Action of phospholipase C on erythrocyte membranes and rauscher virus

147

DISCUSSION AND PRELIMINARY REPORTS

experimental conditions and a new stock solution of DS. In att empts to induc e ds' revertants by exposing t he dsr strain CNAD 3 to N A, HA , and DMS, we did not find any significant decrease in t he proportion of dsr particles afte r treat ment with t hese mu tagens (T able 3). However, it should be mentioned that only gross changes in t he proportion of ds' viru s would hav e been reveal ed by thi s method of testing. The int erpretation of t he abo ve results in terms of the biochemical specificity of action of the three mutagens used meets with some difficulties. Considering only t he results obtained with NA and HA, it might be inferr ed that the ds' -7 ds: mutation could be accounted for by the deamination of adenine (A) and it s substitution by guanine (G) in t he course of successive repli cat ions. The absence of a rever tant (ds r -7 ds') effect of DMS casts some doubt 011 t he occurrence of an A -7 G tra nsition. Furth er investigati ons have to be carried out t o clarify this point. The rela tive independ ence of the variation of t he d character (8) as compared with other mark ers belonging t o the group covarying with neurovirulence (t, ret/40°, E, M 8) , is indirectly supporte d by our results. Four ds r clonal strains obt ained aft er NA treatment (induced mutants) and eight ds: clonal stra ins obtained from untreated LIS virus stocks (spontaneous mutants) wore invest igated for the t and M S markers. No chang es were observed in these charac ters, as compared to their behavior in the parental ds' strain. This suggests that the ds' -7 dsr , like the tl -7 d+ mutation, is a point mutation. ACKNOWLEDGMENTS The aut hors are indeb t ed to Dr . A. G. J'urcanu for critical advi ce in t he s ta tis tical processi ng of the data . Th ey wish t o thank M iss Ana Stroia for her skillful technical assistance .

REFERENCES 1.

A., Virology 9, 691-700 (1959) . 2. CAI'tP , R . 1., and K OP ROWSKI , H ., V i1"ology 17, 99- 109 (1962) . S. G R E ND ON , 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) . B OEYE,

5. 6.

S E HGJESCU ,

D ., H OR OD NICEANU , F. , KLEI N, R.,

and CUAlN IC , n., Arch . Ges. Vi ru sjarsch. 13, 231-243 (1966) . L ORE NZ , R. S., AI·ch. Ges. Virusforsch . 12, 108137 (1902) .

G. W . " St at istic al Methods Ap plied to E xperimen ts in Agriculture an d Bi ology, " 5t h ed . Iowa State Univ , Press, Am es, I owa , 1956. 8. B a In E , A., V iro logy 21, 587- 592 (19ti3) . 7.

SNE DECOR ,

R ICHAR D KLEIN DINA SERG I ESCU MARIUS TEO D Oltl~SC U

Cantacuzino I n stit u te Bucharest , Romania Accept ed Jun e 7, 1966

Action of Phospholipase C on Erythrocyte Membranes and Rauscher Virus

Th e observations reported in this communi cation arose out of an invest igation of the structure of ery thro cyte membranes which involved subjecting t he membranes to the action of phospholip ase C (P LC) . This enzyme ha s recently been applied to preparations of R auscher viru s (1) and influenza A viru s (2) with the object of revealing the internal components of these viruses. In both instances, treatment with the enzy me resul ted in th e appea rance of filamentou s structures in the form of coils and ring s. Both groups of investigators suggested t hat these structures might be derived from nucleoprot ein elements of viral nucleocapsids. It is important to establish whether this is really so, since heretofore it has not been possible to demonstrate a characteristic internal structure in murine and avian leukemia viruses (such as has been observed, for example, in' myxoviruses), and consequently iden tification of su ch viruses in impure preparations has been subject t o difficulty and unc ertainty. In view of the current interest in establishing whether similar viruses are associated with human leuk emia, it be comes all the more impo rtant to determine whether the coiled str uctures reported to be produced or released by PLC treatment 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 a le in di c a t or in e a c h 1ig:lll'C repr ese u ts 0 .1 mi cron, M ngn ific u tin u : Figs. 1-3 , 5, G, X 190 ,000; Fi g . 5 , X 115 ,000. FlU . 1. A segment o f a gl u ta ru lde hy de -fixed sheep ery th r oc y te g host. N o regul ar p a t tern is e v ide n t. 1·'IG. 2 . A seg me n t o f a glutaraldehyde-fixed , phosphol ipase-C t rea ted (2 m innt es ) s h e ep er-yt.hrocy te gh os t showing coils a n d r in gs unaoc iut cd w ith th e me mbrane a nd al s o pr es 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 to form 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 clearly shown. FIG. G. Glutaraldehyde-fixed, phospholipase Cvtrcat ed (2 minutes) Rauscher virus purt.icles. Coils and 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 a more general action of PLC on membranous components of cells and viruses. The observations reported in this paper strongly support the latter alternative. The action of PLO on specimens consisting of cell membrane material only, namely, preparations of erythrocyte ghosts, was first examined. The ghosts were prepared from sheep, bovine, rabbit, and human erythrocytes according to the procedure of Dodge et ol. (3), which produces intact, essentially hemoglobin-free ghosts. Ghosts treated with PLO (Sigma Chemical Company) at a concentration of 1.2 mg/rnl and untreated control ghosts were examined in a Siemens Elmiskop I following negative staining with a 2 % solution of potassium phosphotungstate (pH 6.5). Membranes were examined with 01' without prior fixation in glutaraldehyde (final concentration 0.25 %). The fixed membranes were found to be more resistant to fragmentation during the negative staining procedure than the unfixed, but the types of structure to be described were the same whether the membranes were fixed or not. The effect of treatment of erythrocyte membranes with PLO can be seen by comparing Figs. 1 and 2. Figure 1 shows the typical appearance of an untreated membrane after negative staining. The surface is covered with numerous small granules in more or less random arrangement. After treatment with PLO (Fig. 2) there is an apparent rearrangement of a component- of the membrane that results in the production of partial or complete circular holes of fairly uniform size surrounded by a ridge of width 65-70 A. The total diameter of the ringlike structures ranged from 370 to 460 A. These structures can apparently become detached from the surface either in the form of complete rings or more often in the form of circular arcs (Fig. 2) which may join together as shown in Fig. 3 to form more complex aggregates of coils and loops. Isolated compact aggregates of such struc1 Experiments to be reported elsewhere suggest that 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 to be identical to those considered to be released from the nucleoids of Rauscher (1) and influenza A (2) virus after treatment with the enzyme. The obvious conclusion is that these structures are not in fact associated with the nucleoids or internal components of these viruses but are produced by the action of the enzyme on the viral envelopes or other membranous material in the preparations. Further evidence in support of this conclusion was obtained by examining a purified preparation of Rauscher virus' after enzyme treatment and negative staining. The procedures used were the same as those applied to the cell membrane preparations. The viral suspensions were usually fixed in glutaraldehyde before enzyme treatment to facilitate visualization of the viral nucleoids (4) and differentiation of the nucleoid from the surrounding envelope (Fig. 5). The appearance of Rauscher virus particles after PLC treatment is shown in Fig. 6. The nucleoid is seen to be intact while the viral envelope shows the formations of coils and rings that appear to be characteristic of the action of PLC on membranesin general. It can be concluded from these observations that the coils and rings seen in viral preparations after treatment with PLO are not derived from the viral uucleoids, which seem to be relatively insensitive to the action of the enzyme, but originate in the viral envelopes or other membranous material. It follows that the coils and rings are of no value as a means of detecting or identifying virus particles. ACKNOWLEDGMENTS This study was supported by funds from the National Cancel' Institute of Canada, and the Banting 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 PHEL IMIN ARY HEPORTS RE FERE NCES 1.

P A DGETT ,

F ., an d

L EVI NE ,

A. S ., Vi rology 27,

033--637 (1965).

2.

S I M P SON,

R . W., and

H A USER,

R . E ., Vi rology

27, 642-046 (1905).

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

3.

D O DGE,

4.

L E VY,

(1903) . B O I RQN, J\tI., SI LVJo:S'rRE , D. , and J ., Virol ogy 26, 146-150 (1965) . C. LI NDL E Y KEMP A LLAN F. H OWATSO N Ontario Cancer Institute 500 Sh erbourne Street

J. P .,

B ER NA R D,

T 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 th e Bryan strain of Rous sarcoma virus (RSV) has been reported recently (1). This was interpr eted to indicate a molecular weight of 9.6 X 106, corresponding closely to estimates made by direct chemical analysis (2). As Bryan strain stocks have been r eported to contain Rous-associat ed virus (R AV) (3) in as much as tenfold excess over focusforming Rous par ticles (4), it m ight be questioned whether th e above values characterize RAV, focus-forming RSV, or both. However, since the RNA isolated by the above authors (1) appeared homogeneous with respect to sedimentation velocity, a difference between the nucleic acids of the two ty pes of particle was not suggested. The present report confirms and strengthens these conclusions, since RNA of similar sedimentation constant was isolated by independently developed methods from the immunologically distin ct Harris strain. Indeed, rep orts which have appeared since the presen t experiments were carried out strongly suggest t hat 65 S R NA characterizes most if not all viru ses of the avian leukosis complex (5, 8). The methods herein described permit the isolation of high molecular weight virus 1 Aided by U. S. Public H ealth Ser vice Research Orants HE 09011, OA 02738 -09 and AI 06584-01.

151

specific RNA from infected cells. Evidence suggest s th at a major fractio n of t his material is in the form of RNA-DNA hybrids. Virus parti cle concentrat ions were determined by sediment ation of sonicated and diluted culture fluids onto agar followed by pseudoreplication with collodion using the method originally applied to myeloblasto sis virus (7). Line 15 I RIF-free fertile chick eggs were kindly made available by Dr. B. R. Burmester. Secondary cultures were grown in E agle's medium containing triple concent rations of vit amins and amino acids, 5 % newborn calf serum, and 1 % turkey serum. These were infected when approximately half of the glass surface was covered by cells (approximately 2 X 10 6 cells) with 109 physical particles (about 4 X 106 FFU in 0.5 ml). Aft er a gO-minut e period of adsorpt ion at 37°, the cultures received 5 ml of t he above medium and were incubated for 3 days at 37°. They were t hen subcult ivated by trypsinization and each was divided into four subcultures. One day after subcultiva tion , when cells had again covered about half of the glass surface, trit iated uridine (specific activity 17.4 mCIJ.Lmole) was added t o the culture medium t o give a final activity of 10 ).tClml. After 24 hour s, and at subsequent times, supernatant fluids and cell sh eets were separately frozen at -70° C. for subsequ ent extracti on of RNA and analysis by sucrose density gr adient cent rifugat ion. Extraction of RNA was achieved using a sodium dodecyl sulfate (SDS) procedure which has been employed to isolate high molecular weight RNA from Newcastl e disease virus (8). Virus particles in supernatant fluids were sedimented, either after clarification or, in some cases, together with a few free cells which served to provide ribosomal RNA markers, by centrifugation at 30,000 rpm for 30 minu tes in a No. 40 Spinco rotor. Pellets, Or frozen cell sheets, were taken up in 0.2 ml of 0.5 % SDS made up in pH 5.1 acetate buffer (0.01 M acetate, 0.05 M NaCl, 10- 4 M MgCh ), layered onto 5-20 % linear sucrose gradients in the same buffer, and centrifuged for 90-120 minutes at 35,000 rpm in a Spinco SW-39 swinging-bucket rotor. E qual fr actions were collected, by