DISCUSSION AND PRELIMINARY REPORTS 147

experimental condit ions and a new stocksolut ion of DS.

In att empts to induc e ds' revertants byexposing the dsr strain CNAD 3 to NA, HA ,and DMS, we did not find any significantdecrease in the proportion of dsr particlesafte r treat ment with these mutagens (T able3). However, it should be mentioned thatonly gross changes in t he proportion of ds'virus would have been revealed by thismethod of testing.

The int erpretation of t he above results interms of the biochemical specificity of actionof the three mutagens used meets with somedifficulties. Considering only t he results ob­tained with NA and HA, it might be in­ferr ed that the ds' -7 ds: mutation could beaccounted for by the deamination of adenine(A) and it s substitution by guanine (G) inthe course of successive repli cat ions. Theabsence of a rever tant (dsr -7 ds') effect ofDMS casts some doubt 011 t he occurrence ofan A -7 G transition. Furth er investigationshave to be carried out to clarify this point.

The rela tive independence of the varia­tion of the d character (8) as compared withother mark ers belonging to the group co­varying with neurovirulence (t, ret/40°, E,M8) , is indirectly supported by our results.Four dsr clonal strains obtained aft er NAtreatment (induced mutants) and eight ds:clonal stra ins obtained from untreated LISvirus stocks (spontaneous mutants) woreinvest igated for the t and MS markers. Nochanges were observed in these charac ters,as compared to their behavior in the parentalds' strain. This suggests that the ds' -7 dsr ,

like the tl -7 d+ mutation, is a point muta­tion.

ACKNOWLEDGMENTS

The aut hors are indebted to Dr . A. G. J'urcanufor critical advice in the s ta tis tical processing ofthe data . Th ey wish to thank Miss Ana Stroia forher skillful technical assistance.

REFERENCES

1. B OEYE, A., Virology 9, 691-700 (1959) .2. CAI'tP , R . 1., and K OPROWSKI , H ., V i1"ology 17,

99- 109 (1962) .S. G R ENDON , Yu. Z. Acta Vira l. (Prague) 7, 16--24

(1963) .4. T AKEM OTO , K. K ., and LIEB HABE H, H., Virology

17, 499-501 (1962) .

5. S E HGJESCU , D., H OR ODNICEANU , F. , KLEIN, R.,and CUAlNIC, n., Arch . Ges. Vi rusjarsch. 13,231-243 (1966) .

6 . L ORE NZ , R. S., AI·ch. Ges. Virusforsch . 12, 108­137 (1902) .

7. SNEDECOR , G. W . " St at istical Methods Appliedto Experimen ts in Agriculture and Biology, "5th ed . Iowa State Univ , Press, Am es, Iowa,1956.

8. B a In E , A., V iro logy 21, 587- 592 (19ti3) .R ICHAR D KLEIN

DINA SERGIESCU

MARIUS TEODOltl~SC U

Cantacuzino In stit u teBucharest , Romania

Accept ed June 7, 1966

Action of Phospholipase C on Erythrocyte

Membranes and Rauscher Virus

Th e observat ions reported in this com­munication arose out of an invest igation ofthe structure of ery thro cyte membraneswhich involved subjecting the membranesto the action of phospholipase C (P LC) .This enzyme has recently been applied topreparations of R auscher viru s (1) andinfluenza A viru s (2) with the object of re­vealing the internal components of theseviruses. In both instances, treatment withthe enzy me resul ted in th e appearance offilamentou s structures in the form of coilsand rings. Both groups of investigators sug­gested that these structures might be derivedfrom nucleoprot ein elements of viral nucleo­capsids.

It is important to establish whether thisis really so, since heretofore it has not beenpossible to demonstrate a characteristicinternal structure in murine and avianleukemia viruses (such as has been observed,for example, in' myxoviruses), and conse­quently identification of such viruses inimpure preparations has been subject t odifficulty and unc ertainty. In view of thecurrent interest in establishing whethersimilar viruses are associated with humanleukemia, it becomes all the more impo rtantto determine whether the coiled structuresreported to be produced or released by PLCtreatment are specifically virus- associated,in which case they might be used as a diag­nostic criterion for the presence of virus, or

All the F' igures are elec t ro n mi crographs of preparuuious nega tively s taincd wi th 2% po tassium phos­pho t ungs t u te . The sc ale in di ca t or in ea ch 1ig:lll'C represe u ts 0 .1 mi cron, M ngnificu tin u : Figs. 1-3 , 5, G,X 190 ,000; Fig . 5 , X 115 ,000.

FlU . 1. A segment o f a gl u ta ru lde hy de-fixed sheep ery th roc y te ghost. N o regul ar p a t tern is e v ident.1·'IG. 2 . A segmen t o f a glutaraldehyde-fixed , phosphol ipase-C t rea ted (2 minntes ) s he ep er-yt.hrocy te

ghos t showing coils a n d r ings unaoc iut cd with th e me mbrane a nd al s o pres en t. m\ isol at ed enti t ie s adj a­ce ll t to the m embrane .

148

DIHC(TSSIO~ AND PHELIMINAltY HEPORTS 149

FIG. 3. Sheep erythrocyte ghost similar to that shown in Fig. 2 illustrating coils and loops joined toform elongated filaments.

FIG. 4, Fragment. of a !!;ua.t erythrocyte ghost after treatment with phospholipase C (:30 minutes)showing a compact network of rings and coils.

FIG. 5. Glutaraldehyde-fixed Iiauscher virus particles. The nucleoid and outer membrane are clearlyshown.

FIG. G. Glutaraldehyde-fixed, phospholipase Cvtrcated (2 minutes) Rauscher virus purt.icles. Coilsand rings are appareut in the viral envelope. The nucleoid appears to be unaffected.

150 DISCUSSION AND PRELIMINARY REPORTS

whether such structures are the result of amore general action of PLC on membranouscomponents of cells and viruses. The obser­vations reported in this paper stronglysupport the latter alternative.

The action of PLO on specimens consistingof cell membrane material only, namely,preparations of erythrocyte ghosts, was firstexamined. The ghosts were prepared fromsheep, bovine, rabbit, and human erythro­cytes according to the procedure of Dodgeet ol. (3), which produces intact, essentiallyhemoglobin-free ghosts. Ghosts treatedwith PLO (Sigma Chemical Company) at aconcentration of 1.2 mg/rnl and untreatedcontrol ghosts were examined in a SiemensElmiskop I following negative staining witha 2 % solution of potassium phosphotung­state (pH 6.5). Membranes were examinedwith 01' without prior fixation in glutaral­dehyde (final concentration 0.25 %). Thefixed membranes were found to be moreresistant to fragmentation during the nega­tive staining procedure than the unfixed,but the types of structure to be describedwere the same whether the membranes werefixed or not.

The effect of treatment of erythrocytemembranes with PLO can be seen by com­paring Figs. 1 and 2. Figure 1 shows thetypical appearance of an untreated mem­brane after negative staining. The surface iscovered with numerous small granules inmore or less random arrangement. Aftertreatment with PLO (Fig. 2) there is anapparent rearrangement of a component- ofthe membrane that results in the productionof partial or complete circular holes of fairlyuniform size surrounded by a ridge of width65-70 A. The total diameter of the ringlikestructures ranged from 370 to 460 A. Thesestructures can apparently become detachedfrom the surface either in the form of com­plete rings or more often in the form ofcircular arcs (Fig. 2) which may join to­gether as shown in Fig. 3 to form morecomplex aggregates of coils and loops.Isolated compact aggregates of such struc-

1 Experiments to be reported elsewhere suggestthat cholesterol is the membrane component in­volved in the formation of rings and loops.

tures are frequently observed after pro­longed treatment with the enzyme (Fig. 4).

The rings and coils derived from erythro­cyte ghosts after PLC treatment appear tobe identical to those considered to be re­leased from the nucleoids of Rauscher (1)and influenza A (2) virus after treatmentwith the enzyme. The obvious conclusion isthat these structures are not in fact asso­ciated with the nucleoids or internal compo­nents of these viruses but are produced bythe action of the enzyme on the viral enve­lopes or other membranous material in thepreparations. Further evidence in supportof this conclusion was obtained by examininga purified preparation of Rauscher virus'after enzyme treatment and negative stain­ing. The procedures used were the same asthose applied to the cell membrane prepara­tions. The viral suspensions were usuallyfixed in glutaraldehyde before enzyme treat­ment to facilitate visualization of the viralnucleoids (4) and differentiation of the nu­cleoid from the surrounding envelope (Fig.5). The appearance of Rauscher virus par­ticles after PLC treatment is shown inFig. 6. The nucleoid is seen to be intactwhile the viral envelope shows the forma­tions of coils and rings that appear to becharacteristic of the action of PLC on mem­branesin general.

I t can be concluded from these observa­tions that the coils and rings seen in viralpreparations after treatment with PLO arenot derived from the viral uucleoids, whichseem to be relatively insensitive to theaction of the enzyme, but originate in theviral envelopes or other membranous ma­terial. It follows that the coils and rings areof no value as a means of detecting oridentifying virus particles.

ACKNOWLEDGMENTS

This study was supported by funds from theNational Cancel' Institute of Canada, and theBanting Research Foundation, and by N .I.B.grant number CA04964-06VR. One of us (C.L.K.)is the recipient of a W. P. Caven Memorial Fel­lowship.

2 We are indebted to Dr. F. Rauscher for pro­viding us with the purified virus preparation.

DISCUSSION AND PHELIMIN ARY HEPORTS 151

RE FERENCES

1 . P A DGETT , F ., and L EVI NE , A. S ., Virology 27,033--637 (1965).

2. S I MPSON, R . W., and H A USER, R . E ., Vi rology27, 642-046 (1905).

3. D ODGE, J. T ., MITCHELL, C., and H ANAH AN, D .J ., .Arch. Bio chem, Bioplu)«, 100, 119-130

(1903) .4. L E VY, J. P ., B OI RQN, J\tI., SILVJo:S'rRE , D. , and

B ER NAR D, J ., Virol ogy 26, 146-150 (1965) .C. LI NDLE Y KEMP

A LLAN F. H OWATSO N

Ontario Cancer Institute500 Sh erbourne StreetT oron to 5, Canada

A ccepted May 31, 1966

Characteristics of Rapidly Sedimenting

Ribonudeic Acid from Harris Strain Rous

Sarcoma Virus and Infected Cells1

The isolation of a 64 S R NA from theBryan strain of Rous sarcoma virus (RSV)has been reported recently (1). This wasinterpreted to indicate a molecular weight of9.6 X 106, corresponding closely to estimatesmade by direct chemical analysis (2).

As Bryan strain stocks have been reportedto contain Rous-associated virus (R AV) (3)in as much as tenfold excess over focus­forming Rous par ticles (4), it might be ques­tioned whether th e above values characterizeRAV, focus-forming RSV, or both. However,since the RNA isolated by the above authors(1) appeared homogeneous with respect tosedimentation velocity, a difference betweenthe nucleic acids of the two types of particlewas not suggested. The present report con­firms and strengthens these conclusions,since RNA of similar sedimentation constantwas isolated by independently developedmethods from the immunologically distin ctHarris strain. Indeed, reports which have ap­peared since the presen t experiments werecarried out strongly suggest that 65 SRNA characterizes most if not all viruses ofthe avian leukosis complex (5, 8).

The methods herein described permit theisolation of high molecular weight virus

1 Aided by U. S. Public H ealth Service ResearchOrants HE 09011, OA 02738 -09 and AI 06584-01.

specific RNA from infected cells. Evidencesuggests that a major fractio n of t his ma­terial is in the form of RNA-DNA hybrids.

Virus particle concentrat ions were deter­mined by sedimentation of sonicated anddiluted culture fluids onto agar followed bypseudoreplication with collodion using themethod originally applied to myeloblastosisvirus (7).

Line 15 I RIF-free fertile chick eggs werekindly made available by Dr. B. R. Bur­mester. Secondary cultures were grown inE agle's medium containing triple concent ra­tions of vitamins and amino acids, 5 % new­born calf serum, and 1 % turkey serum.These were infected when approximatelyhalf of the glass surface was covered by cells(approximately 2 X 106 cells) with 109 physi­cal particles (about 4 X 106 FFU in 0.5 ml).After a gO-minute period of adsorpt ion at37°, the cultures received 5 ml of t he abovemedium and were incubated for 3 days at37°. They were t hen subcult ivated by tryp­sinization and each was divided into foursubcultures. One day after subcultivation ,when cells had again covered about half ofthe glass surface, trit iated uridine (specificactivity 17.4 mCIJ.Lmole) was added to theculture medium to give a final activity of 10).tClml. After 24 hours, and at subsequenttimes, supernatant fluids and cell sh eets wereseparately frozen at -70° C. for subsequentextraction of RNA and analysis by sucrosedensity gradient cent rifugat ion.

Extraction of RNA was achieved using asodium dodecyl sulfate (SDS) procedurewhich has been employed to isolate highmolecular weight RNA from Newcastl e dis­ease virus (8). Virus particles in supernatantfluids were sedimented, either after clarifica­tion or, in some cases, together with a fewfree cells which served to provide ribosomalRNA markers, by centrifugation at 30,000rpm for 30 minutes in a No. 40 Spinco rotor.Pellets, Or frozen cell sheets, were taken upin 0.2 ml of 0.5 % SDS made up in pH 5.1acetate buffer (0.01 M acetate, 0.05 M NaCl,10-4 M MgCh), layered onto 5-20 % linearsucrose gradients in the same buffer, andcentrifuged for 90-120 minutes at 35,000rpm in a Spinco SW-39 swinging-bucketrotor. E qual fr actions were collected, by