Characterization of the RNA Mycovirus Infecting Allomyces arbuscula

J. gen. Virol. (I977), 35, 415-424 415 Printed in Great Britain Characterization of the R N A Mycovirus Infecting Allomyces arbuscula By E. W. K H A N D J I A N AND G. T U R I A N Laboratoire de Microbiologie, Ddpartement de Biologie vdgdtale AND H. E I S E N Ddpartement de Biologie moldculaire, University of Geneva, I2I I Geneva 4, Switzerland (Accepted 4 January I977) SUMMARY The mycovirus infecting Allomyces arbuscula has been fractionated into two particle classes designated A and B. Particles A have a buoyant density of I "385 g/cm3, in CsC1, a sedimentation coefficient of 75S and contain one major and two minor polypeptides with molecular weights of 34ooo, 31o00 and 28ooo, respectively. Particles B band at 1.335 g/cm3, sediment with a value of 67S and, in addition to the three polypeptides as in particles A, they contain seven extra polypeptides, six of which are of lower molecular weights. Both particles A and B contain doublestranded RNA which was resolved by electrophoresis on polyacrylamide gels into three components with molecular weights of I"4 (major), 2"5 and I.I × Io 6 (minor). Both particles also contain single-stranded RNA. Particles A contain 27 ~ RNA whereas particles B only contain I4 ~. Evidence is presented that particles A and B belong to the same type of virus and represent two different replicative stages. INTRODUCTION The few fungal viruses (mycoviruses) so far studied in detail are characterized by their small size and morphology, double-stranded RNA, heterogeneous densities in caesium chloride, and especially their lack of pathogenic effects on their host (Hollings & Stone, I97I; Wood, I973; Lemke & Nash, I974). We have previously reported the existence of virus particles in the fungus Allomyces arbuscula (Khandjian et al. 2974). Following a preliminary communication on the general properties of these particles (Khandjian, Roos & Turian, t975), we describe here the purification and characterization of the two classes of particles which were found to contain nucleic acids. They appear to fit the characteristics of two different replicative stages of the same mycovirus. METHODS Buffers. Buffer A: 0"05 M-tris-HCl, pH 7"5 + 5 mM-MgC12+ t mM-EDTA + I mM-dithiothreitol+5 ~ (v/v) glycerol; buffer B: o.o5M-tris-HCl, pH 7"5+5mM-MgC12+I mMEDTA; buffer C: o.oI M-tris-HC1, pH 7"2+ I mM-EDTA; SSC: o'I5 M-NaCI+o'oI5 Msodium citrate, ([Na +] = I95mM), pH 7"0 (Mandel & Marmur, I968); TAE: o'o4 M-tris-HC1, pH 7"6+o'o2 M-sodium acetate +2 mM-EDTA; NaP buffer: stock solution of 4"8 M-sodium phosphate, pH 6.8, consisting of equimolar NaH2PO4 and Na2HPO4 (o.I2 MNaP contains [Na+] = I8o mM). 416 E.W. KHANDJIAN, G. TURIAN AND H. EISEN Virus strains. Strain Bali I (Noack Ioi "33) and strain North Carolina 2 (Hatch Io4"36) o f Allomyces arbuscula were originally obtained from the Centraalbureau voor Schimmelcultures, Baarn, Holland. Preparation of virus particles. Cultures were grown for 62 h in G C Y liquid medium as described by Turian (~963). GCY medium contained (g/l): glucose, 5.o; casein hydrolysate, 3.0; yeast extract, o.I; NaC1, o.I; CaC12.2H2o, o.I; FeC13.6H20, o-o2; K2HPO4, I.o; MgSO~. 7HzO, 0.2. Mycelia were extracted in ten times their weight of buffer A by grinding with quartz sand. The homogenates were then filtered through cheese cloth (mesh, 45 #m) and clarified with the same volume of carbon tetrachloride (Rawlinson et aL I973). The clarified fluids were centrifuged at I2OOOg for 2o min and particles in the supernatants sedimented at I o 5 o o o g for Izo min. The pellets were then resuspended in I/2O of the initial volume of buffer A. This homogenate was again centrifuged for another 20 min at ~2ooog and the unsedimented material was considered the crude preparation. Crude extracts were layered on to 2o ml linear 5 to 35 ~o (w/v) sucrose gradients in buffer A and centrifuged for 90 rain at 8oo0o g. Gradients were pumped through a u.v. analyser (260 rim) connected to a fraction collector. Fractions containing particles revealed by u.v. spectra and electron microscopy were pooled and dialysed against several changes of buffer B. Fractionation of the particles. Preparations obtained from sucrose density gradients were layered on pre-formed gradients of CsC1 0 5 ml, I'27o to I-5oo g/cm 3 of CsC1 in buffer B) and centrifuged at I 3 o o o o g for I8 h. Particles banding between 1.28o and 1.42o g/cm 3 were then subjected to equilibrium density centrifugation by adjusting the initial density to I'4oo g/cm 3 with CsC1. Centrifugations were carried out for 2o to 24 h at I6OOOOg at 4 °C. Gradients were fractionated as described above and the density of fractions was calculated from the CsC1 concentration determined from the refractive index measured at 25 °C. The different classes of particles obtained from the CsCI gradients were dialysed against buffer A and subjected to sedimentation analysis on 5 to 35 ~ sucrose gradients. Ribosomes (8oS) and TMV (I83S) were used as markers. Labelled cultures. 32P-labelled cultures of strain Bali I were prepared in a phosphatefree medium according to Ojha & Turian 0 9 7 0 . Strain North Carolina was labelled with o.4 mCi of 14C-leucine per litre of modified GCY liquid medium containing one quarter of the original amount of casamino acids. Extraction of the RNA. Purified particles were dialysed against o.o48 M-NaP buffer containing 5 mM-MgCI~ and i mM-EDTA. The RNA was extracted according to a modified procedure of Britten, Pavich & Smith (I 969). The particles were lysed by addition of urea and SDS to final concentrations of 4 M and I ~/o (W/V) respectively and the lysates were applied to columns (o.6x6.o cm) of hydroxylapatite equilibrated with o'o48 M-NaP buffer. The columns were washed thoroughly, first with o'048 M-NaP buffer containing 4 M-urea and I ~ SDS and then with o.o48 M-NaP buffer. The nucleic acid was eluted in one step with o'48 M-NaP buffer. Following elution, the R N A was exhaustively dialysed against several changes of buffer C, precipitated with 2"5 volumes of ethanol and redissolved in the same buffer. All glassware and buffers were autoclaved before use. Equilibrium density eentrifugation of the RNA. Columns of 4 ml with an initial density of I-6o g/cm 3 of caesium sulphate (suprapur, Merck) in buffer C and containing approx. 7000 ct/min of 3ZP-labelled virus RNA (0"02 #g RNA) were centrifuged for 62 h at I6OOOOg at 15 °C. Fractions were collected from the bottom of the tubes and acid-insoluble radioactivity [5 ~ (w/v) TCA] was counted on filters. Densities were determined with a refractometer according to Szybalski 0968). Ribonuclease resistance of the RNA. Lability of the labelled R N A was tested against Mycovirus o f A. arbuscula 4~7 bovine pancreatic RNase (o.2#g/ml), DNase 0 o o #g/ml) and alkali. RNase digestion (o-2 #g/ml of bovine pancreatic RNase) was performed in different concentrations of SSC for 3o min at 37 °C. Chromatography on hydroxylapatite columns, a2P-labelled RNA was loaded on a I x 6 cm hydroxylapatite column equilibrated with o'o48 M-NaP buffer. A molarity gradient of o'o48 to o'48 M-NaP buffer (50 ml of each) was applied to the column and 80 fractions of 2o drops each were collected (Pinck, Hirth & Bernardi, ~968). TCA (5 %) insoluble iadioactivity was then counted on filters. Thermal denaturation of the RNA. Hyperchromicity of the RNA preparations was determined using a Gilford Tm Analyser. The R N A eluted from hydroxylapatite columns at 0"22 M-NaP buffer was adjusted by dilution to o-I2 M-NaP. Thermal denaturation of 3~P-labelled R N A was determined by RNase digestion. Samples of R N A containing 3ooo ct/min (o-oI #g) in SSC were heated for 5 rain in sealed tubes at the indicated temperatures, rapidly cooled and digested with 0-2/~g/ml bovine pancreatic RNase for ~o min at 37 °C (Pinck et al. I968 ). Acid-insoluble radioactivity was then counted on filters. Sedimentation coefficient of the RNA. Determinations were made by layering the R N A on to 4 ml columns of 5 to 20 % sucrose in buffer C containing o- 1% SDS and centrifuging for 20 h at 25IOOO g at I5 °C. Fractions were collected from the bottom of the tubes and 5 % TCA-insoluble radioactivity was counted on filters. Sedimentation coefficients were calculated using 4S, 26S and 23S E. coli RNA as markers. Extraction of total double-stranded RNA from the mycelium. Mycelia were ground with sand quartz in o.I M-sodium acetate buffer, pH 5"2, containing 2oo #g/ml polyvinyl sulphate and 2 % SDS. Following pronase digestion (I mg/ml, 2 h at 63°C), the R N A was extracted at 68 °C by extraction buffer-saturated phenol. The R N A was then precipitated with ethanol, resuspended in I x SSC and made to 2 M-LiC1 to precipitate ribosomal R N A (Barlow et al. I963). The RNA in solution was then treated with 0.2/~g/ml of RNase A for 2o min at 37 °C before analysis by gel electrophoresis. RNA gel electrophoresis. Analysis by gel electrophoresis was done in 2.8 % acrylamide0"24 % methylene bis-acrylamide-o.5 % agarose composite gels as described by Peacock & Dingman (I968) in TAE buffer. Electrophoresis was carried out for 4 or 5 h at 6 mA/gel. Double-stranded reovirus RNAs were used as standards. Gels were stained with Toluidine blue and scanned at 630 nm using a Gilford spectrophotometer with a gel scanning attachment. Protein gel electrophoresis. SDS-gels containing I2-5 % acrylamide and 0"33 % methylene bis-acrylamide were prepared as described by Laemmli 0970). The gels were stained with Coomassie brilliant blue and scanned at 55o nm. Molecular weights were calculated using bovine serum albumin (68 ooo), aldolase (40 ooo), lactate dehydrogenase (36 ooo) and trypsin (23 300) as standards. Nucleoprotein determinations. U.v. absorption analyses and spectra were determined with a Beckman DBG or a Gilford 240o spectrophotometer. Proteins were determined by the method of Lowry et al. (295I). R N A and D N A were measured by the orcinol and diphenylamine methods of Dische 0955)RESULTS Purification of the particles The particles extracted from strain Bali I and partially purified by differential centrifugation were subjected to velocity sedimentation on 5 to 35 % sucrose gradients. Two close-spaced bands were present after centrifuging. These two bands were always absent in 28 VIR 35 418 E . W . KHANDJIAN~ G. T U R I A N AND H. EISEN CsC1 density (g/cm3) 1-290 1-335 1.385 I I I 1 "40 1 "36 "r~ g~ ~ ~ 1.32 Strain ~ ~O 1.28 09 "~ I t~ I I ~ 101.33 Frozen e- 0.5 Q 0.3 i ~ I04.36 ! 0-7 i 0.1 10 Fig. I 20 30 40 Fraction number O e-, 7000 ~ ~, "~ O 5000 ~o &~ m.__. 3000 ~ 1000 < b50 Fig. 2 Fig. I. Isopycnic banding of the mycoviruses after centrifugation in CsCI density gradients. Strain Ioi.33 is Bali I which contains the particles; strain IO4.36 is North Carolina which is free of virus particles. Fig. z. Equilibrium density centrifugation on CsCI of particles extracted from strain Bali I and raised with an extract of strain North Carolina labelled with l~C-leucine. Initial density of CsC1 = I'4O g/cm3. Fractions were read at 26o nm (1~ ~,) before TCA precipitation and radioactivity counting ( © - - O ) . • I!, CsCI density. sister gradients loaded with extracts from strain North Carolina, a strain in which no viruslike particles were detected (Roos, Khandjian & Turian, ~976). As the two bands could not be resolved enough to be studied separately, the fractions containing isometric particles of 4o nm (checked by electron microscopic examination and their u.v. spectra) were further purified on CsC1 equilibrium density gradients. When centrifuged to equilibrium on pre-formed CsC1 gradients, two major peaks were clearly resolved having densities of I'385 and I'335 g/cm 3 (bands A and B respectively). With some preparations, an additional minor peak could also be observed at the level of 1.4I to 1.42 g/cm z, but as it was not always present, this peak was not further studied. Extracts from strain North Carolina did not show any of these peaks, while two minor peaks at approximately 1.32 and I-29 g/cm 3 were present (Fig. I). In general, mycoviruses show heterogeneous behaviour with respect to their buoyant densities in CsC1. This is thought to be due to the fact that more than one class of particles can infect the same host (e.g. Van Franck, Ellis & Kleinschmidt, ~97I ; Buck & Kempson-Jones, I973; Buck & Ratti, I975; Rawlinson, Carpenter & Muthyalu, I975). On the other hand, crude preparations of the particles were highly contaminated with the host materials as seen under the electron microscope. To ensure that the peaks detected on the CsC1 gradients represented the particles extracted from strain Bali t and were not contaminating host materials, the following experiment was performed. Mycelium from strain Bali I was mixed with 14C-leucine-labelled mycelium from strain North Carolina and the combined mycelia Myeovirus q/A. arbuscula 1 I I 1 I I I I 419 I 1 '80 1 "70 1"60 "~ ,g 1.5o 700 1.40 d ~g < z 100 Contro II 500 50 300 100 0 0.01 i 1 012 0i5 ii0 2i0 ][SSC] [ 20 30 40 19.5 39 97.5 195 390 [Na +] 1-95 Fraction number Fig. 4 Fig. 3 Fig. 3. Equilibrium density centrifugation of the virus RNA in Cs~SO4 gradient. Initial density = I-6O g/cm ~. Centrifugation was carried out at ] 6oooo g for 62 h at 15 °C. • •, s~p radioactivity; O ©, Cs~SO4 density. Fig. 4. Semi-logarithmic curve of the effect of sodium ion concentrations on RNase efficiency on purified RNA. Digestion was carried out at 37 °C for 3o min in the presence of o.z/zg/ml of pancreatic RNase. The values plotted are recorded as the percentage of the original radioactivity that remains acid-insoluble. 10 were extracted. The crude preparation obtained after differential centrifugations was then examined on CsC1 gradients in the same way as described above. After fractionation of the gradients, the extinction of each fraction was measured at 26o nm, after which T C A was added to a final concentration of 5 ~ and acid-insoluble radioactivity counted. The results are shown in Fig. 2. The acid-insoluble 14C-labelled proteins from the virus-free strain were found at the top of the gradient, while the two peaks at r-38 and 1-33 g/cm3 absorbing at 260 nm were found to be free of any 14C-labelled acid-insoluble products. We therefore conclude that the virus peaks are not appreciably contaminated by host materials. The two bands A and B obtained from the CsCI equilibrium density gradients were found to have the following u.v. absorption values: maximum at 26o nm and minimum at z4o nm and a 26o/28o ratio of r.68 for band A, while band B had a maximum at 260 nm and a minimum at 245 nm with a z6o/28o ratio of 1.47. Based on results of orcinol tests for R N A and Lowry tests for proteins, we estimate peaks A and B to contain 27 and 14 ~o RNA, respectively. Sedimentation analysis by sucrose density gradient centrifugations gave the following values: 75S for particles A and 67S for particles B. Characterization of the RNA The R N A extracted from the particles in bands A (or B) had u.v. spectra typical of nucleic acids with extinction maxima at 258 nm and minima at 232 nm. The ratios 26o/28o nm and z6o/23o nm were r.96 and 2.Io, respectively. The R N A nature of this nucleic acid was confirmed by its positive reaction with orcinol, negative reaction with diphenylamine, alkaline lability and resistance to DNase. 28-2 420 E. W . K H A N D J I A N , G. T U R I A N AND H, EISEN 1 " / 0.48 30 ~ 400 / -" " ~ 0"40 I I I I I _ 27 / / 0"30 ,~ / / .EE 300 0"20 / / / .N 20 0.10 0.05 f -~ 200 cq -/ .o © 10 "T < L) ~- 100 ee 0 20 40 60 Fraction number Fig. 5 80 , 50 60 70 80 90 Temperature (°C) [ -- 100 Fig. 6 Fig. 5. Hydroxylapatite chromatography o f total R N A extracted from particles A purified by CsC1 density gradients. A molarity gradient (o'o48 to o'48 M-NaP buffer, - - - ) was applied at fraction o. • • , TCA-insoluble 32p radioactivity. Fig. 6. Thermal denaturation curve o f the o.22 M fraction eluted from the hydroxylapatite column, and adjusted to o.I2 M of N a P buffer. As mycoviruses commonly contain double-stranded RNA, we have examined the nature of the R N A extracted from our purified particles. (a) Double-stranded R N A has been reported to have an intermediate buoyant density in caesium sulphate, between D N A and single-stranded R N A (Szybalski, I968 ). In our case, the R N A extracted from the purified particles had a buoyant density o f 1.57 g/cm3, which is a first indication of the doublestranded nature of the RNA. A minor shoulder was also observed at the level of ~-69 g/cm a indicating the presence o f single-stranded RNA (Fig. 3). (b) The best criteria to demonstrate the presence of double-stranded R N A is its sensitivity to pancreatic RNase at different ionic strengths. At high ionic strength, double-stranded R N A is stable and at low ionic strength the R N A is digested. We therefore tested the sensitivity of the a2P-labelled R N A in the presence of concentrations of SSC ranging from z x to o.oi x . The results of these experiments are shown in Fig. 4. The RNA remained undigested even alter 4 h incubation at 37 °C in the presence ofo.2 #g/ml of bovine pancreatic RNase in a high ionic environment (~ x SSC and above). In contrast, when this treatment was carried out in o. I x SSC, digestion was complete in 3o min. The resistance of the material to degradation as a function of the ionic environment is a convincing criterion of the doublestranded nature. (c) According to Pinck et al. 0968), double-stranded RNA is eluted from hydroxylapatite columns at a mean molarity of o.22 M buffer, after single-stranded R N A (o-I 4 M) and before D N A (above 0-25 M). The virus RNA studied here was chromatographed on hydroxylapatite column and was eluted as a main peak in a narrow range o f molarities between o.2I M and o'23 M-NaP buffer (Fig. 5). This confirms the double-stranded character of the RNA. A discrete shoulder was also located at the molarity where single-stranded R N A is eluted (o'I4 M). (d) The increase in extinction (at 26o nm) over a small temperature range when the samples are gradually heated is characteristic of double-stranded nucleic acids. Fig. 6 shows Mycovirus of A. arbuscula I I I I I I I I I 421 I - < z I l 16S I I I I [ [ 4S ~- 2 0 0 100 ,.o -5 50 .,.;, lOO < [.. m I 40 I I I I 60 80 100 Temperature (°C) Fig. 7 l0 2O 3O 40 Fraction number Fig. 8 Fig. 7. RNase resistance of the virus R N A which was dissolved in I × SSC and treated with 0"2 #g/ml of bovine pancreatic RNase for Io rain at 37 °C. Before addition of the RNase, the samples were heated at the indicated temperatures, cooled and then digested. The values plotted are recorded as as the percentage of the original radioactivity that remains acid insoluble. Fig. 8. Sedimentation of total virus R N A in sucrose density gradient (5 to 2o ~ in buffer C). The radioactivity of TCA-insoluble R N A was counted on filters. Markers were 4S and t6S E. coli RNA. the behaviour of the 0.22 M-NaP fraction eluted from a hydroxylapatite column. The sample was adjusted to o. I2 M-NaP and subjected to thermal denaturation as described in Methods. The thermal transition mid-point was found to be 90 °C with a calculated hyperchromicity of 27 ~ . We also measured the susceptibility of the 32P-labelled virus R N A after heat denaturation. The labelled R N A was dissolved in I × S S C , heated for 5 min at the different temperatures indicated and rapidly cooled before adding the RNase. Heating to temperatures above 9 o °C rendered the RNA susceptible to the RNase digestion (Fig. 7). The heterogeneity of the total RNA, which was detected on caesium sulphate gradients and hydroxylapatite columns, was examined on sucrose gradients (Fig. 8). A main peak at the I2"5S level and two other minor peaks in the regions of I6S and 4S could be detected [compared to the I8S and 25S found for ribosomal R N A in Allomyces sp. (F/ihnrich, Rothe & Trapp, I975)]. After RNase treatment in I × S S C , the main peak at I2"5S was not affected but the other two peaks disappeared. RNA gel electrophoresis Molecular weight estimates were based on comparisons of the migration of the virus double-stranded R N A with that of reovirus double-stranded RNAs. Three species of RNAs were observed in preparations from both viruses A and B with approximate molecular weights of I"4 (main band) 2"5 and I.I × Io ~ (minor bands). For further comparison, the total double-stranded R N A extracted from the mycelium also showed the same three species (Fig. 9). Virus capsidpolypeptides Gel scans of the polypeptides from purified particles (populations A and B) are shown in Fig. lo. Virus particles A gave one major band with a molecular weight of 34ooo and two minor bands o f molecular weight 3 ~ooo and 28 ooo. Virus particles B also showed the same 422 E.W. KHANDJIAN, G. T U R I A N A N D H. E I S E N (a) ~2 "-d I 6 I I I I I I 5 4 3 2 1 Distance in gel (cm) Fig. 9 I I 23.3 36 40 Mol. wt. x 10 -3 Fig. IO I 68 Fig. 9. Agarose-acrylamide composite gel electrophoresis profile of the virus double-stranded RNA. Gels were scanned at 63o nm after staining with Toluidine blue. Fig. Io. SDS-polyacrylamide gel electrophoresis of virus capsid polypeptides. (a) Proteins from particles 1.385 g/cm 3. (b) Proteins from particles 1.335 g/cm ~. The gels were scanned at 55o nm after staining with Coomassie brilliant blue. three bands, but 7 other bands were found in addition. One of the extra bands had a molecular weight of 38 ooo while the other six had lower molecular weights than the three peaks from virus A. DISCUSSION Like the other mycoviruses, the particles extracted from Allomyces show heterogeneous densities when centrifuged to equilibrium in CsCI. In the case of the viruses infecting A. arbuscula, these particles can be separated, by equilibrium density gradient centrifugation, into two main virus-containing bands. We discount the possible presence of two distinct different types of viruses because: (I) we always found the same three species of double-stranded RNAs in both virus particles A and B. We also found these RNA species when total RNA was extracted from the mycelium and treated to remove ribosomal and single-stranded RNA. The molar ratio o f the R N A species appears to be about ] :8:8 for the species with molecular weights 2"5, 1.4 and 1-I x IO s, respectively. This makes it unlikely that all R N A species can be included in a single particle. On the other hand, separate encapsidation of each RNA species would be expected to give rise to particles with different densities, which does not occur. One possible explanation is that some A particles contain the RNA species of molecular weight 2"5 x Io 6, while others, in greater amounts, contain both species of molecular weights I-4 and I.[ x ~on; both types of A particle would then have the same density as detected in CsC1. An analogous situation is found with brome mosaic virus (Lane & Kaesberg, 197I). (2) Both virus particles Mycovirus o f A. arbuscula 423 contain the three polypeptides with molecular weights of 34ooo, 31 ooo and 2800o. (3) When using a frozen mycelium, the two bands obtained on CsC1 gradients were still present but there was less material in band A, while band B was enriched (Fig. I). This shift to a lower density indicates that virus particles A are susceptible to disruption by physical effects as in the case of Penicillium chrysogenum mycovirus (Nash et al. I973). (4) A detailed ultrastructure study of the infected strain o f A. arbuscula showed that only one type of particle having a diameter of 40 nm was detected. These particles were heterogeneous with respect to their content; full and coreless particles were randomly seen on serial thin sections (Roos et al. 1976). The difference in densities of the two particles might then be explained by the fact that particles B contain short extra polypeptides which could be the capsid protein precursors. In addition, these latter particles contain relatively less RNA; we might suggest then that they represent immature particles or replicative intermediates. The minor band which sometimes appeared at a high density (I "4I to J-42 g/cm 3) was not studied but might represent the very few particles surrounded by an electron-dense envelope described by Roos et al. (I976 , for example see Fig. 3). The majority of the RNA extracted from the purified particles has been proved to possess a double-stranded nature. As also observed by Nash et al. (I 973) and Cox, Kanagalingam & Sutherland (i97o) with the virus double-stranded RNA from Penicillium ehrysogenum, a shouldeT was observed in the pre-melting region; this behaviour, according to Burnett, Frank & Douthart (I975), could be explained by the base composition in certain regions of the double-stranded RNA. Single-stranded RNAs were also detected in the particles extracted from A. arbuscula, as was also reported in some other mycoviruses (Buck & Kempson-Jones, I973 ; Nash et al. I973 ; Buck & Ratti, I975). It is unlikely that these singlestranded RNAs were adsorbed to the external surfaces of the particles because of the different purification procedme used. Mycoviruses appear to be latent and generally have no detectable effects on their hosts although apparently healthy mycelia might contain high amounts of virus particles (Lemke & Nash, 1974). Our infected strain of A. arbuscula does not show any overt cytopathological or morphological disorders. The presence of virus aggregates associated with such a vital component as the ribosomal nuclear cap in the spores (Roos et al. 1976 ) is of high interest. This localization leads us to focus our future investigations on the germination process of the spores when eventual metabolic disorders might be detected. We wish to thank Dr U.-P. Roos, Dr W. E. Timberlake and Dr C. Georgopoulos for helpful discussions and continual encouragement. We also thank Dr M. Paccaud from the Institut d'Hygi6ne de Gen6ve for his gift of reovirus. REFERENCES BARLOW, J. J., MATHIAS, A. P., WILLIAMSON, R. & GAMMAK, D. B. (I963). 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