J. gen. Virol. (1983), 64, 1409-1413. Printedin Great Britain
1409
Key words: measles virus/RNA purification/mol, wt. determination
Purification and Molecular Weight Determination of Measles Virus
Genomic RNA
By K. B A C Z K O , M. B I L L E T E R 1 AND V. T E R M E U L E N *
Institute of Virology and Immunobiology, University of Wftrzburg, D-8700 Wiirzburg, Federal
Republic of Germany and 1Institute of Molecular Biology L University of Zftrich, CH-8093
Zi~rich, Switzerland
(Accepted 10 January 1983)
SUMMARY
A purification procedure for genomic measles virus RNA, free of contaminating
smaller R N A and of DNA, is described. Viral nucleocapsids were prepared from
MA160 cells infected in spinner cultures with measles virus (Edmonston strain).
Nucleic acid was extracted, treated with DNase and R N A sedimenting at about 50S in
sucrose gradients was isolated. This method yielded 0-5 to !"5 ~tg of genomic R N A per
litre of culture. A molecular weight of 4.5 x 106 was determined by gel electrophoresis
under fully denaturing conditions.
Measles virus, a paramyxovirus (Kingsbury et al., 1978) is a human pathogen causing acute
and subacute infections. The association of this agent with a variety of human diseases has
raised great interest amongst immunologists and virologists and many studies have been performed to unravel the pathogenic mechanisms which can lead to these different disease
processes (ter Meulen & Carter, 1982). However, virological approaches have been hampered by
the fact that measles virus grows poorly in tissue culture and the low yield of virus has so far prevented the application of modern molecular virological techniques to the search for measles
virus genetic information present in diseased organs. No reliable method has been available for
the isolation of sufficient quantities of pure, stable genomic R N A to permit cloning of viral
nucleic acids in appropriate vectors. The lack of purified measles virus R N A has also prevented
the exact determination of molecular weight: this has been estimated as 6.2 × 106 from the sedimentation of R N A in sucrose gradients (Schluederberg, 1971), or length measurements of
nucleocapsids (Nakai et al., 1969), but no determination has been performed under fully
denaturing conditions.
In this report, we describe a method for the preparation of genomic R N A free of contaminating smaller RNAs and DNA, and present a molecular weight determination based on
mobility during electrophoresis in fully denaturing methylmercury-agarose gels (Bailey &
Davidson, 1976). Virus RNA was purified from intracellular viral nucleocapsids prepared by a
modification of the method of Compans & Choppin (1967). This approach was used since only a
small percentage of genomic R N A is packaged and released from infected cells as mature
virions.
All buffers and solutions were treated with 0.05~ diethyl pyrocarbonate, autoclaved and
stored frozen before use. Reaction vials were siliconized Corex glass or Eppendorf plastic tubes.
The whole purification procedure was performed under conditions that rigorously excluded
contamination by RNase. The Edmonston strain of measles was a gift from W. Bellini (Bellini et
al., 1979). It was plaque-purified three times and propagated twice in Vero cells at a low
multiplicity of infection (0.1), generating a stock containing few defective particles. Growth of
virus was as described in detail elsewhere (Baczko & Lazzarini, 1979). MA160 cells
(Microbiological Associates) were suspended in spinner culture and adjusted to 2 x 105 to 3 x
105 cells/ml. After 2 days, cells were centrifuged and resuspended in 0.2 vol. for infection at a
multiplicity of 0.1. After 1 h of constant stirring at 37 °C, medium was added to produce a cell
density of 2 x 105 t o 3 x 105 perml.
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0022-1317/83/0000-5517 $02.000 1983 SGM
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Two days later, cells were collected by centrifugation, and resuspended in the original volume.
At 3 days post-infection [3H]uridine (27 Ci/mmol, Amersham Buchler) was added to a concentration of 0-5 ~tCi/ml. Cells were harvested 4 to 5 days post-infection, when the cytopathic effect
was complete, and all subsequent steps leading to the extraction of nucleic acids were carried out
as quickly as possible in a cold room (4 °C). Cells were pelleted at 400g for 10 min, washed with
ice-cold phosphate-buffered saline and sedimented again at 700 g for 5 min. Pellets were resuspended in ice-cold 10 mM-HEPES pH 7.0, 5 mi-MgC12 at a concentration of 3.2 × 107 cells/ml
and the cells were allowed to swell for 10 min on ice. Nonidet P40 was added to a final concentration of 1%. After 5 min on ice cells were carefully disrupted (to avoid destruction of nuclei) by
two slow passages in a Dounce homogenizer. Cellular debris and nuclei were removed by centrifugation for 5 min at 700g. The supernatant was adjusted to 7.5 mM-EDTA and layered onto a
step gradient consisting of 40, 30 and 25 % (w/v) caesium chloride (Serva; ultrapure) in SEH (100
mM-NaC1, 10 mM-HEPES pH 7-0, 1 mM-EDTA). Centrifugation was performed at 36000
rev/min in a SW41 rotor for 120 min at 4 °C. The visible nucleocapsid band in the 30% caesium
chloride region was carefully removed by aspiration in a siliconized Pasteur pipette, diluted at
least threefold with SEH and pelleted onto a 65% (w/v) sucrose/SEH cushion for 20 min at
36000 rev/min in a SW41 rotor at 4 °C. The firm pellet was removed in a shortened siliconized
Pasteur pipette and resuspended in at least five times its volume of SEH containing 2 % SDS, at
37 °C. Pronase (Calbiochem; B grade, 20 mg/ml, self-digested for 2 h at 37 °C in 0.15 M-EDTA)
was added to a final concentration of 1 mg/ml and incubated for 10 min at 37 °C with repeated
shaking, until most of the visible pellet was solubilized. Nucleic acids were extracted four times
with SEH-saturated phenol and the phenol phases re-extracted with the original volume of SEH,
2 % SDS. The aqueous phases were pooled in a siliconized Corex tube and nucleic acids were
precipitated overnight with ethanol at - 20 °C before collection by centrifugation. Pellets were
washed once with 90 % ethanol, dried under reduced pressure and resuspended in 100 mM-NaCI,
10 mM-HEPES pH 7-0, 10 mi-MgC12 in the proportion of 100 p.1 per litre of original spinner
culture.
The nature of the nucleic acids present at this stage was determined by DNase or RNase
digestion followed by electrophoresis under denaturing conditions. For each test, 1 to 2 ~tg of
nucleic acids from the nucleocapsid preparation was mixed with 0.1 ~tg of a marker D N A as an
internal control for successful nuclease function. One sample was kept on ice, one was digested
with 1000 U/ml DNase I [Worthington; treated with Macaloid to remove any trace of RNase
(Schaffner, 1980)] and one was treated with pancreatic RNase A (Worthington; heated 10 min
at 90 °C to inactivate DNase) at a concentration of 1 ~tg/ml. After incubation for 5 min at 37 °C,
samples were adjusted to 20 mM-rnethylmercury hydroxide, heated for 5 min at 50 °C, cooled,
adjusted to 10% glycerol, and 0-025% bromophenol blue and subjected to electrophoresis
through a vertical 1% agarose gel (10 × 10 × 0.2 cm) in denaturing buffer (0-05 M-boric acid,
0.005 M-sodium borate, 0.01 M-sodium sulphate, 0.001 M-sodium EDTA, 6 mM-methylmercury
hydroxide). After a run of 1 to 2 h at 30 mA, 70 V, gels were stained with ethidium bromide (1
~tg/ml) in 0-2 M-ammonium acetate, 18 mi-2-mercaptoethanol, and photographed. As seen in
Fig. 1 (a), the nucleocapsid preparation contained considerable amounts of D N A and smaller
than genome-size RNAs. The larger band was not abolished entirely by either RNase or DNase
and therefore contained both R N A and D N A which co-migrate in the gel.
The amount and size of contaminating nucleic acids were found to vary in different preparations. Apparently they are only loosely associated with the nucleocapsid protein. Treatment
of nucleocapsid preparations with DNase or RNase degraded the D N A and the low molecular
weight R N A species respectively, whereas the genomic R N A was only slightly affected by
RNase (results not shown). Repeated banding in caesium chloride reduced the contaminating
nucleic acids; however, the yield of genomic R N A was also decreased. Therefore, we routinely
used only one density gradient step. The contaminating RNAs were mostly of ribosomal origin;
the prominent bands co-migrated precisely with the 28S and 18S bands obtained in cytoplasmic
extracts from uninfected cells. The nature of the D N A remains unclear and was not investigated
further; on some occasions a D N A band was observed migrating even above the genomic
measles virus RNA. We assume that the contaminating D N A consists of relatively high moleDownloaded from www.microbiologyresearch.org by
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(b)
1
(a)
1
2
3
4
5
I[ 50S
50S--
28S-m 28S
18S--
i
18S
Fig. 1. Characterization of nucleic acid preparations isolated from measles virus nucleocapsids using
electrophoresis in 1~ agarose under fully denaturing conditions. (a) Lane 1, nucleic acids extracted
from nucleocapsids; lane 2, marker DNA (pBR322 linearized; 4362 base pairs); lane 3, mixture of
material in lanes 1 and 2; lane 4, mixture of material in lanes 1 and 2, digested with DNase I; lane 5,
mixture of material in lanes 1 and 2, digested with RNase A. (b) Lane 1, RNA from nucleocapsid preparation (after DNase I treatment); lane 2, 50S RNA isolated from RNA shown in lane 1 by centrifugation in a sucrose gradient.
cular weight fragments of different sizes which run artefactually as one band (Lehrach et al.,
1977). This D N A invariably contaminated the viral R N A after sucrose gradient sedimentation
(see below) unless it had been previously removed by DNase I digestion.
For the further purification of measles virus genomic R N A in routine preparations, the
nucleic acids extracted from nucleocapsid preparations derived from caesium chloride, without
RNase or DNase treatment, were resuspended in 100 ~tl of 100 mM-NaCI, 10 mM-HEPES pH 7-0
and 2 ~ SDS per litre of original spinner culture. Any contaminating heavy metals were removed
using a Chelex 100 (Bio-Rad) column (0.3 x 0-3 cm) (omission of this step led to degradation of
the R N A , even in the presence of ethanol at - 20 °C). Nucleic acids from the flow-through were
phenol-extracted three times, ethanol-precipitated, collected by centrifugation, washed with
9 0 ~ ethanol and dried under reduced pressure. The pellet was resuspended in 25 ~tl per litre of
original spinner culture of 100 mM-NaC1, 10 mM-HEPES pH 7.0, 10 mM-MgC12, and D N a s e I
was added to a final concentration of 1000 U/ml.
After incubation for 5 min at 37 °C, the reaction mixture was adjusted to 1 ~ SDS and 15 m~tEDTA, Pronase was added to 1 mg/ml and the solution incubated for 10 min at 37 °C. R N A was
extracted three times with phenol, ethanol-precipitated, collected by centrifugation, washed
with 9 0 ~ ethanol and dried under reduced pressure. The pellet was resuspended in 100 ~tl of 10
mM-HEPES pH 7.0, 1 mM-EDTA per litre of original culture and heated for 1-5 min in a boiling
water-bath. [Denaturation of R N A was necessary, since genomic R N A was partly doublestranded at this stage (30% RNase-resistant) and, consequently, would have been lost as
material sedimenting more slowly than 50S during the following sucrose gradient.]
Centrifugation was carried out in polyallomer centrifugation tubes containing 10 to 30 ~ (w/v)
sucrose in 100 mM-NaC1, 10 mM-HEPES pH 7.0, formed as a step gradient (five steps) in a
Spinco SW55 rotor at 50000 rev/min and 15 °C for 100 min. Fractions were collected by a sterile
syphon system and aliquots were scintillation-counted. Fractions containing genomic R N A
sedimenting around 50S were pooled, passaged through a Chelex 100 column (0.3 x 0.5 cm),
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(a)
1
2
3
(b)
5.0
4.0
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
t
I
3.0
~-- 2.0
x
~ 1-5
0.8
0.6
7
11
15
19
23
Distance migrated (mm)
t
27
Fig. 2. Molecular weight determination of measles virus genomic RNA. (a) Electrophoresis in 1
agarose under fully denaturing conditions. Lane 1, VSV RNA; lane 2, RNA from nucleocapsids (after
DNase I treatment); lane 3, mixture of material in lanes 1 and 2. (b) Calibration curve for the molecular
weight determination of measles virus genomic RNA, using data from (a).
adjusted to 1 mM-EDTA and ethanol-precipitated. As seen in Fig. 1(b) it is possible by this
method to isolate intact genomic RNA. This R N A remains intact after incubation for 1 h at
41 °C in 10 mM-HEPES pH 7-0, 1 mM-EDTA or for several weeks as an ethanol precipitate at
-20
°C; it is fully sensitive to RNase digestion (results not shown). In addition to genomic
minus strands, it contained considerable amounts of full-length plus strands and was 50%
RNase-resistant after self-annealing. The yield of genomic R N A varies from infection to
infection, but was of the order 0.5 to 1-5 pg per litre of spinner culture. This is low compared to
yields of other negative-strand viruses, e.g. vesicular stomatitis virus (VSV), but it is enough for
cloning experiments, which are in progress (K. Baczko, M. Billeter & V. ter Meulen, unpublished results).
To determine the molecular weight of measles virus genomic RNA, nucleocapsid R N A (after
treatment with DNase I) was mixed with VSV genomic RNA, denatured and electrophoresed
into a 1% agarose gel containing methylmercury hydroxide (Bailey & Davidson, 1976) for 3 h at
30 mA, 70 V (Fig. 2a). Migration distances were plotted against the logarithm of the molecular
weights ofVSV (Repik & Bishop, 1971) and 28S and 18S ribosomal reference RNAs (Stewart &
Letham, 1973). The graph (Fig. 2b) shows a clear linear relationship in three experiments between the known molecular weights of the marker RNAs and their distance of migration. Therefore, we feel confident in our estimation of the molecular weight of measles virus genomic R N A
as 4.5 x 106. The relationship between log of molecular weight and distance migrated under the
conditions used is known for RNA molecules up to 6-67 x 106 (Wege et al., 1981). The value of
4.5 x 106 for measles virus genomic R N A was also obtained (results not shown) using coronavirus RNAs (a gift from H. Wege) as size markers. The molecular weight found here differs
significantly from the published value of 6.2 x 106 (Schluederberg, 1971). However, that figure
was derived from sedimentation in zonal sucrose gradients, which may be subject to some
inaccuracy, due to the non-linearity of the gradient and the particular conformation of the RNA.
Measles virus R N A has a molecular weight intermediate between that of other negative-strand
viruses, e.g. VSV 3.8 x 106 (Repik & Bishop, 1971) and respiratory syncytial (RS) virus 5 × 106
(Stewart & Letham, 1973) which is in agreement with the estimated number of virus-coded
proteins: five for VSV (Wagner et al., 1972), six for measles (Graves et al., 1978; Stephenson &
ter Meulen, 1979) and seven for RS virus (Huang & Wertz, 1982).
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1413
We wish to thank Mike Carter for critical reading of the manuscript, Jeanette Hintermeier for excellent
technical assistance and Helga Kriesinger for typing the manuscript. This work was supported by the Hertie
Foundation and grant no. 3.045.81 of the Schweizerische Nationalfonds.
REFERENCES
BACZKO, K. & LAZZARINI, R. A. (1979). Efficient propagation of measles virus in suspension culture. Journal of
Virology 31, 854-855.
BAILEY, J. M. & DAVIDSON, N. (1976). Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Analytical Biochemistry 70, 75-88.
BELLINI, W. J., TRUDGETT,A. & McFARLIN,D. E. (1979). Purification of measles virus with preservation of infectivity
and antigenicity. Journal of General Virology 43, 633-639.
COMPANS, R. W. & CHOPPIN, P. W. (1967). Isolation and properties of the helical nucleocapsid of the parainfluenza
virus SV 5. Proceedings of the National Academy of Sciences, U.S.A. 57, 949-956.
GRAVES, M. C., SILVER, S. M. & CHOPPIN, P. W. (1978). Measles virus polypeptide synthesis in infected cells. Virology
86, 154-163.
HUANG, Y. T. & WERTZ, G. W. (1982). The genome of respiratory syncytial virus is a negative stranded R N A that
codes for at least seven m R N A species. Journal of Virology 43, 150-157.
KINGSBURY, D. W., BRATT, M. A., CHOPPIN, P. W., HANSON, R. P., HOSAKA, Y., TER MEULEN, V., NORRBY, E.,
PLOWRIGI-rr, W., ROTT, R. & WUNNER, W. n. (1978). Paramyxoviridae. lntervirology 10, 137-152.
LEHRACH, H., DIAMOND, D., WOZNEY, J. M. & BOEDTKER, H. (1977). R N A molecular weight determinations by gel
electrophoresis under denaturing conditions, a critical re-examination. Biochemistry 16, 47434751.
NAKAI, T., SHAND, F. L. & HOWATSON,A. F. (1969). Development of measles virus in vitro. Virology 38, 50-67.
REPIK, P. & BISHOP, D. rI. L. (1971). Determination of the molecular weight of animal R N A viral genomes by
nuclease digestion. I. Vesicular stomatitis virus and its defective T particle. Journal of Virology 12, 969-983.
SCHAFFNER, W. (1980). Direct transfer of cloned genes from bacteria to m a m m a l i a n cells. Proceedings of the
National Academy of Sciences, U.S.A. 77, 2163-2167.
SCHLUEDERBERG,A. (1971). Measles virus R N A . Biochemical and Biophysical Research Communications 42, 10121015.
STEPHENSON,J. R. & TER MEULEN,V. (1979). Antigenic relationships between measles and canine distemper viruses.
Comparison of immune response in animals a n d h u m a n s to individual virus-specific polypeptides. Proceedings of the National Academy of Sciences, U.S.A. 76, 6601-6605.
STEWART, R. R. & LETHAM,D. S. (1973). The Ribonucleic Acids, p. 124. Berlin, Heidelberg & N e w York: SpringerVerlag.
TER MEULEN,V. & CARTER,M. J. (1982). Morbillivirus persistent infections in animals and man. In Virus Persistence,
pp. 97-132. Edited by B. W. J. Mahy, A. C. Minson & G. K. Darby. Cambridge: Cambridge University
Press.
WAGNER, R. R., KILEY, M. P., SNYDER, R. M. & SCHNAITMAN,C. A. (1972). Cytoplasmic compartmentalization of the
proteins and ribonucleic acid species of vesicular stomatitis virus. Journal of Virology 9, 672-683.
WEGE, H., SIDDELL,S., STURM,M. & TER MEULEN,V. (1981). Coronavirus J H M : characterization of intracellular viral
R N A . Journal of General Virology 54, 2 1 3 ~ 1 7 .
(Received 9 November 1982)
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