The uncoating of alfalfa mosaic virus by its own RNA

vmomciy

48, 699-708 (1972j

The Uncsating

of Alfalfa

Mosaic

LOGS VAN VLOTEX-DOTING

AND 31i;M.J. JASPARS

Upon addition of alfalfa mosaic virus (A&IQRN.4 to AMV particles :he latter are rapidly changed into highly RXase-sensitive, slow-sedimenting structures. These structures still contain intact RNA but they band at a higher density in CsCl gradrents than virus particles and lack the typical bacilliform virus structure. The disintegration of the particle structure is thought to be due to the free RNA comhirringwith some of the prot,ein subunits. The presence of protein subunits on HKA molecules was demonstrated ‘by means of biological assay. The reaction is dependent on the concentrations of virus and Rn’A, and on the time of incubation. When particles of two other viruses were brought in contact with their own KPiA or with A&IV-RNA no reaction was observed.Addition of other RNAs to AMV particles also had no effect,. Because of the specificity of the reaction, we postulate the existence of sites with a, high affinity for AMV coat protein on the AMV-RXAs. These sites may be related LO the biological role of the coat protein. The fact that the AMiw structure becomes disturbed upon xldition of its own RNA confirms the idea thst in this virus part.icle the protein-protein interactions are of minor importance.

Alfalfa mosaic virus (AMV) has a genome consisting of three pieces of RNA encapsulated in three different components, the bottom component (B), middle component (3%) and top component b (Tb). For infectivity the coat protein is required. In its absence infect~ion can be induced onIy by the addition of the RKd from the accessory top eomponent a (Ta): which presumably represents the message for the coat protein (Bol, Van Vloten-Doting, and Jaspars: 1971). Recently we have observed that nucleoprotein particles are t,wo orders of magnitude more eff e&ive in activating the AMV genome than equimolar amount,s of coat protein. This suggests t’hat nucleoprotein particles may distribute their protein subunits among a large number of RNA molecules. In this paper sucrose density gradient analysis of mixtures of nucieoprotein and RNA is used te study t,his interaction. 1 Dedicated to Professor II. Veldstra on the occasion of his retirement from the Chair of Biochemist,ry of the Universit,y of Leiden.

IsolatiorL

of viral

~nt&oprtlteins.

3Wmid

of t’he 4MV strains 425 and YSMV (gellriw spot mosaic virus) was isoiated ati foi!o~: Each 200 g of icaves was first homogenized in a Waring Blendor with 200 ml of 0.1. X &HPO+ 0.1 M ascorbic acid, and 0.02 31 acid (EDTAj ethylencdiaminetetraacetic adjusted to pH 7.1 with KOH. The siurry was rehomogenized with 200 ml of 1 : I chloroform-butanol for P min, and t,he emulsion was broken by centrifugatlon (Hull, Rees, and Short, 1969). To the water layer, a solution of 30 % polyethyiene glycol (Serva, 1M. W. 2@,000) m distilled wa,ixr WA”; added to a finai concentration of 5 5.: to precipitate the virus (Clark, 19%). ‘3~ pellets obtained after iobI~-speed cemrifugation were resuspended in about 40 ml of 0.01 M NaHJ?Q.~ adjustzed to pR 7.0 with KaOH (phosphate buffw). Pellets obtained after two cycles of low- and high-speed centrifugation were dissolved in phosphate buffer. Top component a was scparatec? from the

699

700

VAN VLOTEN-DOTING

heavier components by a slight modification of the method of Kelly and Kaesberg (1962). An equal volume of a solution of 0.06 M MgS04 and 0.01 M NaH2P04, adjusted to pH 7.0 with NaOH was added to the virus solution (25 mg/ml). After standing for 2 hr, the milky suspension was centrifuged for 30 min at 20,000 rpm in a Spinco Model L 30 rotor through a layer of 12 ml of 100 mg sucrose/ml in 0.03 M MgS04 and 0.01 M NaH2P04 (pH 7.0). The pellets were redissolved in 0.01 M NaH2P04, 0.001 M EDTA and 0.001 M NaN3 adjusted to pH 7.0 with NaOH (PEN buffer). This solution (bottom fraction) as well as the supernatant (top a fraction), was dialyzed overnight against PEN buffer and concentrated by high-speed centrifugation. AMV nucleoproteins were stored and handled at 4” in PEN buffer, unless stated otherwise. Bottom fraction was freed from contaminating top component a by centrifugation in sucrose density gradients in an SW 27 rotor. Tubes were loaded with 100 mg bottom fraction each and were spun for 3.5 hr at 24,500 rpm in a gradient of 60-300 mg of sucrose/ml. The lower opalescent band was sucked out. From this material preparations of purified B were obtained by one cycle of density gradient centrifugation in a Spinco B IV zonal rotor as described previously (Bol, VanVloten-Doting, and Jaspars, 1971). Turnip yellow mosaic virus (TYMV) isolated from Chinese cabbage (Brass& chinensis L. var. Wong Bok) by the procedure of Dunn and Hitchborn (1965) was a gift from Mr. C. W. A. Pley. Cowpea mosaic virus (CPMV) isolated from Vigna unguiculata (L.) Walp. as described by Van Kammen (1967) was a gift from Ir. C. P. de Jager, Laboratory of Virology, Wageningen. Labeling of AA/IV nucleoprotein with SHuridine. To each tobacco plant (lo-15 cm high) 0.5 ml of a solution containing 0.33 mCi of [3H]uridine was administered by means of a small Pasteur pipet, which was inserted into the stem directly after inoculation (Van Ravenswaay Claasen, 1967). Extraction of RNA. AMV nucleoprotein was dissociated into protein and RNA by dropwise addition of an equal volume of

AND JASPARS

1 M MgClz (sometimes containing 5 % 2mercaptoethanol) to a virus suspension (25 mg/ml) under vigorous stirring (Moed, 1966). The mixture was centrifuged for 15 min at 1600g. The pellet, containing the RNA contaminated with about 5 % protein, was washed with 0.05 M MgC12 and resuspended in about one fourth of the original volume of PEN buffer which contained 10 times more EDTA than indicated. These RNA solutions were further deproteinized with phenol as described previously (Van Vloten-Doting and Jaspars, 1967) except that pyrophosphate was omitted. As judged by gel electrophoresis and infectivity, the quality of RNA prepared by this method was comparable to that of RNA extracted directly with phenol, while the yield was about 20 % higher. Tritiated AR/IV-RNA was prepared directly from bottom fraction with phenol. RNA from healthy tobacco plants was isolated in the following way: Leaves (X g) were homogenized with McX ml of a solution containing 0.1 M NaHZP04 and 10 % w/v Na4P207. 10HzO adjusted to pH 7.0 with HCl and X ml of phenol. The homogenate was centrifuged for 10 min at 10,OOOg. The water layer was stirred twice with phenol. To the final solution 2 vol of cold isopropanol were added. After 1 hr at -2O”, the solution was centrifuged for 10 min at 10,OOOg.The pellet was resuspended in PEN buffer and dialyzed overnight against the same buffer. The RNA solution was cleared by low-speed centrifugation. It is possible that these RNA preparations contain a few percent DNA, as no attempt was made to eliminate it. TYMV-RNA was prepared according to Stols and Veldstra (1965). Tobacco mosaic virus (TMV)-RNA was extracted as described by Gierer and Schramm (1956). CPMV RNA and phage MS2-RNA were donated by Miss J. M. H. Assink, Laboratory of Virology, Wageningen and by Mr. C. Vermeer, respectively. AMV protein. AMV protein was prepared as described by Kruseman et al. (1971). In some cases, the 2-mercaptoethanol was omitted. Sap from bean leaves. Sap from bean leaves

UNCOATING

OF AMV

was freshly prepared just prior to inoculation. Primary bean leaves were ground in a mortar and the homogenate was filtered through one layer of cheesecloth. The sap was diluted with PEK buffer as indicated in the legends. Chenzicnls. Poly (U) and transfer RNA were obtained from Schwartz and General Biochemicals, respectively. All chemicals used were analytical grade except CsCl which was suprapure from Merck. Suwose clenxity gradient centrifugation. Mixtures of RXA and nucleoprotein were centrifi;ged in an SW 27 rotor, equipped with either !arge (SW 27) or small (SW 27.1) buckets for 13 hr and 20 min at 21,000 rpm

BY ITS

OWS

RKA

7131

and 4”, unless stated otherwise. Density gradients of 150-350 mg sucrose/ml were used in both kinds of tubes. A!1 gradients contained PEN buffer, unless stated ot’herwise. From some gradients fractions were taken by collecting droplets from the bottom of the tubes. Fractions from these gradients were used alternat.ively for optical densit,y measurements or for infectivity assay as indicated in the figure legends. From mo.& gradients the transmission at 254 nm was measured continuously. A long narrowgauge needle was placed on the bottom of the tube and the contents were pumped through a flow cell (optical pat,h 3 mm) of

FIG. 1. Effect of increasing amounts of AMV-RNA on the properties of AWV nucleoprotein bottom fract.ion. (0) Optical density patterns of sucrose density gradients (SW 27) in which 0.5 mg AMV (a); 0.5 mg AMV + 0.1 mg AMV-RNA (B), 0.3 mg (C), 0.7 mg (D) and 1.5 mg A&W-RNA (E); and 1.5 mg AZ/IV-RSA alone (F) were centrifuged. (X) Infectivity of fractions after a IO-fold dilution (A and D), a Z-fold dilution (E) and undiluted (B and F). (0) Infectivity of fractions after incubation for 15 mia at 24” wit.h sap frxn bean leaves (final dilution of the latter 160 times). Final dilntions of the fractions as indicated above. The RNA used was a preparation containing B-RN-4, M-Rx-4 and Tb-RNA. Sedimentation from right to left.

702

VAN VLOTEN-DOTING

an LKB Uvicord. The transmission pattern was recorded by a Unicam AR 25 recorder. Infectivity assay, ultraviolet absorption and polyacrylamide gel electrophoresis. The methods described earlier were used (Van Vloten-Doting and Jaspars, 1967; Van Vloten-Doting, Kruseman, and Jaspars, 1968; Bol, Van Vloten-Doting, and Jaspars, 1971). For TYMV and CPMV A:;% was taken as 5.9 and 8.1 according to Kaper (1968) and Van Kammen (1967), respectively. Measurement of [3H]zcridine. Fractions of 15 droplets (about 0.4 ml) were collected in glass counting vials containing 10 ml scintillation solution (600 ml toluene, 300 ml Triton X100, 100 ml distilled water, 90 mg POPOP and 4.95 g PPO) and the radioactivity was determined in a liquid scintillation counter (Philips). Analytical ultracentrifugation (performed by Dr. J. F. Bol). Fractions from sucrose density gradients were dialyzed against 1O-4 M NaH2P04, lo+ M NaN3, and 1% formaldehyde (adjusted to pH 7.0). To 0.5 ml solution 0.31 g CsCl was added. Centrifugation was for 21 hr at 4” in a Spinco Model E analytical ultracentrifuge equipped with a photoelectric scanner. Electron microscopy. Fractions from sucrose density gradients were dialyzed against 10e4 M NaH2P04, lOA df NaN3, 1% formaldehyde (adjusted to pH 7.0), stained with 2% phosphotungstic acid (PTA) (pH 7.0), and observed in a Philips EM 200 electron microscope. RESULTS

Physicochemical Interactions Between AMV Nucleoprokin and A&TV-RNA Figure 1 shows the effect of increasing amounts of AMV-RNA on the properties of AMV nucleoprotein. (Bottom fraction was used in this experiment.) It is evident that small amounts of AMV-RNA can alter the sedimentation behavior of AMV nucleoprotein, even when they have been in contact for only a short time. When the amount of AMV-RNA increases the sedimentation velocity of the virus-derived material decreases. Patterns of optical density flatten gradually. Furthermore, the infectivity be-

AND JASPARS

100 1

0

10 FRACTION

j-0

20 NUMBER

FIG. 2. Infectivity of RNA purified from an RNA-virus mixture by sucrose density gradient centrifugation and rerun. Transmission patterns of sucrose density gradients (SW 27.1) run with 0.07 mg AMV-RNA (upper curve) or with 0.07 mg RNA obtained from a gradient run with a mixture of 0.4 mg AMV (bottom fraction) and 0.28 mg AMV-RNA. The RNA used was a preparation containing B-RNA, M-RNA, and TbRNA. Lesion numbers are totals from 5 haIf leaves. (-) Transmission; (---) infectivity. Sedimentation from right to left.

comes sensitive to incubation with diluted sap from bean leaves (Fig. lD, E), while the original material (Fig. IA) is about 90 % resistant to the RNase action of the very dilute sap. At the same time, the infectivity in the RNA peak increases. The infectivity of the peak fraction from Fig. lB, is seven times higher than that of the peak fraction from Fig. IF, although it contains only 5 % as much RNA as this fraction. The effects described above were also found when instead of bottom fraction, purified bottom component (B) was mixed with AMV-RNA. To exclude the possibility that the infectivity in the RNA peak was due to the tail-

UNCOATING

OF SMV

ing of the faster sedimenting nucleoprotein, all fractions of the RXA peak from a gradient comparable to that of Fig. 1C were pooled and part of the solution was diluted to 10% sucrose with PEN buffer and rerun. The infectivity was located at the heavy side of the RNA peak (Fig. 2) as is found after one cycle (Fig. ID). No infectivity was found at the position of nucleoprotein. The increase in infectivity in the RNA region

BY ITS

OWX

RNA

703

suggests t,he presence of activating prot4n subunits. AMV-RNA was incubated iw 24 hr with AMV nucleoprotein and then separated from the latter by sucrose density gradient centrifugation. This RNA no innger had the capacity to alter the properties o:i AMV nucleoprotein. The results shown were obtained with material of AMV strain 425. C~rn~~r~~~e results were foUnd when material from strain

FRACTION

3. ‘Time dependence of the effect of AMV-RKA

NUMBER

on A%tV nucleoprotein. Transmission pstterm of sucrose density gradients (SW 27) in which 0.3 mg A&IV (A); a mixture of 0.3 mg AL&IV and 0.42 mg AkIV-RT’U’A after 50 min (B), 3 hr 18 min (C), and 29 hr (D), incubation at 4”; and 0.42 mg AMV-Z),K.P. (E) were centrifuged. Bottom component (B) was used as AMV. The R.NA used wss a prepsrnt~iancontaining B-RNA, M-RKA, and Tb-RNA. Sedimentation from right to left. FIG.

704

VAN

VLOTEN-DOTING

YSMV was used, or when nucleoprotein from one strain was mixed with RNA from the other. When, instead of a mixture of B-RNA, M-RNA, and Tb-RNA, purified Ta-RNA was added to AMV nucleoprotein similar effects appeared. Substitution of PEN buffer in the gradient by 0.05 M sodium acetate (pH 6.0) did not change the results. However, 0.01 M t,ris(hydroxymethyl)aminomethane (Tris), 0.06 M NH&l, adjusted to pH 7.8 with HCl, decreased somewhat the shift in the sedimentation patterns. From the gradient shown in Fig. 3 it becomes evident that the effect of AMV-RNA on AMV nucleoprotein is a time-dependent process. There is an initial very quick change followed by a slower process. Finally, all material sediments like RNA. The latter process isnot due to action of RNases upon the virus-derived nucleoprotein because fractions from the upper part of gradient 3D were highly infectious (not shown in the figure). Nature of Virus-Derived

Nucleoprotein

Why should virus particles sediment slower after having been in contact with RNA than before? Results obtained by polyacrylamide gel electrophoresis exclude the possibility that this is due to fragmentation of the RNA of the particles.

AND

JASPARS

FIG. 5. Cesium chloride equilibrium density gradients containing 5 pg control virus (A) and 5 pg virus-derived nucleoprotein to which 5 fig control virus was added as a marker (B). Virus and virus-derived nucleoprotein were obtained from gradients comparable to Fig. 1A and D, respectively. Meniscus is at the left.

Furthermore, the possibility that the RNA and the virus formed a slower sedimenting complex was excluded because upon addition of [3H]AMV-RNA to AMV particles no significant label was found in the position of the virus-derived nucleoprotein (Fig. 4). The most likely explanation is that the sedimentation velocity decreases as a result of a change in conformation triggered by the loss of protein. This is in accordance with the high sensitivity to RNases and the presence of protein subunits in the RNA peak (compare Fig. 1). This is confirmed by the increase in buoyant density (Fig. 5) and the less-defined appearance on electronmicrographs (Fig. 6). E$ect of other RNAs on AMV

10

20 FRACTION

30

LO

NUMBER

FIG. 4. Distribution of 3H Iabe in gradients (SW 27.1) run with a mixture of AMV nucleoprotein (0.1 mg bottom fraction) and [3H]AMVRNA. [3H]AMV-RNA (0.42 mg) was extracted from bottom fraction. Untreated bottom fraction was run in a sister tube (upper curve). Sedimentation from right to left.

Nucleoprotein

Experiments were performed to determine the specificity of the RNA-virus interaction. Figure 7 demonstrates the effect of tobacco RNA on AMV. The addition of a high concentration of tobacco RNA has no effect on the sedimentation velocity. The peak, however, is somewhat lower, probably due to aggregation of virus particles with RNA. The virus remains resistant to incubation with sap from bean leaves. In the lighter half of the gradient some infectivity is found when a mixture of B-RNA, M-RNA, and Tb-RNA is added to these fractions suggesting the presence of a few protein subunits. Similar patterns were found when one of the following RNAs was added to AMV nucleoprotein: TYMV-RNA, TMVRNA, CPMV-RNA, MS2-RNA, a mixture

FiiACllCN

FIG. 6. Electron micrographs of virus (A) and virus-derived mat.erial (B), obtained from sucrose density gradients run with 0.5 mg bottom component and with 3 mixture of 0.5 mg bottom component and 0.5 mg RNA, incubated for 24 hr at 4”. Scale 100 nm.

kJMBER

FIG. 7. Effect of BXA from healthy tobacco plank on AMV nucleoprotein. Optical density patterns of gradients (SW 27) run with 0.3 mg AMV (-4); a mixture ol” 0.3 mg AMV and ;).I8 (B) and 1.14 (6) mg tobacco RXA. Bottom component was used for AMV nucleoprotein. (X) Infectivity measured after a lo-fold di8utio:l in the presence of 20 pg/ml $1 and 20 &ml Tb. (u) Infectivity after incubat.ion with sap from bean leaves (final dilution 200 times). Final concentration o?’ B, XI, and 7% as above. (a) Infee.. tivit,y after addiCon of a preparation containing B-RNA, M-E.NA, and Tb-RNA (25 &ml:. Se&mentation from right to ?eft

706

VAN VLOTEN-DOTING

of tRNAs or poly (U). The integrity of all RNAs was checked by polyacrylamide gel electrophoresis. Thus, we conclude that the effect of AR/IVRNA on AMV nucleoprotein is highly specific.

,000 w!3 AND JASPARS

0

0

O+

0

0

oo”

E$ect of RNAs on Other Viruses To investigate whether other viruses are also degraded by their own RNA or by AMV-RNA the following combinations were assayed: TYMV/TYMV-RNA; TYMV/ CPMV/ CPMV-RNA and AMV-RNA; CPMV/AMV-RNA. None of these nucleoproteins was converted to slower sedimenting material. In addition CPMV remained resistant to incubation with sap from bean leaves. DISCUSSION

Addition of AMV-RNA to AMV particles causes an extensive structural disintegration of the latter. All results described above seem best fitted by the model represented in Fig. 8. An early stage of the interaction seems to involve the movement of a few protein molecules from the particles (I) to the RNA (II), causing drastic structural changes in the particles as judged by their sedimentation behavior. This virus-derived nucleoprotein (III) still has about the same RNA : protein ratio, but is more sensitive to RNases, and its structure seems partly perturbed as seen on electron micrographs. The sedimentation velocity of the RNA molecules which have accepted protein subunits (IV) is scarcely changed. The protein present in the RNA peak could not be measured by the Folin method. However, when a mixture of B-RNA, M-RNA, and Tb-RNA was used for the experiments, the increase of infectivity clearly showed the presence of protein. When the virus is left for a longer time in contact with the RNA, or when a large excess of RNA is added, the peak of virus-derived nucleoprotein becomes flattened and shifts into the direction of the RNA peak, until finally both peaks merge. These further stages of III clearly differ from virus in buoyant density and in appearance on electron micrographs.

P FIG. 8. Proposed model of successive stages of interaction between AMV particles and AMVRNA. (-) RNA; (0) protein subunit; (I) high affinity site.

It is uncertain if in the final stage the protein molecules are divided statistically among the RNA molecules present (V). Experiments with a series of RNAs from different origin showed that AMV is sensitive only to its own RNA. The four AMV-RNAs may differ in their effectiveness. In view of the specificity of the reaction we postulate that AMV-RNA has a few sites with a strong affinity for AXIV coat protein. In the competition for coat protein between free RNA and RNA in the virion, those protein molecules occupying less favorable positions in the virion can move to the free RNA molecules, thereby neglecting their structural role. As a consequence, the virus adopts a conformation (III) comparable to that of the RNA protein complex (IV). Equi-

UNCOATING

OF &XV

librium would be reached when the protein molecules are distributed statistically over the RNA molecules. The fact that addition of RNA which had been preincubated with virus had no effect on AMV particles, is in accordance with this hypothesis. In this casethe high affinity sites on the RNA molecules are already associated with protein subunit8s. The postulated high-affinity sites have probably to do with secondary or tertiary structure of the RNA, since its capacity to destroy the structure of BMV is very quickly lost upon incubation with. dilute solutions of R,Nase (J. A. M. Van Boxsel, personal communication). The high-affinity sites may be comparable to t,he specific sites in the RNA of small bacteriophages, which are responsible for the formation of the so-called complex I at low protein:RNA ratios. Complex I has a negative translational control function (review article by Hohn and Hahn, 1970). The highaffinity sit,es in AMV-RNA might be connected with the positive biological role of the coat protein (Bol, Van Vloten-Doting, and Jatpars, 1971). ‘The sites in the RNA, of simple R.NA viruses, that are involved in particle structure have less afEnity, and are less specific for their own coat protein, as is evident from the existence of protein RNA hybrid particles [reviewed by Hohn and Hohn (1970) for simple RNA bacteriophages, acd by ancroft (1970) for spherical plant viruses]. According to Kaper and Geelen (1971) simple RNA viruses may be arranged in a series according to the degree to which their stability is derived from, primarily proteinprotein interactions to predominantly protein-RNA interactions. AMV is similar to cucumber mosaic virus (CMV) with regard to a number of criteria: sensitivity to RNase (Bol and Veldstra, 1969), to alkaline pH $01, 1969) and to high ionic strength (Bol and Bruseman, 1989). Both viruses lack nat,urally occurring empty capsids. Kaper and Geelen (1971) placed CMV at that end of the range where t.he protein-RNA interactions predominate. Possibly the nonicosahe&al AMV has even less stable prot’einprot,ein interactions than CXV in view of the

BY

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OWN

y\Qy

RNA

fact that free RNA can compete with t,he virion RNA for t,he coat protein. ACKNOWLEDGMENTS We are indebted to all members of the plar;t virus unit of our laboratory for their contribut,ions to the experimental part of the work as well as for their stimulating discussions, Thanks are due co Dr. Enid Newell for reading the manuscript. This work was supported, in part, by the Netherlands Organization of Pure Research (Z.W.O.), REFEREXCES

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L.,

and

.laS-

equiva\e;iee of top component ci R.NA and coat, protein in the initiatiorl of infection by alfalfa mosaic virus. yirologv 46, 73-85. CL~RX, NI. F. (1968). Purification and frac:ionation of aIfnlf a mosaic virus with polyetby!e!?e glycol. J. Gen. Viral. 3,427~432. DUNN, D. B., and MITCEIRORX, 5. pi. !.l%Sj. The use of bentonite in the purificabion of plant viruses. Virology 25, 171-192. GIERER, A., and SCHUMU, G. (1956j. Die in:‘&tiositgt der NukleinsHure aus Tabaksmosaik. virus. 2. Nuturjor~orsch. 11, 138-142. HULL, R., REES,M.W., and &XQRT,?il. 1. (1969). Studies on alfalfa mosaic virus. I. The prct<+rrl and nncleic acid. Trti,ology 37, 404-4%. HORN, T., and Hoax, B. (1970). Strucr:!re nud assembly of simple RNA bact,eriophages. fi/~ “Advances in Virus Research” (M. %. Smi:;h and M. A. Lauffer, eds.), Vol. 16, pp, -h3-%’ Academic Press; New Pork. &PER, J. X. (1968). The small RNA virwes oc animals and bacteria. A. Physlcai plants, properties. In i’Molecular Basis of ITirology” (I%. Frael:keI-Conrat, ed.), pp. 60-61 Reinb~>id, New York.

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and properof cowpea mosaic virus.

VAN RAVENSWA.~Y CLAASEN, J. C. (1967). Synthesis of a plant cell-free system Univ. of Leiden.

viral specific of Escherichia

protein coli.

in the Thesis,

VAN VLOTEN-DOTING, L., and J~SPARS, E.M.

J. (1967). Enhancement of infectivity by combination of two ribonucleic acid components from alfalfa mosaic virus. Virology 33, 684-693. VAN VLOTEN-DOTING, L., KRUSEMAN, J., and JASPARS, E. M. J. (1968). The biological function and mutual dependence of bottom component and top component a of alfalfa mosaic virus. Virology 34, 728-737.