, 1964. 2. S~ITH, K. O., and RASMUSSEN, L., J. Bacteriol. 88, 1319-1325 (1963). 3. RUSSELL, W. C., and CRAWFOR~),L. V., Virology 21, 353-361 (1963). 4. MARMUR, J., J. Mol. Biol. 3, 208-218 (1961). 5. MESELSON, M., STAHL, ]~. W., and VINOGRAE, J., Proc. Natl. Acad. Sci. U. S. 43, 581-588 (1957). 6. SVEoKa, N., J. Mol. Biol. 3, 31-40 (1961). 7. ANDREWE8, C. I-I., Advan. Virus Res. 9, 271296 (1962). 8. GOODHEART, C. R., FILBERT, J. E., and McALLISTER, R. M., Virology 21, 530-532 (1963). 9. THOMAS,C. A., JR., and BERNS, K. I., J. Mol. Biol. 3, 277-288 (1961). L. V. CRAWFOI~D1

AvRIL J. LEE2 Institute of Virology University o~ Glasgow Glasgow, Scotland Accepted February 5, 1964

1Member of the Medical Research Council Experimental Virus Research Unit. -~Registrar in Clinical Virology, Western Infirmary, Glasgow.

Aphid Transmission of a Purified Stylet-Borne Virus Acquired through a Membrane

Unanswered questions regarding the transmission of styler-borne plant viruses include the nature of vector specificity and of the forms in which the viruses are transmitted. Studies on these problems are complicated by the fact that the virus must be

107

acquired from a host plant, in which its form and location are not known with certainty. Previous attempts to simplify the system by presenting partially purified virus 4o the insects through membranes have been unsuccessful (1, 2). This paper reports successful transmission of purified alfalfa mosaic virus (AMV), a stylerborne plant virus (3), acquired by aphids through an artificial membrane. A strain of AMV (ATCC 105), obtained from R. W. Fulton, University of Wisconsin, was purified from systemically infected tobacco ( N i c o t i a n a t a b a c u m L. var. Havana 425) by the procedure described by Kuhn and Bancroft (4). The aphids, M y z u s persicae (Sulz.), were reared o n young healthy tobacco plants, at 20-22°C under a 14-hour photoperiod. The insect feeding cages followed the design of I~ochow (5). They were made by cutting a hole, with a cork borer, through sponge rubber ½ inch thick and 2 inches in diameter. The bottom of the hole was covered with fine-mesh screen. About 10-15 nonviruliferous aphids, starved for 1 hour, were placed in the hole, and the top was capped with a thin membrane of Parafilm (Marathon Corporation, Menasha, Wisconsin) stretched over the end of an 18-mm diameter glass tube about 2 cm long. The hole in the cage was slightly smaller than the glass tube, so that the tube was held firmly in the top of the hole. Inside the tube, and on top of the membrane, was placed 0.6 ml of purified, concentrated (ca. 4 mg/ml) AMV in 0.01 M phosphate buffer, pH 7.0, containing 5% sucrose. Probing and feeding of the aphids was observed with a dissecting microscope. Since transmission of AMV has been reported to be of the nonpersistent type (i.e., styler-borne) (3, 6), aphids were allowed to probe the membrane for 40-80 seconds in most experiments. The period of probing was measured from the time that the labium was lowered to make contact with the membrane. Aphids were not tested unless they had probed for at least 40 seconds. Those which had not stopped probing at the end of 80 seconds were induced to do so by prodding with a camel's