A New Double-stranded RNA Virus from Gaeumannomyces graminis

A New Double-stranded RNA Virus from Gaeumannomyces graminis

J. gen. Virol. (1984), 65, 987-990. Printedin Great Britain
987
Key words: mycovirus/RNA, double-stranded/Gaeumannomyces graminis/serology
A New Double-stranded RNA Virus from
Gaeumannomycesgraminis
By K. W. B U C K
Department of Pure and Applied Biology, Imperial College of Science and Technology,
London SW7 2BB, U.K.
(Accepted 14 February 1984)
SUMMARY
An isometric virus, obtained from an isolate of Gaeumannomyces graminis, had the
following properties: diameter 29 nm; sedimentation coefficient 127S; buoyant density
in CsC1 1.40 g/ml; one double-stranded R N A component of 1.80 kilobase pairs; one
capsid polypeptide species of mol. wt. 66000; A2~o/A28o 1-7. The virus was unrelated
serologically to three other viruses present in the same fungal isolate and to 12
pt'eviously described viruses belonging to G. graminis virus groups I, II and III. On the
basis of its distinctive properties the virus is assigned to a new G. graminis virus group
IV.
Isometric virus particles in the wheat take-all fungus, Gaeumannomyces graminis var. tritici,
were first discovered by Lapierre et al. (1970). Subsequently, Rawlinson et al. (1973) and
Rawlinson & Muthyalu (1975) found that virus particles were of common occurrence in field
isolates of this fungus. They found three sizes of isometric particles with diameters of 27 nm,
35 nm and 40 nm. Frick & Lister (1978) found considerable serotype variability among virus
particles with modal diameters of 35, 39 and 41 to 42 nm obtained from 19 isolates of G. graminis.
Later, 13 viruses from eight isolates of G. graminis were classified, on the basis of their
serological and physical properties, into three groups, two of 35 nm viruses and the third of
40 nm viruses (Buck et al., 1981). However, up to now none of the 27 nm particles has been
purified or characterized. In the present communication I report the isolation of a G. graminis
virus of 29 nm diameter and that its properties are distinct from those of previously described
viruses in groups I, II and III. A fourth G. graminis virus group is proposed to accommodate this
new virus.
G. graminis isolate 45/101, a spontaneous mutant of isolate 45/10 (Romanos et al., 1980) which
had previously been shown to contain particles of 35 nm and 27 nm diameter (C. J. Rawlinson,
personal communication), was grown in 60 litre fermenters and virus particles were isolated and
partially purified by polyethylene glycol precipitation and differential centrifugation as
described by Buck et al. (1981). Further purification and fractionation of the virus particles were
effected by sucrose density gradient centrifugat]on as described by Buck & Girvan (1977). Five
fractions, shown by electron microscopy to contain isometric virus particles, were designated 1
to 5 in order of increasing rate of sedimentation and dialysed against 0.03 M-sodium phosphate
buffer pH 7.6 containing 0.15 M-KC1 (PK buffer).
Virus RNA was prepared from each sucrose fraction and shown to be double-stranded (ds) as
described by Buck & Ratti (1975). Analysis and molecular weight determinations of dsRNA
components by polyacrylamide gel electrophoresis (PAGE) and of capsid polypeptides by SDSPAGE were carried out as described by Buck & Ratti (1977) and Buck et al. (1981). Particles in
fraction 3 contained seven dsRNA components of 2-33, 2.27, 2.08, 2.02, 1.80, 1.70 and 1.53
kilobase pairs (kbp) (Fig. 1a, lane 2) and four polypeptide species with mol. wt. 73 000, 68 000,
66000 and 55000 designated P73, P68, P66 and P~5 respectively (Fig. lb, lane 1). Fraction 1
contained only the two smallest dsRNAs and predominantly P55, while fraction 5 contained
predominantly the four largest RNAs and the two largest polypeptides. These results suggested
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988
(a)
(b)
2
(c)
1
3
--73
--2.33
--68
--2.02
--55
-
-
1-44
'~:
Fig. 1. (a) P A G E of viral dsRNA. Lane 1, virus D; lane 2, sucrose fraction 3; lane 3, CsCI band 1 ; lane
4, virus C. (b) S D S - P A G E of viral polypeptides. Lane 1, sucrose fraction 3; lane 2, virus C. In both (a)
and (b) electrophoresis was from top to bottom. (c) CsC1 isopycnic sedimentation of sucrose fraction 3.
Sizes in kbp (a) and mol. wt. × 10 -3 (b) were determined by co-electrophoresis with d s R N A (a) and
polypeptide (b) markers from other G. graminis viruses (Buck et al., 1981) and are indicated on the righthand side of the gels.
that P~3, P~s, P66 and Ps5 may belong to four distinct viruses, designated A, B, C and D
respectively, assuming one capsid polypeptide species per virus.
For isolation of virus C, sucrose fraction 3 was mixed with CsC1 to give a density of about
1-37 g/ml and centrifuged at 35000 rev/min for 21 h at 4 °C in a Beckman SW50.1 rotor.
Examination of the gradient by overhead illumination revealed four light-scattering bands (Fig.
1 c). Fractions (0.05 ml) were collected from the bottom of the gradient and the density of each
was measured by refractometry (Ifft et al., 1961). Small amounts of each fraction were spotted
onto an agarose plate containing 6 mg/1 ethidium bromide and examined for fluorescence on a
u.v. transilluminator. No fluorescence was obtained from fractions in the top part of the
gradient, indicating that the uppermost band contained no nucleic acid. Strong fluorescence was
obtained in fractions corresponding to the other three bands which were designated 1, 2 and 3
(Fig. 1 c). Peak fractions were dialysed against PK buffer and examined by PAGE, SDSPAGE, electron microscopy and sedimentation analysis. For electron microscopy, samples were
negatively stained in 1 ~ potassium phosphotungstate, pH 7-0 and examined in a Siemens
Elmiskop 1A electron microscope which had internal calibration with a 69.6 ~tm aperture. Band
3 contained isometric particles of diameter 29.3 + 1-9 nm (standard error, 115 particles), which
had a similar appearance in electron micrographs to previously reported particles of 27 nm
diameter from G. graminis (Rawlinson et al., 1973). The particles had a sedimentation
coefficient (s,°0 in PK buffer) of 127S, a buoyant density in CsC1 of 1-40 g/ml, Az6o/Azso ratio of
1.7, a single dsRNA component of 1-80 kbp (Fig. 1 a, lane 4) and a single polypeptide species of
tool. wt. 66000 (Fig. 1 b, lane 2); they thus correspond to virus C which is therefore the virus with
particles of 27 nm diameter previously detected in G. graminis isolate 45/10.
Bands 1 and 2 from the CsC1 gradient both contained P73 and P~8 and dsRNA components of
2.33, 2.27, 2.08 and 2.02 kbp in similar proportions (Fig. 1 a, lane 3) and particles of about 35 nm
diameter. Both bands evidently contained viruses A and B. The reason for formation of two
bands is unknown. For isolation of virus A, bands 1 and 2 were combined and dialysed against
0.01 M-Tris-HCI pH 8.0. This caused virus B and a proportion of virus A to precipitate out of
solution. Virus A in the supernatant gave a single band of buoyant density 1-37 g/ml when
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989
Table 1. Properties of virus particles in G. graminis virus groups I to 1V
Size of dsRNA Mol. wt. of capsid
Buoyant density
Particle diam.
s%
c o m p o n e n t s polypeptides
in CsC1
Group
(nm)
(Svedbergs)*
(kbp)t
( x 10- 3)
A2~o/A2so~
(g/ml)
I
II
III
IV
35
35
40
29
109-128
133-140
159-163
127
1-59-1-89
2.02-2.33
5. i-6.0
1.8
54-60
68-73
78-87
66
1-4-1.5
1.4-1.5
1.6
1.7
1.36-1.37
1.35-1-37
1.40-1.41
1.40
* Measured in PK buffer.
t A value of 687 for the mol. wt. of a base pair was used to convert mol. wt. values (Buck et al., 1981) to kbp.
Measurements were made at low concentrations (A26oabout 0.5) and were not corrected for light scattering.
centrifuged in a CsC1 gradient and consisted of particles with diameter 35-4 +__2-1 nm (standard
error, 85 particles), s°o (in PK buffer) of 140S, A26o/A2so of 1.5, two dsRNA components of 2.33
and 2.27 kbp and one polypeptide species, P~3- Virus B was deduced by elimination to contain
the two dsRNA components of 2.08 and 2-02 kbp and the polypeptide P6s.
Virus D was unstable in CsC1, but could be isolated from sucrose fraction 1 by isopycnic
centrifugation in a Cs2SO, density gradient (40000 rev/min, 15 °C, 24 h, Beckman SW50.1
rotor). It had particles of diameter 34.8 + 1.8 nm (standard error, 76 particles), buoyant density
in Cs2SO4 of 1.29 g/ml, s°0 (in PK buffer) of 113S, A26o/A2so of 1.4, two dsRNA components of
1.70 and 1.53 kbp and one polypeptide species, Pss.
Relationships of viruses A, B, C and D with each other and with previously described G.
graminis viruses were examined by serology. An antiserum to viruses A, B, C and D was
prepared by immunizing rabbits with sucrose fraction 3 and gel diffusion analyses were carried
out as described by Buck et al. (1981). When allowed to diffuse against this antiserum viruses A,
A + B mixture, C and D gave single precipitin lines with antiserum titres of 1 : 2048, 1 : 2048,
1:512 and 1 : 1024 respectively. When pairs of viruses were placed in adjacent wells virus C gave
crossing precipitin lines with virus A, virus D and A + B mixture. Virus A gave a crossprecipitin line with virus D. It was concluded that virus C is serologically unrelated to viruses A,
B and D and that virus A is serologically unrelated to virus D. Virus C did not react with any Of
six antisera containing antibodies to a total of 12 G. graminis viruses from groups I, II and III
(Buck et al., 1981 ; McFadden et al., 1983). However, virus A reacted with antiserum to virus T 1A (group II) and virus D reacted with antiserum to virus 019/6-A (group I).
The properties of viruses A and B are similar to those of group II viruses, while virus D has the
properties of a group I virus (Buck et al., 1981). The properties of virus C, however, are
significantly different from viruses in groups I, II and III. The particles are smaller and have a
dsRNA in the size range for a group I virus but with a capsid polypeptide size closer to that of a
group II virus. Furthermore, the A26o/A~8o ratio and buoyant density in CsC1 are significantly
greater than those of virus A, suggesting a higher dsRNA : protein ratio. Since data on these
properties were not available for groups I, II and III viruses described previously (Buck et al.,
1981) they were determined for the following viruses. A26o/A2so ratios were as follows. Group I:
019/6-A, 1.4; 01-1-4-A, 1.4; 38-4-A, 1.5; OgA-B, 1.4. Group II: T1-A, 1-4; OgA-A, 1.4; 3bla(B1 + B2), 1.4. Group III: 3bla-A, 1.6. Buoyant densities in CsC1 (g/ml) were as follows. Group
I: 019/6-A, 1.36; 01-1-4-A, 1.36; 38-4-A, 1.37; OgA-B, 1-36. Group II: T1-A, 1.35; OgA-A, 1.36.
Group III: 3bla-A, 1-40 and 1-41 for particles containing dsRNAs of 5100 and 6000 bp
respectively. It is clear that the A26o/A2s o ratio and buoyant density in CsC1 of virus C are
significantly higher than those of groups I and II viruses, although similar to those of the one
group III virus measured. Virus C, however, has a much smaller particle diameter and smaller
dsRNA and capsid polypeptide species than the group III viruses. It is therefore proposed to
assign virus C to a new group, designated IV. Properties of the viruses in the four groups are
compared in Table 1.
It is noteworthy that virus C apparently has only one dsRNA component. Furthermore, the
full coding capacity of this RNA would be needed to encode the capsid polypeptide of mol. wt.
66000. By analogy with other dsRNA viruses it would be expected that at least one other gene
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w o u l d b e n e e d e d to e n c o d e t h e R N A - d e p e n d e n t R N A p o l y m e r a s e n e e d e d for d s R N A
t r a n s c r i p t i o n a n d r e p l i c a t i o n . I t is t h e r e f o r e p o s s i b l e t h a t (i) v i r u s C is a satellite v i r u s d e p e n d e n t
o n o n e o f t h e o t h e r viruses in t h e s a m e isolate for r e p l i c a t i o n , (ii) v i r u s C h a s t w o d s R N A
c o m p o n e n t s o f t h e s a m e size b u t d i f f e r e n t s e q u e n c e o r (iii) v i r u s C h a s a single d s R N A
c o m p o n e n t w i t h t w o or m o r e g e n e s e n c o d e d in d i f f e r e n t r e a d i n g f r a m e s . I f v i r u s C is t y p i c a l o f
G. graminis viruses o f a b o u t 27 n m d i a m e t e r it is u n l i k e l y t o b e a satellite s i n c e m a n y G. graminis
isolates c o n t a i n o n l y 27 n m p a r t i c l e s ( R a w l i n s o n et al., 1973). H o w e v e r , f u r t h e r studies o n t h e
m o l e c u l a r biology o f v i r u s C a n d o t h e r 27 n m viruses will b e r e q u i r e d to resolve t h e s e
possibilities.
I am grateful to Miss M. B. Fitzgerald for assistance, Mr R. D. Woods for electron microscopy and Dr C. J.
Rawlinson for stimulating discussions.
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