J. gen. ViroL (I97O), 8, I45-I48
Printed in Great Britain
~45
Molecular Weight of Rhinovirus Ribonucleic Acid
( A c c e p t e d 22 April I97O)
The picornaviruses have a wide range of buoyant densities in caesium chloride. Whereas
the density of the pH 3-stable viruses is I'34 g./ml. (Mattern, I962; Schaffer & Frommhagen,
I965), the acid-sensitive rhinoviruses and foot-and-mouth disease viruses have densities of
~.38-i.4I g./ml. (Dans, Forsyth & Chanock, 1966; Chapple & Harris, ~966; McGregor,
Phillips & Mayor, I966; Gerin et al. 1968) and I'43 g./ml. (Trautman & Breese, ~962; Wild
& Brown, 1967). Although the reason for this difference in density is not understood, it
seems likely that the higher values obtained with the acid-labile group are due to reaction
of the caesium ions with the more accessible RNA of these viruses (McGregor et al., I966).
Recently, however, McGregor & Mayor (I968) suggested, on the basis of comparative
measurements of the strand lengths of the ribonucleoproteins isolated from strains of
poliovirus and rhinovirus, that the higher buoyant density of the rhinovirus was due to
the high molecular weight (4 × Ion) of the virus RNA.
The molecular weight of the RNA of foot-and-mouth disease virus is about 2-8 x io 6
(Wild & Brown, i97o), a value only slightly greater than those obtained for the RNAs of
poliovirus (2-6× ~o6; Granboulan & Girard, 1969) and EMC virus (2.7 x lO6; Burness,
I97o). The higher density of foot-and-mouth disease virus (t.43 g./ml.), compared with that
for poliovirus and EMC virus (I'34 g./ml.) is not due to the different proportions of RNA
in the viruses since each contains 30 to 32 % (Bachrach, Trautman & Breese, I964; Schaffer,
Moore & Schwerdt, 196o; Burness, ~97o). Preliminary work on human rhinoviruses (Brown,
Newman & Stott, nnpublished observations) also indicates that these contain not more
than 30 % RNA.
The similar values obtained for the molecular weights and RNA contents of poliovirus,
EMC virus and foot-and-mouth disease virus suggested that the high molecular weight of
rhinovirus RNA obtained by McGregor & Mayor 0968) was incorrect, especially as van
Elsen, Boey6 & Teuchy 0968) cast doubts on the identity of the ribonucleoprotein strands
which McGregor & Mayor observed in the electron microscope. We have attempted to
resolve this question by comparing, in the same laboratory and under identical conditions,
the sedimentation characteristics of the RNAs isolated from two strains of rhinovirus, one
strain of foot-and-mouth disease virus and a pig enterovirus (ITALIANI/66) recently examined
at this Institute (Nardelli et al. I968). The pig enterovirus is acid-stable and has a buoyant
density in caesium chloride of 1.34 g./ml.
Each virus was grown in the presence of actinomycin D (o.t to I.O #g./ml.) and either
[aH]uridine or [a~P]phosphate. When a2p was used, the virus-infected cells were incubated in
medium containing tris or N-2-hydroxyethyl piperazine-N'-ethanesulphonic acid instead of
phosphate. Human rhinovirus, type 2 (strain HGP), was grown in suspended L I32 cells,
equine rhinovirus (strain N~t I1) in RK I3 cells, pig enterovirus (ITALIAN1/66)in the pig
kidney cell line IB-RS-2 and foot-and-mouth disease virus, type O (strain I), in BHK 2I
cells. Each virus was purified by the method described for foot-and-mouth disease virus
(Brown & Cartwright, I963).
Virus RNA was usually extracted with cold phenol, but in some experiments the RNA of
the acid-labile viruses was obtained by mixing with o., vol. I.og-acetate, pH 5"0, containing
I'o % sodium dodecyl sulphate. Each RNA preparation was mixed with BHK cell RNA,
II-2
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I46
Short communications
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Fig. I. Centrifugation in 5 to z5 % sucrose gradients of the RNAs extracted from (a) human
rhinovirus, strain ~oe; (b) equine rhinovirus, strain yM t I ; (e) enterovirus, strain zTat.taN 1/66;
(d) foot-and-mouth disease virus, type O, strain t. The samples were mixed with BHK 2I cell RNA
and centrifuged for J7 hr at ~8,ooo rev./min, in the SW z5"I rotor of the Spinco ultracentrifuge.
The gradients were prepared in o.1 N-acetate, pH 5"0, containing o.1% sodium dodecyl sulphate.
•-• , radioactivity; •
• , absorbency at 260 nm.
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Fig. z. Centrifugation in 5 to z5 % sucrose gradients of formaldehyde-treated RNAs. Mixtures of
the RNAs from [3H]uridine-lahelled foot-and-mouth disease virus and (a) a~P-labelled human
rhinovirus, strain Hoe, or (b) 32P-labelled enterovirus, strain ITALIAN I/66, were treated with 6 %
formaldehyde in o.oIM-EDTA at 70 ° for 5 rain., then made to o.I ~-NaC1 and centrifuged for
17 hr at 18,ooo rev./min, in gradients prepared in 6 % formaldehyde, o.oi M-EDTA and o.z M-NaCI,
pH 7-o. • - - • ,
foot-and-mouth disease virus RNA; m - - - i ,
human rhinovirus RNA;
• . . . . • , enterovirus RNA.
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I47
precipitated with 2 vol. cold ethanol and stored at - 2 o ° overnight. The precipitates were
then dissolved in O'IM-acetate, p H 5"0, containing o-~ % sodium dodecyl sulphate and
filtered through Sephadex G-2oo in equilibrium with the same solution. A portion of each
filtrate was used directly for determining the sedimentation coefficient of the R N A in 5 to
25 % sucrose gradients prepared in o.1 ~ acetate containing o.I % sodium dodecyl sulphate
or in O-IM-NaC1, o.OlM-EDTA, p H 7-0. The remainder of the G-2oo filtrates were precipitated at - 2 0 ° with 2 vol. ethanol and these precipitates were redissolved in a freshly
prepared solution of 6 % formaldehyde in o.o~ M-EDTA, p H 7.0. The solutions were heated
at 7 °° for 5 min., made to o.~ M-NaC1 and centrifuged in 5 to 25 % sucrose gradients in 6 %
formaldehyde, O.IM-NaC1 and o.O~M-EDTA, p H 7.0. Under these conditions the configurational differences were reduced to a minimum, and enabled a more valid comparison
of relative molecular weights to be made (Fenwick, I968).
Table I. Some physico-chemical properties of rhinovirus strain ~cP and the virus RNA
Sedimentation coefficient of virus
Diameter
Buoyant density in caesium chloride at pH 7'6
Absorbency 26o/28o nm.
Sedimentation coefficient of RNA
(a) in o'iM-acetate+o-i % SDS, pH 5"o
(b) in 6 % formaldehyde, o'oI M-EDTA,
o-I M-NaCI, pH 7o
Base composition (5 determinations)
Adenylic acid
Cytidylic acid
Guanylic acid
Uridylic acid
I5os
22 11Ill.
I'4o g./ml.
I "67
30 to 32s
i6s
34"o±I'7
20"2±I' 5
I9'5±o'9
26-3±0'7
The sedimentation profiles of the four RNAs in 5 to 25 % s u c r o s e gradients prepared in
o.I M acetate, o-1% sodium dodecyl sulphate, p H 5"o, are shown in Fig. I. The RNAs from
each of the rhinovirus strains and the enterovirus gave sharp peaks with sedimentation
coefficients in the range 30 to 35s relative to values of 28s and I6s for ribosomal RNA.
The R N A from foot-and-mouth disease virus gave a typically heterogeneous profile with
a distinct peak at 35s. The heterogeneity was less pronounced when the R N A was released
from the virus by mixing with o-i M-acetate, o. ~ % sodium dodecyl sulphate, p H 5"o, but was
still greater than that for the other virus RNAs. The profiles of the phenol- and acid-released
R N A from the human rhinovirus strain were identical.
The sedimentation profiles of the R N A s from the human rhinovirus, pig enterovirus
and foot-and-mouth disease virus after heating in 6 % formaldehyde were very similar. The
rhinovirus R N A had a relative sedimentation coefficient of I6s and the enterovirus and
foot-and-mouth disease virus RNAs of x7s. Under the same conditions the larger ribosomal
RNA, of sedimentation coefficient z8 s in o.I ~-acetate, sedimented at I9 s, a value obtained
by Fenwick (I968) using the analytical ultracentrifuge. The small difference between the
relative sedimentation coefficients of the rhinovirus R N A and the enterovirus and foot-andmouth disease virus RNAs was confirmed by centrifuging mixtures of the RNAs in the same
tube (Fig. 2).
The data presented indicate that the size of the R N A in rhinovirus strain HCP is in the
range 2. 4 to 2.8 x io 6 found for other picornaviruses. The value of 4 x io 6 obtained by
McGregor & Mayor (~ 968) for rhinovirus strain a 632 is thus open to question. It is possible,
however, that the molecular weights of the RNAs of the different rhinoviruses vary widely.
Although this is unlikely, the value obtained by McGregor & Mayor should not be dismissed
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I48
until more information is available on the physico-chemical properties of several members
of the rhinovirus group. A summary of our findings with strain x-iat, is given in Table ~, so
that a comparison can be made with information on other strains.
We wish to thank Mr D. Goodridge for providing us with the equine rhinovirus and
enterovirus.
Animal Virus Research Institute
Pirbright, Surrey
F. BROWN
J. F. E. NEWMAN
W.H.O. International Reference Laboratory
for Respiratory Virus Diseases,
Common Cold Research Unit,
Salisbury, Wiltshire
E. J. STOTT
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