J. gen. ViroL (I978), 4I, 443-446
Printed in Great Britain
443
P r e s e n c e o f a C o v a l e n t l y L i n k e d P r o t e i n o n Calicivirus R N A
(Accepted 21 August I978)
SUMMARY
The infective RNA of the calicivirus, vesicular exanthema virus, has been
shown to contain a protein which is apparently linked to the RNA by a covalent
bond. The protein remained bound to the RNA after boiling with SDS-mercaptoethanol-urea or treating with formamide-dimethylsulphoxidebut was removed by
incubating with proteinase K. The tool. wt. of the protein was estimated to be
about IO× Io 3 by electrophoresis in highly cross-linked polyacrylamide gels.
The infectivity of the RNA was destroyed by removal of the protein with
proteinase K.
The RNA extracted from three picornaviruses, poliovirus, foot-and-mouth disease virus
(FMDV) and encephalomyocarditis virus is infectious and possesses messenger activity
in vitro. However, none of these RNA species contains the 5'-terminal 'cap' structure
m~G(5')ppp(5')Nmp found in most eukaryotic mRNA molecules but instead a small
covalently linked protein has been found at the 5' end (Flanegan et al. 1977; Lee et al.
1977; Sangar et aL I977; Hruby & Roberts, r978).
In preliminary experiments on the RNA of vesicular exanthema virus (VEV) we had
been unable to demonstrate the presence of a cap when asP-labelled VEV-RNA, digested by
the method of Adams & Cory 0975), was fractionated on a DEAE-Sephadex column
with a NaC1 gradient (o'o5 to o'4 M in 7 M-urea, o'o3 M-tris, HC1, pH 7"6). Digestion and
fractionation of 32P-labelled Semliki Forest virus RNA by the same method showed the
presence of a cap structure. Having failed to detect a cap structure in VEV-RNA by this
method, we then tried to determine whether the RNA contains a covalently bound protein
instead.
VEV (type D-53) was grown in Vero cells in the presence of 14C-uridine (o'5/zCi/ml)
and 3H-lysine (50/zCi/ml) and purified by the method of Wawrzkiewicz et al. 0968). The
purified virus, in o.1% SDS, o-I M-tris, pH 9"0, was extracted twice with phenol and the
aqueous phase washed three times with ether. All the 14C counts, together with about o'o3 %
of the ~H counts, remained in the aqueous phase. The RNA in this phase was mixed with
carrier ribosomal RNA and precipitated overnight at - 2 o °C with z vol. of ethanol. The
RNA was resuspended in o.I M-tris, 0"I M-NaC1, oq % SDS, pH 7"5, and sedimented
through a 5 to 25 % sucrose gradient in o.I M-acetate-o" 1% SDS, pH 5"0, for 3 h at zooooo g
in a Beckman SW4I rotor at 2o °C. Samples from each fraction were dried on to glass fibre
discs and counted in a liquid scintillation counter. The 3H and x4C profiles roughly coincided; unlike the 3H peak, which was sharp and had a sedimentation coefficient of 37S, the
~C profile was very broad (Fig. I a) and more typical of previously published profiles of
VEV-RNA (Wawrzkiewicz et al. I968; Schaffer & Soergel, I973). This indicates that not
all the virus RNA molecules were associated with 3H-lysine labelled material. The 3H
counts were still associated with the RNA after heating at IOO °C for 2 rain in 1% SDS,
1% mercaptoethanol and o'5 M-urea (Fig. I b) although the sedimentation profile of the
RNA was altered. Treatment at 2o °C with 5o % formamide-25 % dimethyl sulphoxide
also did not separate the 3H and 14C counts. However, after treatment with proteinase K
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444
Short communications
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Fig. t. Sucrose gradient centrifugation in 5 to 25 ~o sucrose density gradients in o.I M-acetate,
o'I % SDS, pH 5"0, of 3H-lysine (© . . . . O) and a4C-uridine ( •
• ) labelled VEV-RNA (a)
untreated; (b) after heating at ioo °C for 2 rain with 1% SDS, 1% mercaptoethanol, 0"5 u-urea;
(c) after heating at 37 °C for 30 min with proteinase K (0"5 mg/ml) in the presence of o'5 % SDS.
In each gradient the positions of BHK cell 28S and 18S ribosomal RNA are marked.
(0"5 mg/ml) in the presence of o"5% SDS, 3H counts were no longer associated with
the 14C peak, but were all found at the top of the gradient (Fig. I c), suggesting that the
3H-labelled material is a protein, covalently linked to the RNA.
To estimate the mol. wt. of this protein, aH-lysine labelled R N A was digested with a
mixture of T1, T 2 and pancreatic ribonucleases at 37 °C for 24 h. This digest was then
heated at IOO °C for 5 min with ~ % SDS, I °fo mercaptoethanol and 8 M-urea and examined
on ~2"5 % cylindrical polyacrylamide gels (acrylamide:bisacrylamide ratio of IO: I) by
the method of Swank & Munkres 0 9 7 I ) . The SDS-disrupted material was co-electrophoresed with SDS-disrupted insulin and aSS-methionine labelled F M D V polypeptides to
act as tool. wt. markers in addition to the ribonucleases. Electtophoresis was performed at
z'5 mA/gel and 4o V for I5 h. After fixing and staining with Coomassie blue the gel was
cut into I mm slices, digested with o'5 ml NCS and counted in a liquid scintillation counter.
The 3H-labelled material migrated as a peak with an apparent mol. wt. of about Io × io 3
which compares with 4 to 5 × Ioa for the protein on picornavirus R N A (Fig. 2). The aH
counts found at the top of the gel are probably due to incomplete ribonuclease digestion.
To determine whether the protein was required for infectivity, virus labelled with aHlysine and x~C-uridine as previously described was mixed with unlabelled virus grown in a
single growth cycle in 2o Roux flasks each containing about Io 8 Vero cells. To minimize the
breakdown of R N A within the virus particle, harvests were kept at - 2o °C until processed
and a more rapid purification procedure was used. Pelleted virus was immediately resuspended and subjected to sucrose gradient centrifugation rather than being allowed to
resuspend overnight at 4 °C. Using such procedures, homogeneous R N A has been obtained
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Short communications
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Fig. z. Electrophoresis in a highly cross-linked polyacrylamide gel of the aH-lysine labelled protein
isolated from VEV-RNA by digestion with pancreatic, Tt and T2 ribonucleases. Markers of insulin
and 35S-methionine labelled FMDV polypeptides were mixed with the protein before electrophoresis.
I: T~ and pancreatic ribonucleases; II: 3~S-methionine labelled VP4 of FMDV; III: Tt ribonuclease;
IV: insulin B chain; V: insulin A chain.
from F M D V (Denoya et al. I978 ) which otherwise resembles the caliciviruses in yielding
heterogeneous RNA. The purified virus in o.5% SDS, o.I M-tris, pH 7"6, was extracted
once with phenol-chloroform, the R N A mixed with ribosomal R N A and precipitated
overnight at - z o °C with z vol. of ethanol. The RNA was then washed with cold 67%
ethanol and resuspended in o'5% N-Iauroyl sarcosine in o.I M-tris, p H 7'6. One half was
heated at 37 °C for 3o rain and the other half treated with proteinase K as previously
described. The two samples were sedimented through 5 to 15 % sucrose gradients in o-1%
N-lauroyl sarcosine - N T E (o.I2 M-NaCI, o.ooi2 M-EDTA, o'o5 M-tris, p H 7"6) at zooooo g
in a Beckman SW4I rotor at 4 °C. The 3H and laC profiles obtained were similar to those
shown in Fig. ~(a, c) demonstrating that the RNA remained intact after the enzyme
treatment. Fractions from each gradient containing the RNA sedimenting at 35 to 37S
were pooled, ethanol precipitated and then assayed for infectivity by inoculating monolayers of IBRS-2 cells in I oz bottles in the presence of zooy/ml DEAE-dextran. The
untreated R N A had a titre of IO5"0 IDs0/ml but the proteinase K treated R N A was
non-infectious.
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446
Short communications
Picornavirus RNA was the first RNA species shown to be distinct from most eukaryotic
mRNA in possessing a protein linked to the 5' end rather than a cap. A genome-linked
protein has now been demonstrated on the RNA of the nepoviruses (Harrison & Barker,
1978) and, in the present work, on the RNA of VEV. VEV is a member of the Calieiviridae
(Burroughs & Brown, 1974) and has a different strategy from that of the picornaviruses.
Whereas the replication of picornaviruses takes place through successive proteolytic
cleavages of a very large precursor polypeptide, the virus RNA being translated as a monocistronic message, in calicivirus-infected cells the only major induced protein has a mol. wt.
identical with that of the virus structural polypeptide (Black & Brown, I975/76). Two
minor polypeptides are also found; but the three polypeptides are synthesized independently
and there is no precursor-product relationship between them (Black & Brown, I978). In
addition, calicivirus replication involves the synthesis of sub-genomic RNA molecules
which probably function as messengers (Ehresmann & Schaffer, 1977; Black et al. I978 ).
The present work shows that VEV differs further from the picornaviruses in that the
protein covalently linked to the RNA is required for infectivity. In this respect VEV-RNA
resembles that of the nepoviruses (Harrison & Barker, 1978). This result suggests a different
role for the RNA linked protein in VEV compared with that of the picornaviruses.
Animal Virus Research Institute
Pirbright
Surrey
J.N. BURROUGHS
F. BROWN
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