J. gen. Virol. (1984), 65, 1245-1251.
Printed in Great Britain
1245
Key words: vaccinia virus~expression~transcription~initiation
Expression of Cloned Vaccinia Virus DNA Sequences Introduced into
Animal Cells
By C L A U D E T T E
B O N I , l t M A R I A N O E S T E B A N 2. A N D
ANGEL PELLICER t
1Kaplan Cancer Center and Department of Pathology, New York University Medical Center,
New York, New York 10016 and 2Department of Biochemistry, Microbiology and Immunology,
Downstate Medical Center, Brooklyn, New York 11203, U.S.A.
(Accepted 21 March 1984)
SUMMARY
Individual cloned HindlII fragments of vaccinia virus D N A were introduced into
cells by DNA-mediated gene transfer. The presence of individual fragments in the
different transformants was confirmed by Southern blot hybridization analysis. Several
of the transformants were found to express viral sequences at various levels. The sizes
of the transcripts containing vaccinia virus sequences were highly heterogeneous, with
no discrete species of RNA. Positive clones contained vaccinia virus sequences in both
the poly(A) ÷ and poly(A)- RNA fractions, although the prevalence of these sequences
was variable in the two fractions. The S1 nuclease map of the 5' end of the transcripts
from transformants containing the HindlII-J fragment revealed a unique 5' end,
similar to RNA from virus-infected cells• In contrast, analysis of the 3' end of RNAs
from these transformants showed a high degree of heterogeneity, which might explain
the heterogeneity found in Northern blot patterns. In this report, it is shown for the first
time that in cells transformed with vaccinia virus D N A there is proper initiation for, at
least, the viral thymidine kinase gene.
Vaccinia virus is a cytopJ~t~lmc virus wmcu contains the enzymes that are involved in
transcription and processing of viral RNAs. These include DNA-dependent R N A polymerase,
poly(A) polymerase, guanyl- and methyltransferases, topoisomerase, endoribonuclease and
phosphohydrolases (Moss, 1974; Dales & Pogo, 1981). These unique properties of vaccinia virus
suggest that the regulatory sequences of the viral genome might have evolved in a different way
to those of the host cell. Indeed, several regions of the vaccinia genome have been sequenced
(Venkatesan et al., 1981, 1982; Baroudy et al., 1982; Pickup et al., 1983), one of which contains
the viral thymidine kinase (tk) gene (Weir & Moss, 1983; Hruby et al., 1983), and it was found
that the consensus sequences normally found at the 5' and 3' end regions of prokaryotic and
eukaryotic genes are not present in vaccinia virus DNA. We have previously shown that whole
vaccinia virus D N A can be introduced, stably maintained and its presence be associated with
some of the changes in the biochemical properties of the transformed cells (Petlicer & Esteban,
1982). To characterize the expression of vaccinia virus sequences in such cells, we have
established cell lines containing discrete regions of the viral genome and studied the
transcriptional levels and fidelity of their viral sequences.
In all cases, the recipient cells were mouse L t K ( - ) , adenine phosphoribosyltransferase ( - )
[aprt(-)] fibroblast cells (Wigler et al., 1979a) which were maintained in Dulbecco's modified
Eagle's medium containing 10 % calf serum and antibiotics. The WR strain of vaccinia virus was
used as a source of D N A which was prepared as previously described (Cabrera et al., 1978). To
facilitate the analysis of different regions on the viral genome, we cloned HindlII restriction
fragments of vaccinia virus DNA. Briefly, intact viral and pBR322 DNAs were digested with
HindlII and ligated under appropriate conditions to favour the formation of recombinants. The
i Present address: Laboratoire de Neurobiologie, Institut de Microbiologie, B~timent 409, Universit~ ParisSud, Orsay, France.
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bacterial plasmid pBR322 had been treated with bacterial alkaline phosphatase to prevent recircularization. Bacterial clones resistant to ampicillin were screened by hybridization to nicktranslated whole vaccinia virus D N A using the Grunstein & Hogness technique (1975). Positive
colonies were isolated, the D N A obtained by a miniprep protocol (Birnboim & Doly, 1979),
digested with HindIII and analysed by Southern (1975) blots to confirm that it contained viral
sequences and in addition to identify the recombinants from different viral fragments. Selected
recombinants were grown and D N A was purified by centrifugation in ethidium bromide-CsC1
(Maniatis et al., 1982). By this approach we obtained recombinant plasmids containing the
HindIII fragments E, F, G, H, I, J, L and M.
It has been reported that sequences exist (generically referred to as 'enhancers') that can
increase the expression of D N A which is in their proximity (see Weiss et al., 1982). Because our
aim is to study expression of vaccinia virus D N A in mammalian cells, we wanted to determine if
such enhancers might have a role in the expression of vaccinia virus DNA. To this end, we
cloned in the proximity of the HindIII-J fragment the long terminal repeats (LTR) of Moloney
murine leukaemia virus. To avoid the 'poison' sequences of pBR322, the LTR was subcloned in
pAT153 (Twigg & Sherratt, 1980). Subsequently, the HindIII-J fragment was introduced into
the HindIII site of the pAT153-LTR plasmid, following the same procedures as described
above. Recombinants containing the HindIII-J fragment in each orientation were isolated.
Plasmids containing the various HindIII restriction fragments of vaccinia virus D N A were
independently introduced into mouse L t k ( - ) a p r t ( - ) cells using the calcium phosphate
precipitation technique (Graham & van der Eb, 1973). We have used three different vectors to
co-transform with the vaccinia sequences: these were clones of herpes simplex virus (HSV)
thymidine kinase (Wigler et al., 1977; Maitland & McDougall, 1977; Pellicer et al., 1978),
hamster adenine phosphoribosyltransferase (Lowy et al., 1980) and the bacterial transposon
gene that confers resistance to the antibiotic G418 (Colbere-Garapin et al., 1981). All of the
HindlII fragments, except HindIII-J, were alternatively introduced with tk or aprt in order to
determine if different vectors could influence the biological properties of the transformants.
Since the HindIII-J fragment contains the vaccinia virus tk gene (Hruby & Ball, 1982; Weir et
al., 1982), this fragment was introduced into recipient cells using G418 selection because this
marker is not involved in nucleic acid metabolism.
To determine the physical presence and distribution of the cloned HindIII fragments of viral
D N A in transformed cells, Southern blot hybridization analyses were carried out. High
molecular weight DNA isolated from a series of transformants was digested with HindIII, then
blotted and hybridized to nick-translated isolated HindIII fragments of vaccinia virus DNA.
Representative transformed clones are indicated in Fig. 1. Most of the transformants (lanes c to
f ) were found to contain bands of the same size as the fragment of viral D N A that had been
introduced into them (see marker fragments, lane a). This shows that the transformants
contained the entire D N A fragments used for transfection.
Since vaccinia virus replicates in the cytoplasm of infected ceils and carries within its particles
all of the enzymes required for transcription (Moss, 1974; Dales & Pogo, 1981), it was of interest
to determine whether the viral sequences now introduced into the nucleus could also be
expressed. To that end, total RNA was extracted from transformants, fractionated by oligo(dT)cellulose chromatography and both poly(A)- and poly(A) + R N A fractions were analysed for
the presence of viral sequences by Northern blot hybridization with appropriate D N A
restriction fragments that had been purified from recombinant clones. Total RNA prepared
from virus-infected cells at early (E, 2 h) and late (L, 4 h) times post-infection served as controls.
Five species of RNA have been shown to hybridize with the 5 kb fragment J of vaccinia virus
DNA, one of which (590 bp) codes for the viral tk enzyme (Bajszar et al., 1983). Fig. 2 shows a
representative autoradiogram of Northern blots from various transformants corresponding to
cloned HindII! fragments J and I. RNAs from infected cells appear as discrete bands on gels
when isolated at early times post-infection, while at late times we found them to be much more
heterogeneous. Upon examination of poly(A)- and poly(A) + RNA fractions from biochemical
transformants, hybridization was observed in a number of clones. The pattern and extent of
hybridization was highly heterogeneous, ranging in size in some cases up to about 10 kb, with
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(a)
(b)
(c)
(d)
(e)
(f)
E-
I-
JL-
Fig. 1. Detection of cloned vaccinia virus D N A sequences in transformed cells. The co-transformation
experiments were performed as described by Wigler et al. (1979b) using as vectors 1 ng of: (a) ptk2, a
pBR322 plasmid carrying a 3.6 kb HSV-1 BamHI fragment that contains the HSV tk gene (Wigler et al.,
1977), (b) pAprt, a pBR322 plasmid carrying the hamster aprt gene (Lowy et al., 1980), or (c) pNeo, a
pBR322 plasmid carrying the bacterial gene that confers resistance to the antibiotic G418 (ColbereGarapin et al., 1981). Cloned HindlII fragments of vaccinia virus DNA (5 ktg/plate) were used as cotransforming sequences, with 20 ~tg/plate of cell DNA [Ltk(-) aprt(-)] as carrier. After 2 weeks in
selective medium [HAT (Littlefield, 1965), adenine-azaserine (Lowy et al., 1980) or G418 (ColbereGarapin et aL, 1981)],clones were picked and grown into mass culture. Twenty p,gof D NA from several
transformants obtained by co-transformation with individual cloned HindllI fragments of viral DNA
were digested with HindlII, run on a 0.7~ agarose gel, blotted (Southern, 1975) and hybridized with a
mixture of agarose-purified HindlII fragments E, I, J, L obtained from recombinant clones labelled
with 32p by nick translation. (a) 100 ng of total vaccinia virus DNA mixed with 20 ktgof Ltk(- ) DNA
as carrier and digested with HindlII. (b) Clone E1A. (c) Clone I1A. (d) Clone J3A. (e) Clone JLTR 2B.
(f) Clone L2A. The origin of the various clones is indicated in Table 1.
some clones giving strong signals while others were negative. The distribution of relevant
sequences between the poly(A) ÷ and poly(A)- R N A fractions from positive clones can also be
seen in Fig. 2. As an example, clone J3A revealed much higher levels of expression in the
poly(A)- fraction (Fig. 2a). In contrast, clone 13 showed the opposite (Fig. 2b). Table 1
summarizes the distribution of transformants corresponding to the various restriction
fragments. All of the transformants were found to express a heterogeneous pattern o f R N A
similar to those seen in Fig. 2, In most cases the abundance of the viral R N A was higher in the
poly(A)- fraction and, as expected, most of the transformants with positive expression
contained vaccinia virus sequences in both fractions. In transformants containing the H i n d l I I - J
fragment (in either orientation) closely linked to the enhancer sequences (LTR), we have been
unable to measure a greater expression of viral R N A . There is a rough correlation between gene
dosage and the extent of vaccinia virus R N A expression. Nevertheless, a n u m b e r of
transformants that contained viral D N A sequences did not reveal detectable levels of viral
sequences. Appropriate controls showed that these heterogeneous molecules are resistant to
DNase and sensitive to RNase, which eliminates the unlikely possibility that this hybridization
was due to contaminating D N A .
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1248
(a)
Poly(A) +
E
3A
L
Poly(A)I!
5A LTR12 LTRI5 3A 5A LTR12 LTR15~
bp
3840--
1790-1140 ~
540--
Poly(A) +
(b)
Poly(A)iI
E
L
2
3
4
1
2
3
Fig. 2. Detection of vaccinia virus sequences in RNAs from transformed cells. RNAs obtained from
infected Ltk(-) cells or from several transformants were run on a 1% formaldehyde-agarose gel,
blotted (Thomas, 1980) and hybridized with 32p-labelled HindlIl fragments of viral DNA. The probes
were agarose-purified cloned HindllI-J fragment (a) and HindlII-I fragment (b). The amount of RNAs
employed were 100 ng for the control (infected), 0.5 #g for the poly(A) + and 20 ~tg for the poly(A)
fractions. The origin of the RNAs is indicated on top of the autoradiogram. E refers to early RNAs prepared at 2 h from L t k ( - ) cells infected with 10 p.f.u./cell of purified vaccinia virus, L to late RNAs
(4 h). Extraction of RNAs was by the guanidine thiocyanate method (Maniatis et al., 1982). The code
names of independent clones labelled as indicated in Table 1 are given on top of each autoradiogram.
In o r d e r to investigate the possible cause o f R N A heterogeneity, we d e c i d e d to e x a m i n e the
initiation and t e r m i n a t i o n sites o f viral R N A present in t r a n s f o r m e d cells. Since a detailed
transcriptional m a p for the v a c c i n i a virus HindIII-J f r a g m e n t has been d e s c r i b e d (Weir & Moss,
1983 ; Bajszar et al., 1983), we analysed the 5' ends of transcripts m a p p i n g on the left portion o f a
H i n d I I I - E c o R I f r a g m e n t of J. In infected cells, only one initiation site for t r a n s c r i p t i o n has been
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1249
Table 1. Expression o f vaccinia virus R N A sequences in transformants*
Sequences present
h
HindlII fragment
E
F
I
J
L
Poly(A)+
E2A, E3A
F1
I3, I1A, I5A
J7, J9, J3A, J4A,
J5A, J6A, JLTR12,
JLTR(2C, 2D, 2E)
L2A, L5A
Poly(A)E2A, E3A
F1
I3
J7, J9, J10, J3A,
JLTR(2C, 2D, 2E)
Sequences not detected
E3, E4, E5, EIA, E5A
F2, F3, F1A
I1, 12, I4, I2A, I3A
J3, J4, J5, J6, J8, JLTRI5,
JLTR(1A, 1B, 1C, 2A, 2B)
L5A
L4A
* Total RNA isolated from clones of cells shown to contain several copies of individual HindlII fragments of
vaccinia virus DNA were run on a formaldehyde-agarose gel, blotted and hybridized with the corresponding
fragments of viral DNA (as in Fig. 2). Clones were scored as negative by the absence of any hybridization signal
after 1 week's exposure with intensifying screens, using probes of sp. act. > 108 c.p.m./p.g DNA. Where indicated,
the poly(A)÷ and poly(A)- RNA fractions were analysed. Clones containing restriction fragments E, F, I and L
(denoted by first letter) were co-transformed with either cloned aprt (where the last letter of the coding name is A)
or with cloned HSV tk. Clones containing vaccinia virus HindlII-J were co-transformed with aprt (coded as
above) or with pNeo (G418 resistance) with or without LTR sequences. Numbers represent randomly isolated
clones.
located, at 290 bp from the EcoRI site (Bajszar et al., 1983). To identify the 5' and 3' ends of
RNAs, S1 nuclease mapping was carried out (Berk & Sharp, 1977). The total R N A s from several
clones showing different levels of expression in cells transformed with the HindlII-J fragment
were hybridized with the EcoRI-digested pBR-J plasmid, then digested with S1 nuclease,
electrophoresed on an alkaline 1.5 ~o agarose gel, blotted and probed first with isolated HindlIIJ-EcoRI fragment (for 5' ends). Thereafter, the hybrid was melted, removed by washing and
used to characterize the 3' ends. This filter was hybridized with the isolated 5 kb HindIII-J
fragment. As can be seen in Fig. 3(a), only one fragment, 290 bp, appeared in nearly all
clones. As mentioned above, this size corresponds to the distance between the EcoRI site and
the 5' end of the tk message. When the 3' end of the R N A was examined, a high degree of
heterogeneity was observed (Fig. 3b), which is direct evidence for improper termination.
In this study, we have constructed a number of cell lines that contained cloned HindIII
restriction fragments, E, F, G, H, I, J, L and M, of vaccinia virus D N A . The most significant
findings of this report are (i) that viral sequences are represented in the poly(A) + and poly(A)R N A fractions, (ii) that the levels of expression are variable, (iii) that these levels are not
increased by LTR enhancers cloned adjacent to the 5 kb HindIII-J fragment, and (iv) that the
R N A s in the two fractions are highly heterogeneous in size (up to 10 kb) with no discrete species
of R N A . The fidelity of transcription was determined by S1 nuclease mapping of the 5' and 3'
ends of the vaccinia tk R N A present in transformants. Other workers have determined the
precise ends of the viral tk gene by this approach (Bajszar et al., 1983) and by nucleotide
sequence analysis (Weir & Moss, 1983 ; Hruby et al., 1983). As indicated in Fig. 3, the 5' end of
R N A s from various transformants was the same as the 5' ends of R N A s from infected cells.
Interestingly, the transformants that contain the L T R sequences have the same 5' end; thus, the
mechanisms by which the host transcribes the viral sequences are not affected by the promoter
present in these enhancer elements. In contrast, similar analysis showed a high degree of heterogeneity at the 3' ends of R N A s from these transformants. The most intriguing observation is the
proper transcription initiation of the viral tk gene in transformants. It will be of interest to find
out if R N A polymerase II is responsible for this transcription, since the viral tk gene lacks the
consensus sequences known to be required for transcription by this polymerase. Alternatively,
R N A polymerase I or III might be responsible for transcription of the viral sequences.
Appropriate in vitro transcription systems will be required to distinguish these possibilities. The
heterogeneity of the transcripts at their 3' ends is less surprising, since these transformed cells
will probably lack the virus-specific poly(A) polymerase. Nevertheless, many of the transcripts
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1250
6
7
8
9
3A 4A
5A
6A
2C
2D
2E
2B
E
(a)
(b)
Fig. 3. S1 nuclease mapping. (a) Characterization of the 5' ends. A slight modification of the Berk &
Sharp (1977) technique was used. Briefly, 20 ng of the vaccinia virus HindlII-J DNA digested with
EcoRI (Bajszar et al., 1983) was mixed with 100 vtg of total RNA from the different transformants in
300 mM-NaCI, 1 mM-EDTA, 10 mM-P1PES pH 6.4, 80~ formamide. The mixture (100 ~tl) was heated
at 80 °C for 10 min, and hybridized at 45 °C for 16 h, Thereafter, the volume was increased to 400 ~tl
with S 1 buffer, at final concentrations of 50 mM-sodium acetate pH 4, 150 mM-NaCI, 3 mM-ZnCIz, and
digested with 5000 units S1 (Boehringer) at 37 °C for 5 min. The reaction was terminated by chilling on
ice and with the addition of 10 ~tl of 0.25 M-EDTA and 2 p.g of sonicated calf thymus DNA. The
reaction products were precipitated with 2 vol. ethanol, pelleted by centrifugation, dried, resuspended,
and run on alkaline 1.5~ agarose gels (Corces et al., 1981). The gel was blotted and hybridized to the
purified left Hindlll-EcoRI fragment of HindllI-J DNA labelled by nick translation. (b)
Characterization of the 3' ends. The experiment was carried out as above but the probe was the isolated
HindlII-J DNA fragment. The size of the RNAs from this fragfiaent, found in virus-infected cells at 2 h
post-infection, are indicated at the right (neutral gel). Numbers above (a) are the code names of
independent clones isolated from cells transformed with the fragment (alkaline gels in a and b). E, early
viral RNAs isolated from virus-infected L t k ( - ) cells at 2 h p0st-infection.
are polyadenylated even though they lack the eukaryotic polyadenylation sequences. Whether
the vaccinia virus transcripts in these transformants are present in the cytoplasm of the cells and
can be translated into virus-specific proteins remains to be established.
We thank Pablo Calzada and Victoria Jimenez for skilled technical assistance. We thank R. Bablanian for his
critical reading of the manuscript. This investigation was supported by NIH grants AI 16780 (M.E.) and AM 29725 (A.P.) A.P is a recipient of an Irma Hirschl Monique Weill-Canlier Award.
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