Journal of General Virology (1990), 71, 775-783.
775
Printed in Great Britain
Host cell proteins required for measles virus reproduction
Sue A. Moyer,* Susan C. Baker t and Sandra M. Horikami
Department of Immunology and Medical Microbiology, University of Florida College of Medicine, Gainesville,
Florida 32610, U.S.A.
We have developed a cell-free system derived from
measles virus-infected cells that supported the transcription and replication of measles virus RNA in vitro.
The data suggest that tubulin may be required for these
reactions, since an anti-fl-tubulin monoclonal antibody
inhibited viral RNA synthesis and the addition
of purified tubulin stimulated measles virus RNA
synthesis in vitro. Tubulin may be a subunit of the viral
RNA polymerase, since two different anti-tubulin
antibodies, one specific for the t - and another specific
for the ~-subunit of tubulin, coimmunoprecipitated the
measles virus L protein as well as tubulin from extracts
of measles virus-infected cells. Other experiments
further implicated actin in the budding process during
virus maturation, as there appeared to be a specific
association of actin in vitro only with nucleocapsids
that have terminated RNA synthesis, which is presumably a prerequisite to budding.
Introduction
Richardson et al., 1985; Rima et al., 1986; Shioda et al.,
1983, 1986). We previously described in vitro RNA
synthesis systems for both Sendai virus and vesicular
stomatitis virus (VSV), a negative-strand RNA virus of
the rhabdovirus family, which utilize cytoplasmic
extracts of virus-infected cells permeabilized with
lysolecithin (Carlsen et al., 1985; Peluso & Moyer, 1983).
These extracts accurately transcribe viral mRNAs and
can also support genome RNA replication and encapsidation. An advantage of the cell-free transcription
system is that it can be used in reconstitution experiments to identify and purify both viral and host proteins
required for RNA synthesis (Baker & Moyer, 1988;
Peluso & Moyer, 1988). We have adapted this methodology to prepare a cell-free system from measles virusinfected cells and show that it supports viral mRNA
synthesis and genome RNA replication in vitro.
We have been interested in understanding the role of
the host cell in negative-strand RNA virus infections.
Our laboratory (Moyer et al., 1986), as well as that of Hill
et al. (1986), has reported the requirement of components
of the microtubule system for VSV and Sendai virus
RNA synthesis. We have recently shown that tubulin is
required for RNA synthesis by both Sendai virus and
VSV (New Jersey), whereas the microtubule-associated
proteins are required for VSV of both the New Jersey
and Indiana serotypes, but not for Sendai virus (S. M.
Horikami, R. Williams, Jr & S. A. Moyer, unpublished
data). We report here that, like Sendai virus, measles
virus also appears to require tubulin for RNA synthesis.
In addition previous studies (Bohn et al., 1986; Stallcup
et al., 1983) have suggested that actin, another cytoskele-
The paramyxoviruses, measles and Sendai viruses, each
contain a single-stranded RNA genome of the negativestrand sense. These viruses package an RNA-dependent
RNA polymerase consisting of the L and P protein
subunits, which catalyse transcription and replication of
the nucleocapsid template containing the genome RNA
encapsidated in the major nucleocapsid protein N and
NP, respectively (Ray & Fujinami, 1987; Seifried et al.,
1978; Stallcup et al., 1979; Stone et al., 1971). The
envelope proteins, F, H or HN and M, constitute the
remaining structural proteins of the virion (Mountcastle
& Choppin, 1977; Wechsler & Fields, 1978). There are,
in addition, viral non-structural proteins whose functions
are unknown: the C protein, which is synthesized in
infected cells from an overlapping reading frame of the P
mRNA (Bellini et al., 1985; Dethlefsen & Kolakofsky,
1983; Dowling et al., 1986; Shioda et al., 1983) and the V
protein, which is translated from a measles virus RNA
polymerase-edited transcript of the P gene (Cattaneo et
al., 1989).
Transcription of the paramyxovirus genome RNA
occurs from the 3' end to the 5' end of the genome,
sequentially yielding the leader RNA, followed by the N
and NP, P + C, M, F, H and HN and L mRNAs for
measles and Sendai viruses, respectively (Dowling et al.,
1986; Glazier et al., 1977; Leppert et al., 1979;
t Present address: Department of Microbiology, University of
Southern California School of Medicine, Los Angeles, California
90033, U.S.A.
0000-9108 © 1990 SGM
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776
S. A. Moyer, S. C. B a k e r and S. M . H o r i k a m i
tal c o m p o n e n t , m a y be i n v o l v e d in measles virus
m a t u r a t i o n by b u d d i n g from the p l a s m a m e m b r a n e .
A c t i n is, in fact, p a c k a g e d into completed virions
(Tyrrell & N o r r b y , 1978; W a n g et al., 1976). W e report
here that there was a tight association of actin with
measles nucleocapsids in vitro, specifically u p o n the
t e r m i n a t i o n of R N A synthesis, w h i c h is p r e s u m a b l y a
prerequisite for b u d d i n g .
Methods
Cells and viruses. Measles virus (Edmonston strain) was grown in
spinner HeLa cells (both generous gifts from Dr Peter Dowling,
UMDNJ-New Jersey Medical School, Newark, N.J., U.S.A.), and a
high titred virus stock was prepared as described by Udem & Cook
(1984). The measles virus experiments were carried out in the human
cell line A549 (American Tissue Culture Collection).
Sendai virus (Harris strain) was grown in embryonated chicken eggs
and purified as described previously (Carlsen et al., 1985). The
preparation of Sendai virus-infected cell extracts and conditions for in
vitro Sendai RNA synthesis were as described by Carlsen et al. (1985).
Measles virus in vivo RNA synthesis. Confluent 100 mm dishes of
A549 cells were infected with measles virus at an m.o.i, of 5 at 37 °C.
The cultures were labelled with [3H]uridine (50 ~tCi/ml; ICN) in the
presence of actinomycin D (2 ~tg/ml)at various times after infection, as
indicated in the text. At the end of the labelling period a cytoplasmic
extract was prepared, after permeabilizing the infected cells with 2 ml
lysolecithin (250 ~tg/ml)for 1 min at 4 °C as previously described by
Carlsen et al. (1985), except the cells were scraped in HND buffer,
containing 0.1 M-HEPES pH 8-1, 0.05 M-NH,CI and l mM-DTT.The
cytoplasmic extracts were digested with Proteinase K, the RNA was
purified by phenol-chloroform extraction and then analysed by
electrophoresis on acid/8 M-urea/1"5% agarose gels and fluorography as
described previously (Peluso & Moyer, 1988).
Measles virus in vitro RNA synthesis. Cytoplasmic extracts of measles
virus-infected A549 cells for use in RNA synthesis were prepared as
described above, except that the cells were scraped into a transcription
reaction mixture containing 0.1 M-HEPES pH 8.1, 0.05
M-NH4C1, 7 mM-KCI, 4.5 mM-magnesium acetate, 1 mM each of
spermidine, DTT, CTP and GTP, 10 ~tM-UTP, 2 mM-ATP, 2 ~tg/ml
actinomycin D, 3.3 mg/ml creatine phosphate and 40 units/ml creatine
phosphokinase. The extracts were incubated at 30 °C for 3 h in the
presence of [3H]UTP (250 ~Ci/ml; ICN). The RNA was purified as
described above and analysed by electrophoresis on acid/urea/agarose
gels. To assay genome replication in the in vitro RNA synthesis
reactions the 3H-labelledRNA products were digested with micrococcal nuclease prior to Proteinase K digestion and processed as described
previously (Carlsen et al., 1985).
To prepare components for reconstitution experiments the infected
cell extract was centrifuged in 0.7 ml Beckman ultraclear tubes that
contained 100 ixlof 30% (v/v) glycerolin HD buffer (10 mM-HEPES pH
8.1 and 1 mM-DTT)on a 25 ~tl cushion of 96% (v/v) glycerol in HD
buffer at 54000 r.p.m, for 65 min at 4 °C in a Beckman SW55 rotor. The
viral nucleocapsid fraction, which contained the RNA-N template and
the associated RNA polymerase (L and P), was collected from the top
of the 96% cushion; the remaining supernatant fluid constituted the
measles soluble protein fraction. Purified bovine brain soluble tubulin
was kindly provided by Dr Robley Williams Jr, Vanderbilt University.
Analysis of viral proteins. Measles virus-infected A549 cells were
labelled separately with either [3H]leucine (25 ~tCi/ml; ICN) or
[35S]methionine(25 p.Ci/ml, New England Nuclear) in the presence of
actinomycin D (2 p.g/ml) from 15.5 to 17 h post-infection. The soluble
protein fraction was prepared from the combined cytoplasmic extracts
prepared in HND buffer as described above. Samples were immunoprecipitated with various antibodies (2 ~tl),the immunocomplexeswere
collected by binding to Staphylococcus aureus (Cowan strain) and
analysed by electrophoresis on 11~ polyacrylamide-SDS gels (Peluso
& Moyer, 1983). The anti-measles virus serum no. 2676 was from a
subacute sclerosing panencephalitis (SSPE) patient and was the
generous gift of Dr Peter Dowling. The anti-•-tubulin, anti-actin and
anti-SP-2 antibodies used in viral protein and RNA experiments were
described previously (Moyer et al., 1986). The c~-tubulin-specific
peptide antibody was a generous gift of Dr J. Bulinski (Gundesen et al.,
1984). The radiolabelled soluble protein fractions from either measles
virus-infected cells or uninfected cells were also used for reconstitution
experiments with the viral nucleocapsid fraction, either in the presence
of all four nucleoside triphosphates to allow RNA synthesis, or in the
absence of the nucleosidetriphosphates with the addition of EDTA (20
mM), which prevented RNA synthesis (data not shown). The product
nucleocapsidswere purified by banding in 20 to 40 % CsCI gradients, as
previously described (Simonsen et al., 1979). The nucleocapsids were
collected, diluted and pelleted at 50000 r.p.m, for 2.5 h at 4 °C in the
SW55 rotor and the associated proteins were analysed by SDS-PAGE
and fluorography, as described above.
Results
R N A synthesis in measles virus-infected A 5 4 9 cells
T h e first steps in developing a n in vitro R N A synthesis
system for measles virus were to find the o p t i m a l cell line
a n d d e t e r m i n e the kinetics of viral R N A synthesis in
vivo. A m o n o l a y e r cell line is preferred for the methodology we employ for extract p r e p a r a t i o n . W e tested
several lines, i n c l u d i n g h u m a n cells ( m o n o l a y e r H e L a ,
A431 a n d A549) as well as m o n k e y cells (Vero a n d
L L M C K - 2 ) a n d the A549 cells were far superior (greater
t h a n fivefold) in terms of the total a m o u n t of measles
virus R N A s synthesized in vivo. However, the yield of
progeny virus from this cell line is very poor (data n o t
shown). W e d e t e r m i n e d that measles virus R N A
synthesis in A549 cells p e a k e d at 17 to 18 h after
infection a n d all of the viral m R N A s , as well as g e n o m e
R N A , were synthesized in the infected cells (data n o t
shown). I n addition, several r e a d t h r o u g h R N A products,
which are larger t h a n the F m R N A , were also
synthesized in vivo, as was previously observed (Dowling
et at., 1986; W o n g & H i r a n o , 1987; Y o s h i k a w a et al.,
1986) (See Fig. 1, lane 1).
I n vitro measles virus R N A synthesis
T o develop a cell-free t r a n s c r i p t i o n system, cytoplasmic
extracts of measles virus-infected A549 cells were
prepared in reaction m i x t u r e (see Methods) at 17 h after
infection, the time of m a x i m a l R N A synthesis in vivo. By
systematically v a r y i n g the reaction c o m p o n e n t s (data not
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Measles virus reproduction
1
2
3
4
1
2
777
3
Nj
1
Fig. 1. Measles virus in vitro RNA synthesis. Measles virus RNAs
synthesized in vitro in two different experiments (lanes 2 and 3) are
compared with either 3H-labelledRNAs from measlesvirus-infected
A549 cells (lane 1) or 3H-labelled RNAs from Sendai virus-infected
BHK cells (lane 4).
shown) the optimal conditions stated in Methods for in
vitro measles virus R N A synthesis were determined, and
include the key conditions of 0.1 M-HEPES pH 8-1 and
0-05 M-NH4C1, with an incubation period of 3 h at 30 °C.
Utilizing this reaction mixture in two different experiments shown in Fig. 1, the cytoplasmic extract was
shown to synthesize measles virus RNAs (lanes 2 and 3)
identical to those synthesized in the infected cell (lane 1).
By comparing the in vivo and in vitro synthesized measles
RNAs with marker Sendai virus mRNAs (Fig. 1) and
VSV mRNAs (data not shown), we calculated the sizes
of the measles virus RNAs as 1900, 1900, 1500, 2200,
2000 and about 6000 nucleotides for the N, P, M, F, H
and L mRNAs, respectively, and greater than 12000
nucleotides for the genome RNA. These values are in
reasonable agreement with the sizes [nucleotides minus
poly(A)] obtained by sequencing cDNA clones of several
of the mRNAs; 1688, 1657, 1472 and 1949 for N, P + C ,
M and H, respectively (Alkhatib & Briedis, 1986; Bellini
Fig. 2. Measles virus in vitro genome RNA synthesis. Micrococcal
nuclease-resistantmeaslesvirus RNAs synthesizedin vitro (lane 3) and
in vivo (lane 2). Lane 1 shows marker 3H-labelledmeasles virus total
RNA synthesizedin vivo.
et al., 1985, 1986; Billeter et al.,
et al., 1986; Rozenblatt et al., 1985).
1984;
Gerald
Since genome length R N A also appeared to be
synthesized in vitro, we wanted to confirm that this
product was encapsidated as it is in vivo. Cytoplasmic
extracts from measles virus-infected A549 cells were
prepared at 17 h post-infection, incubated in reaction
mixture with [3H]UTP and the products were treated
with micrococcal nuclease to digest any unencapsidated
RNA. The micrococcal nuclease-resistant R N A species
synthesized in vitro (Fig. 2, lane 3) and in vivo (lane 2)
comigrated in the agarose-urea gel, with the largest
product synthesized being greater than 12000 nucleotides. The encapsidation reaction did not require de novo
protein synthesis, since no translation occurred in these
reactions, but utilized the preformed proteins present in
the cell extract. The micrococcal nuclease-resistant in
vitro and in vivo product RNAs also banded in the same
position as nucleocapsids at 1.31 g/ml on CsCI density
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778
S. A. Moyer, S. C. Baker and S. M. Horikami
1
gradients (data not shown). It should be noted that, in
addition to the full-length genome RNA, there were
smaller nuclease-resistant RNAs that were also synthesized, particularly in vitro. Although the replication and
encapsidation of the large measles virus genome R N A
occurred in vitro, it appeared to be relatively ineffÉcient
with premature stop sites, a finding we also reported for
wild-type Sendai virus genome R N A replication in vitro
(Carlsen et al., 1985).
2
3
4
Inhibition o f measles virus R N A synthesis by an
anti-tubulin antibody
We previously reported that tubulin was necessary for
Sendai virus, as well as VSV R N A synthesis in vitro
(Moyer et al., 1986). It was therefore of interest to
determine whether measles virus, a member of a
different genus of the paramyxovirus family, also had a
similar requirement. Extracts of measles virusqnfected
A549 cells were incubated with [3H]UTP in reaction
mixture in the presence of individual monoclonal
antibodies and the products were analysed by agarose gel
electrophoresis. Compared with the control the anti-fltubulin antibody completely inhibited all measles virus
R N A synthesis (Fig. 3, lanes 1 and 2, respectively). In
contrast, neither an anti-myeloma ascites fluid nor an
anti-actin antibody had any effect on viral R N A
synthesis (Fig. 3, lanes 3 and 4, respectively). In
experiments using dilutions of the anti-fl-tubulin antibody the amount of inhibition of measles virus R N A
synthesis decreased with decreasing amounts of the
antibody (data not shown). These data suggested that the
host cell-derived tubulin might play a role in the
synthesis of measles virus RNA.
Effect o f tubulin on measles virus R N A synthesis in vitro
Following the separation of the measles virus-infected
cytoplasmic extract into nucleocapsid and soluble protein fractions as described in Methods, one can assay the
effect of purified components on transcription. The
nucleocapsid fraction alone was capable of some viral
R N A transcription in vitro, since it contained the RNAN template, the viral R N A polymerase (Fig. 4, lane 1)
and probably some tubulin (see Discussion). The
addition of increasing amounts of the soluble protein
fraction from infected cells stimulated synthesis of all the
viral m R N A s (Fig. 4, lanes 2 to 5). The stimulation (2-9fold increase) appeared to be saturated when the soluble
protein fraction added to nucleocapsids was from an
equivalent number of infected cells (Fig. 4, lane 3). These
data suggested that one or more proteins from the
infected cell were required for efficient measles virus
R N A synthesis. It should be noted that measles genome
Fig. 3. The effect of monoclonal antibodies on measles virus in vitro
RNA synthesis.Samples(100~tl)of an extract of measlesvirus-infected
A549 cells were incubated in reaction mixture at 4 °C for 30 min and
then at 30 °C for 2 h in the presence of 980 units/ml of RNasin
(PromegaBiotech)as describedpreviously(Moyeret at., 1986)with no
additions (lane 1), 2 ~tlanti-fl-tubulinmonoclonalantibody(lane 2), 2 p.1
anti-SP-2 myelomaascites fluid (lane 3) and 2 ~tlanti-actin monoclonal
antibody (lane 4). The measles virus RNAs are identifiedon the left.
R N A replication seemed to occur only inefficiently in
the reconstituted reactions in comparison with the
unfractionated extracts (Fig. 1 and 2).
Since the anti-~-tubulin antibody appeared to inhibit
measles virus R N A synthesis, a confirmation of this
result would be to show that the addition of tubulin had a
stimulatory effect. In fact, purified tubulin alone did
stimulate (2.3-fold) measles virus transcription, although
the effect was primarily on the comigrating N and P
mRNAs, with a smaller stimulation of the other m R N A s
(Fig. 4, lanes 6 to 9). These data suggested that tubulin
may be one, but perhaps not the only, factor in the
soluble protein fraction utilized for measles virus R N A
synthesis in vitro.
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1
2
3
4
5
6
7
8
9
1
2
3
779
4
--Tb
Fig. 4. Effect of cell protein and tubulin on measles virus in vitro R N A
synthesis. Extracts of measles virus-infected cells were separated into
nucleocapsid and soluble protein fractions as described in Methods.
The measles virus intracellular nucleocapsids from 3 × 106 cells (3.75
mg]ml) were added to transcription reactions containing no additions
(lane 1) or the soluble protein fraction at concentrations of approximately 0.625 (lane 2), 1.25 (lane 3), 2.50 (lane 4) and 3.75 (lane 5) mg/ml,
respectively, or tubulin at 42 (lane 6), 107 (lane 7), 212 (lane 8) and 318
(lane 9) ~tg/ml for 3 h at 30 °C. The measles virus RNAs are identified
on the left.
Tubulin appeared to stimulate measles virus transcription so we wanted to determine whether there might be
an association of tubulin with either of the viral
polymerase subunits, as we previously reported for VSV
(Moyer et al., 1986). The 3H and 35S double-labelled
soluble protein fraction from measles virus-infected cells
was incubated with various antibodies and the immunoprecipitated proteins were analysed by SDS-PAGE.
Compared with the control anti-myeloma ascites fluid
(Fig. 5, lane 4), the anti-fl-tubulin monoclonal antibody
and an anti-~-tubulin peptide antibody (Gundesen et al.,
Fig. 5. Immunoprecipitation of measles virus soluble protein with
various antibodies. The combined soluble protein fraction from
[3H]leucine- or [3sS]methionine-labelled measles virus-infected cells
was incubated with 2 ~tl antibody no. 2676 from an SSPE patient
(lane 1), 2 ~tl anti-fl-tubulin monoclonal antibody (lane 2), 2 Ixl anti-cttubulin peptide rabbit antibody (Gundesen et al., 1984) (lane 3) and 2 lal
anti-SP-2 myeloma cell ascites fluid (lane 4). The measles virus proteins
L and N are identified on the left; Tb and Ac indicate tubulin and
actin, respectively.
1984) both specifically coimmunoprecipitated tubulin
and the measles virus L protein (Fig. 5, lanes 2 and 3,
respectively). The L protein was identified on the basis of
its size and its comigration with the presumptive viral L
protein immunoprecipitated with serum from an SSPE
patient (Fig. 5, lane 1). The position of tubulin was
identified by its immunoprecipitation from an uninfected cell extract with the anti-fl-tubulin antibody
(data not shown). The anti-tubulin antibodies also
immunoprecipitated another, apparently host protein,
with a much lower Ms of approximately 30K. These data
suggested that tubulin was associated with the measles
virus L protein in the soluble protein fraction of the
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S. A. Moyer, S. C. Baker and S. M. Horikami
780
(a) 1
2
3
4
5
(b) 1
2 3
4
5
p--
HNJ
Fo•
NP-,Ac
M m
Fig. 6. The association of actin with Sendai and measles virus
nucleocapsids in the absenceof RNA synthesis. Sendai (a) or measles
(b) virus nucleocapsids isolated from extracts of infected cells were
incubated in the appropriate reaction mixtures containing the
[3H]leucine and PsS]methioninedouble-labelledsoluble protein fraction from Sendai virus-infectedcells(a, lanes 2 and 3) or measles virusinfectedcells(b, lanes 1and 2) or uninfectedcells(a, lanes 4 and 5 and b,
lanes 3 and 4) in the presence (a, lanes 3 and 5 and b, lanes 2 and 4) or
absence (a, lanes 2 and 4 and b, lanes 1 and 3) of RNA synthesis (see
Methods and text). Lanes 1 (a) and 5 (b) are 3H-labeUedpurifiedSendai
virus and measles virus, respectively,with the viral proteins identified.
Ac indicates the 43K actin protein.
infected cell and therefore may be a subunit of the
measles virus R N A polymerase.
Association of proteins with RNA during replication
We demonstrated above that measles virus genome
R N A replication and encapsidation occurred in the cellfree system (Fig. 2). To identify the viral protein(s)
associated with the nucleocapsid product, unlabelled
measles virus nucleocapsids isolated from infected cells
were incubated in reconstitution experiments with the
3H- and 3sS-labelled soluble protein fraction from
uninfected or virus-infected cells either with or without
R N A synthesis. The inhibition of the R N A polymerase
activity required removal of the nucleotide substrates
from the reaction mixture as well as the addition of
EDTA, since there appeared to be a pool of nucleoside
triphosphates in the soluble protein fraction. The
product nucleocapsids were purified by banding on CsC1
gradients and the nucleocapsid-associated proteins were
analysed by S D S - P A G E . In the presence of substrates
and the measles virus soluble protein fraction R N A
transcription (Fig. 4) as well as limited R N A replication
occurred, since there was a two-fold increase in the N
protein (as well as the M protein) associated with
nucleocapsids (Fig. 6b, lane 2) compared to reactions
where replication was inhibited (Fig. 6b, lane 1). The low
level of N protein binding in the absence of replication
(lane 1) probably represents some exchange of the soluble
radiolabelled N with the template-bound N protein.
Genome R N A replication in the reconstituted system
was so low, as we noted earlier, that it was hardly
detectable by radiolabelling (Fig. 4), although it was
clearly detectable as measured by N protein association.
Interestingly, under conditions where R N A synthesis
was inhibited there was a specific, CsCl-stable association of actin with the template nucleocapsid, which
disappeared when R N A synthesis occurred (Fig. 6b,
lanes 1 and 2). We identified the 43K protein as actin by
its comigration with actin immunoprecipitated from
uninfected cells-with an anti-actin monoclonal antibody
and by the immunoprecipitation of the nucleocapsids
with bound actin by the anti-actin antibody (data not
shown). This specific association of actin with nucleocapsids when R N A synthesis was inhibited was not
mediated by other viral proteins or by an effect of R N A
replication, since a similar experiment with radiolabelled, uninfected cell-soluble protein showed the same
result (Fig. 6b, lanes 3 and 4). In the latter case,
transcription of the nucleocapsid template occurred in
the presence of nucleotide substrates, although R N A
replication did not, since N protein was not present.
In view of the apparent importance of actin in
paramyxovirus maturation by budding (Bohn et al.,
1986; Stallcup et al., 1983), we wanted to study actin
binding to nucleocapsids with other negative-strand
R N A viruses. In analogous experiments with Sendai
virus the unlabelled viral nucleocapsid and radiolabeUed
virus-infected or uninfected radiolabelled soluble protein
fractions (Carlsen et al., 1985) were reconstituted as
above with similar results (Fig. 6a). Actin in either
infected or uninfected cell soluble protein fractions
bound in a CsCl-stable association only to nucleocapsids
that were not synthesizing R N A (Fig. 6a, lanes 2 and 4,
respectively). In identical experiments with VSV, which
does not require actin for maturation, we detected no
binding of actin to nucleocapsids under any conditions
(data not shown). Thus, these data suggest that stable
actin binding occurred in vitro to paramyxovirus nucleocapsids specifically when R N A synthesis was inhibited.
Discussion
We have described here the development of a cell-free
transcription/replication system to study the viral and
host components required for measles virus R N A
synthesis. The basic methodology involving lysolecithin
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Measles virus reproduction
permeabilization of infected cells, which had previously
been successful for the establishment of in vitro replication systems for both VSV (Peluso & Moyer, 1983) and
Sendai virus (Carlsen et al., 1985), also worked very well
for measles virus. Perhaps the key feature yielding this
transcription system with our methodology was the
selection of a monolayer cell line, A549, which gave a
high level of viral RNA synthesis in vivo. This appeared
to be achieved, in part, by the fact that progeny measles
virus formation was very limited, resulting in the
accumulation of viral nucleocapsid templates within the
infected cell. The cell-free system appeared to synthesize
all the measles virus mRNAs with the correct sizes and
supported at least some replication of the genome RNA
and its encapsidation with N protein (Fig. I, 2 and 6).
Although specific activities cannot be determined in cell
extracts, on a per cell basis the relative levels of RNA
synthesis in the various systems is VSV >>measles
virus > Sendai virus.
Measles virus RNA replication in vitro did not require
de novo protein synthesis, but utilized the preformed
proteins present in the infected cell at the time of extract
preparation. It should be noted that the ability to
replicate genome RNA appeared to be substantially
reduced in the reconstitution reactions, although transcription could be fully restored (Fig. 2, 4 and 6). Perhaps
a labile component required for R N A replication was
lost in the period required for the separation of the
subfractions. By analogy with the VSV and Sendai virus
replication systems (Carlsen et al., 1985; Peluso &
Moyer, 1983) in vitro measles virus R N A synthesis (Fig.
I) probably did not represent initiation, but rather the
elongation of mRNAs and genome R N A already
initiated in vivo. However, this point needs to be further
investigated.
While this work was in progress, Ray & Fujinami
(1987) reported the in vitro synthesis of measles virus
mRNAs employing intracellular nucleocapsids isolated
from infected HeLa cells. Although the reaction conditions in the two systems are generally similar there are
some differences with regard to various components and
incubation conditions. These differences are perhaps
attributable to the different methodologies employed in
each case. As in our reconstitution experiments, Ray &
Fujinami (1987) did not observe in vitro R N A replication
from the isolated nucleocapsids by R N A labelling
experiments.
We have been particularly interested in understanding
the role of the host cell, particularly the components of
the cytoskeleton, in the reproduction of negative-strand
RNA viruses. As we previously reported for VSV and
Sendai virus (Moyer et al., 1986) tubulin also appeared to
be an essential component for measles virus R N A
synthesis, since an anti-fl-tubulin antibody inhibited
781
both viral transcription and replication in vitro (Fig. 3)
and the addition of purified tubulin stimulated viral
transcription (Fig. 4), although the level was not
impressive (about threefold). The inhibition did not
appear to be non-specific, since this anti-fl-tubulin
antibody did not inhibit transcription by the poxvirus
R N A polymerase (data not shown). The reason the
stimulation with tubulin was not greater, as expected
from the antibody result, appeared to be that endogenous
tubulin was present in the nucleocapsid fraction. Some
polymerized tubulin co-pelleted with the intracellular
nucleocapsids during the fractionation procedure (unpublished data) and some tubulin was probably attached
to the nucleocapsid via its association with L protein
(Fig. 5). Thus it is likely that these sources of endogenous
tubulin in the reaction were responsible for both the level
of RNA synthesis seen in the absence of any additions
(Fig. 4, lane 1), as well as the low level of stimulation
observed with the addition of soluble protein or purified
tubulin(Fig. 4). F u t u r e experiments employing purified
measles virus should be of interest in this regard.
In contrast to our results (Fig. 4) Ray & Fujinami
(1987) reported no stimulation of measles R N A synthesis
by an extract of uninfected cells, as monitored by the
incorporation of [32p]UMP into TCA-precipitable
material. The reason for this discrepancy is unclear,
although their assay may not be as sensitive as
monitoring individual mRNAs. The suggested involvement of tubulin we observed in measles virus R N A
synthesis was particularly intriguing, since both VSV
and Sendai virus also required microtubule components
for R N A synthesis (Moyer et al., 1986; Hill et al., 1986)
and in addition both rhabdoviruses (Chatterjee et al.,
1984) and paramyxoviruses (Hamaguchi et al., 1985)
seemed to require positioning of the subviral components
on the cytoskeleton during the productive infection in
vivo.
To understand how tubulin could be exerting its effect
we tested for its possible interaction with viral proteins.
In fact two different anti-tubulin antibody preparations
coimmunoprecipitated both tubulin and the measles
virus L protein (Fig. 5). These same antibodies also
coprecipitated tubulin and the VSV L protein (Moyer et
al., 1986 and data not shown). In contrast we detected no
such interaction with the Sendai virus L protein and
tubulin in a similar experiment (data not shown). Similar
data with two different antibodies with non-overlapping,
unique epitopes strengthens our conclusion that tubulin
and the measles virus L protein must interact in a
relatively stable fashion. It seems unlikely that two
different tubulin epitopes would be contained in both the
measles virus and VSV L proteins. The anti-0t-tubulin
peptide antibody immunoprecipitated less tubulin and
measles virus L protein, since it apparently had a much
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782
S. A. Moyer, S. C. Baker and S. M. Horikami
lower titre than the monoclonal anti-fl-tubulin antibody.
It should also be noted that the anti-SSPE sera
immunoprecipitated only small amounts of the measles
virus L protein and no detectable tubulin (Fig. 5, lane 1).
Tubulin was probably not detectable in the latter case,
since the anti-L activity in this serum was weak and
tubulin (and all host cell proteins) was poorly labelled late
in measles virus infection.
Previous experiments have suggested that another
cytoskeleton component, actin, was essential for the
reproduction of paramyxoviruses, specifically for budding. First, actin was found to be packaged within the
virion of both measles and Sendai viruses (Tyrrell and
Norrby, 1978; Wang et al., 1976) and biochemical data
showed that actin appeared to bind specifically to the M
protein of Sendai and Newcastle disease viruses (Giuffre
et al., 1982). Stallcup et al. (1979) showed that treatment
of infected cells with cytochalasin B, which disrupted
actin filaments, prevented virion formation and resulted
in the accumulation of viral nucleocapsids within the
infected cell. From these data it was postulated that actin
filaments might be essential for the transport of the
nucleocapsid from the cytoplasm to the cell surface
where budding occurred. Further support for this idea
came from electron microscopic studies by Bohn et al.
(1986), who showed that in cytoskeleton preparations of
measles virus-infected cells the growing end of the actin
filament protruded into budding virus particles and was
in close association with viral nucleocapsids. These
authors suggested that the vectorial growth of the actin
filaments was involved both in the transport of the
nucleocapsid to the surface of the cell and for budding
itself.
The results presented here suggested that actin was
tightly bound to both Sendai and measles nucleocapsids
in vitro specifically when RNA synthesis had been
terminated (Fig. 6). The actin binding was extremely
stable, since the CsCI centrifugation step used for
purification of the nucleocapsids normally removed all
the viral proteins except N from the nucleocapsid. It
should be noted that we were able to inhibit RNA
synthesis only by the addition of EDTA to the reactions
because of endogenous nucleotides in the cell extracts.
EDTA may potentially cause some aberrant proteinR N A interactions, but similar experiments with VSV
did not result in actin bound to VSV nucleocapsids,
suggesting that our observation is specific for
paramyxoviruses.
We propose that it is the transcriptionally silent
paramyxovirus nucleocapsids that bind to the actin
filaments for transport and budding, and the actin is
incorporated into progeny virions. The actin would then
be released when new R N A synthesis begins upon
subsequent infection with the virus. How is viral R N A
synthesis terminated in the cell prior to budding? In the
case of VSV the M protein appeared to function, in part,
to inhibit transcription both in vivo and in vitro (for a
review see Banerjee, 1987). However, VSV did not
appear to utilize actin filaments for maturation, since
actin was not packaged in the virion, cytochalasin B had
no effect upon virus yield (Gentry & Busserau, 1980) and
finally we found no actin binding to VSV nucleocapsids
under any conditions in vitro (data not shown). Indirect
evidence suggested that the paramyxovirus M protein,
which was known to interact with actin in vitro (Giuffre
et al., 1982) may have also inhibited R N A synthesis in
vitro (Marx et al., 1974). However, our data suggested
that the M protein need not necessarily be present, since
when RNA synthesis was artificially inhibited in vitro
actin bound tightly to silent nucleocapsids in the
presence of uninfected cell protein (Fig. 6). Further
experiments are in progress to understand the mechanisms by which these various cytoskeletal components
participate in the reproduction of paramyxoviruses.
The authors would like to thank Dr Peter Dowling for helpful
discussions and for providing the measles virus, HeLa cells and measles
antisera; Dr Jeanette Bulinski for the anti-ct-tubulin peptide antibody
and Dr Robley Williams Jr for the tubulin. We would also like to thank
Sharon ToUefson for dedicated technical support and Pat Austin for
excellent secretarial assistance. This work was supported by Public
Health Service grant AI 14594 from the NIH.
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