J. gen. Virol. (1982), 60, 99-113.
99
Printed in Great Britain
Key words: A dl 2~early genes~mapping~cellular transformation
Expression of Early Viral Gene Products in Adenovirus Type 12-infected
and -transformed cells
By H E L M U T
ESCHE*
AND B E A T E
SIEGMANN
Institute of Genetics, University of Cologne, Weyerta1121, 5000 Cologne 41, Federal
Republic of Germany
(Accepted 11 December 1981)
SUMMARY
We have analysed early viral gene products expressed in adenovirus type 12
(Adl2)-infected cells as well as in two Adl2-transformed hamster cell lines, and
Adl2-induced rat tumour cell lines by cell-free translation of virus-specific RNA
which was selected by hybridization to cloned restriction endonuclease fragments of
virus DNA. Proteins synthesized in vitro were analysed by one- and twodimensional gel electrophoresis. It was found that RNA encoded by early region
E1A directs the synthesis of at least eight polypeptides with apparent mol. wt. 38K,
36K, 30K, 28K, 26K, 25K, 24K and 22K. All these proteins are related to each
other. E1B-specific RNA directs the synthesis of three proteins: 59K, 19K and 17K.
Early region E2a codes for a 61K polypeptide which probably represents the
single-strand DNA-binding protein of Adl2. RNA complementary to region E3
directs the synthesis of a 16K protein, and RNA transcribed from region E4 the
synthesis of polypeptides with mol. wt. 20K, 18K and l l.5K. We have mapped a
67K polypeptide into the region within 11 to 28 map units (E2b). The analysis of
proteins directed by virus-specific RNAs prepared from two Adl2-transformed
hamster cell lines (T637, HA12/7) and one Adl2-induced rat tumour line
(RBT12/3) showed that early regions E1 and E4 are expressed in all three
Adl2-transformed cell lines. RNA transcribed from early regions E2 and E3 have
been detected in lines T637 and RBT12/3. The virus RNA prepared from the
Adl2-transformed cell lines directed synthesis of polypeptides with mol. wt. very
similar to those of early virus proteins from infected cells. However, in all three
Adl2-transformed cell lines mentioned above we have found RNAs which directed
the synthesis of additional polypeptides of early regions E1 (34K) and E4 (25K,
24K) not detected in infected cells. The DNA sequence between 11 and 28 map units
(coding for the 67K protein) is not expressed in the Ad 12-transformed cells.
INTRODUCTION
Human adenoviruses, especially types 2, 5 and 12, have been the subject of extensive
studies because of their ability to cause morphological transformation of cultured cells, or in
the case of Adl2, to induce tumours in animals. Transformation of cells with purified
restriction endonuclease fragments of virus DNA (Graham et al., 1974; van der Eb et al.,
1977; Sekikawa et aL, 1978; Shiroki et aL, 1977) as well as studies on the effect of host-range
and deletion mutants on the transforming activity of the virus (Graham et al., 1978) have
located the virus genes responsible for cellular transformation to (approx.) the left-most 12 %
of the virus genome. This transforming region constitutes one out of five gene blocks
expressed at early times after infection. RNAs transcribed from the five early regions encode
at least 17 virus-specific proteins, which were characterized by several means, at least for the
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100
H. ESCHE
AND
B. S I E G M A N N
non-oncogenic adenovirus types 2 and 5 (for review, see Flint & Broker, 1980). The gene
products of the highly oncogenic adenovirus type 12, however, have not been sufficiently
characterized so far. Neither all proteins encoded by early region E1 nor those of regions E3,
E4 and E5 have been identified unambiguously. Also, less is known about the virus proteins
in cells transformed with Adl2. Studies on transcription of virus genes in Adl2-transformed
hamster cells have shown that, just as in Ad2- and Ad5-transformed cells, exclusively early
virus genes are transcribed (Ortin et al., 1976; Smiley & Mak, 1978; Ibelgaufts et al., 1980).
Moreover, in vitro translation of virus-specific RNA from Ad5- and Ad2-transformed cells
has recently indicated that essentially the same virus proteins are expressed in lytically
infected and transformed cells, at least from the transforming region (J. H. Lupker, personal
communication; Esche, 1982).
To compare the products of the transforming genes of the non-oncogenic adenoviruses
with those of the highly oncogenic adenovirus type 12, we have at first identified and
characterized more precisely the Adl2-encoded proteins in infected cells. Therefore, we
describe in the present report the identification and mapping of Adl2-specific proteins
expressed in productively infected cells. Some of these proteins have been observed for the
first time. Our analysis of the virus proteins has been based on the in vitro translation of
preselected Adl2-specific RNAs prepared from the infected cells, using a cell-free system
derived from rabbit reticulocytes. Applying this method we also looked for functional virus
mRNAs and proteins expressed in two Adl2-transformed hamster cell lines and one
Adl2-induced rat brain tumour cell line. It is shown that in comparison with the proteins
found in infected cells, all three Adl2-transformed cell lines investigated express additional
proteins from early regions E 1 and E4.
METHODS
Cell lines and virus. Human KB cells (CCC 17) were obtained from the American Type
Culture Collection and were propagated in monolayer or suspension cultures in Eagle's
medium supplemented with 7.5 % calf serum (Seromed, Munich, F.R.G.). Human embryonic
kidney (HEK) cells (obtained from Seromed) were grown in Dulbecco's medium
supplemented with 10% foetal calf serum. Cell cultures were periodically screened for
mycoplasma contamination (Hayflick, 1965). Human adenovirus type 12 (Adl2), a gift of
W. Rowe (National Institutes of Health, Bethesda, Md., U.S.A.), was propagated in
monolayer cultures of HEK cells and in spinner cultures of KB cells. The purification of the
virus has been described elsewhere (Doerfler, 1969). The origins as well as the media and
methods of propagation of the Adl2-transformed hamster cell lines T637, HA 12/7 and the
Ad12-induced rat brain tumour line RBT12/3 have been described previously (Fanning &
Doerfler, 1976; Ibelgaufts et al., 1980). Some of the properties of the Ad 12-transformed lines
used in this study are summarized in Table 1.
Virus DNA. Ad12 DNA was prepared from CsCl-purified virions by SDS-Pronase
B-phenol method described elsewhere (Doerfler et al., 1972). All Adl2 DNA preparations
used were identified by the specific BamHI restriction endonuclease pattern of Adl2 DNA
(Ortin et al., 1976).
Preparation of restriction endonuclease fragments of A d12 DNA. The specific fragments of
Ad12 DNA that we used had been cloned in the plasmids pBR322 (fragments HindlII I, F;
B a m H I C, B) or pBR325 (fragments EcoRI D, F and parts of fragment A) by S. Vogel, D.
Eick and M. Brrtz (Vogel et al., 1981). The plasmid containing the EcoRI C fragment was a
gift from Dr A. J. van der Eb (Sylvius Laboratories, Leiden, The Netherlands). Plasmid DNA
was isolated from Escherichia coli X 1776 by slight modification of the procedure of Tanaka
• & Weisblum (1975).
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A d12 proteins in infected and transformed cells
101
Table 1. Properties of the Ad12-transformed hamster and Ad12-induced rat tumour cell lines
Cell
line
T637
HA12/7
RBT12/3
Cells used for
transformation
BHK21
PrimarySyrian
hamster
Multiplicityof
Ad 12 used in
Locusof the
T
Oncotransformation t u r n o u r antigen g e n i c i t y
Reference
350
+
+
Strohl et al. (1970)
10
+
+
zur Hausen (1973)
Brain
+
+
Ibelgauftset al.
(1980)
RNA preparation. KB cells grown in suspension cultures were infected at a density of
5 × 106 cells/ml with 20 to 30 p.f.u./cell of Adl2. After adsorption for 1.5 h, infected cells
were diluted to approx. 5 × 105 cells/ml with medium supplemented with 10% calf serum
and incubated at 37 °C for 9 h. When indicated, 25 pg cycloheximide or 20/~g cytosine
arabinoside (araC) per ml were present from 4 h post-infection throughout infection.
Cytoplasmic RNA was isolated from infected or Adl2-transformed cells as described
previously (Esche et al., 1979).
Selection and assay of virus RNA. RNA complementary to a given region of the Adl2
genome was selected by hybridization of approx. 3000/~g cytoplasmic RNA to approx. 30 pg
of appropriate fragments of Adl2 DNA immobilized on nitrocellulose filters (Ricciardi et
aL, 1979). Hybridization was carried out in 50% formamide, 0.01 M-PIPES pH 6.8,
0.4 M-NaC1 for 3 h at 50 °C. Upon hybridization, filters were washed extensively with 1 ×
SSC, 0.1% SDS and 0.002 M-EDTA, preheated to 55 °C. Ad 12-specific RNA was eluted by
heating the samples to 100 °C for 1 min and precipitated with 2.5 vol. ethanol at - 2 0 °C.
The RNA was then translated in a micrococcal nuclease-treated reticulocyte lysate (Pelham
& Jackson, 1976). Polypeptides synthesized in the presence of [35S]methionine (Amersham
International) were analysed on 10 to 15 % (w/v) polyacrylamide gels (Laemmli, 1970) and
treated for fluorography (Bonner & Laskey, 1974).
Two-dimensional gel electrophoresis. Isoelectric focusing was performed as described by
O'Farrell (1975) and O'Farrell et al. (1977)with minor modifications. Proteins synthesized in
vitro were diluted with 3 vol. 9.5 M-urea, 2 % (w/v) Nonidet P40, 5% mercaptoethanol and
2% ampholines (1.4% pH range 5 to 10; 0.6% pH range 3-5 to 10) and incubated at 37 °C
for 30 min. Aliquots were isoelectric focused for 4 h at 500 V (non-equilibrium pH gradient
electrophoresis), or for 14 h at 400 V (equilibrium isoelectric focusing) on 11 × 0.4 cm
cylindrical gels prepared with pH 3.5 to 10 and pH 5 to 7 ampholytes. The gels were then
equilibrated in sample buffer (0.065 M-tris-HC1 pH 6.8, 2.5% (w/v) SDS, 5% (w/v)
mercaptoethanol, 10% (w/v) glycerol for SDS-gel electrophoresis for 30 rain and applied to
15 % polyacrylamide gels. After electrophoresis gels were processed for fluorography.
Peptide maps. Spots of [35S]methionine-labelled polypeptides were identified after
two-dimensional electrophoresis on dried gels by autoradiography and peptides were mapped
essentially as described by Elder et al. (1977). The protein spots cut out from the gels were
washed with 10% methanol, dried and digested with 100 ~1 freshly prepared trypsin in
50 mM-NHaHCO 3 at 37 °C for 4 h, and further incubated with 0.5 ml in water overnight.
The lyophilized supernatants were dissolved in 10~1 electrophoresis buffer (acetic
acid:formic acid:water; 15:5:80, by vol.) spotted on a 10 × 10 cm cellulose-coated
thin-layer plate and electrophoresed at 1000 V for 20 min. Ascending chromatography was
performed in the second dimension in butanol : pyridine : acetic acid : water (32.5 : 25 : 5 : 20,
by vol.). Plates were coated with molten 0.4% PPO in 2-methylnaphthalene (Bonner &
Stedman, 1978) and fluorographed at --80 °C.
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102
H. E S C H E A N D B. S I E G M A N N
j
92.5K
~ Z,-72KE2 6 K
" =¢/ 58K')
9
; ...~__~54~LEI A
~ __~-48K ¢
~45KJ
46K
~ 1
W i [ ~
I m
30I~-xL
E I A ~ 28K--x\
126/25K-'X,,_~_
| 24K-..-~
[,. 22KJ - E4 20K.
E1B 1 9 K ~
E4 18K- -~-'-- //E1B 17K--'/rE3 16K-v
E4 1 1 . 5 K ~
~:
30K
-24K E4
~ ,~m,~-~
19K E4
14K
E3
E1
E2b
c
G
Marker
proteins
E 67K-N
E2 61K--,,~ !
EIB 5 9 K - r- 38K-x
[ 36K-~\
!
Ad2
(a) (b)(c) (d) (e) (f) (g) (h) (i) (j) (k) (1) (m) (n)
Adl2
E2a
D
I
F
B
A
1'0
.
C
[ G IHI
210
I
30
E
]
D
410
E4
A
D ~v~lJ[J~) ~
A
]II F I
[
I
50
610
C
I
70
A,
[H K
I FcoRI
E
[ HirtdlII
BIHi!I ~IVl,L I PstI
810
910
I
100
Map units
Fig. 1. Translation in vitro of Adl2-specific RNA prepared from infected KB cells by hybridization to
cloned restriction endonuclease fragments of the Adl2 genome. Specific RNAs were selected by
hybridization to whole Ad12 DNA (c) and the Ad12 DNA fragments EcoRI C (d), HindIII I (e),
HindIII F (f), EcoRI D (l), EcoRI F (g), BamHI C (h), BamHI B (l), EcoRI A (j) or PstI I (k). These
experiments used RNA prepared 9 h post-infection from cells that had been treated with 25 gg/ml
cycloheximide from 2 h after infection. The products of cell-freetranslation of the selected RNAs were
analysed by electrophoresis through 15% polyacrylamide gels which were dried and exposed for
fluorography for 2 to 4 days. The results of cell-free translation with no added RNA (b) or
unfractionated cytoplasmic RNA used for RNA selections (a) are shown for comparison. (m) In vitro
synthesized early Ad2 proteins; (n) size marker proteins (phosphorylase b, 92.5K; bovine serum
albumin, 69K; ovalbumin, 46K; carbonic anhydrase, 30K; lysozyme, 14K).
RESULTS
We have employed a translation-based assay to analyse early viral mRNAs and proteins
present in cells infected and transformed with adenovirus type 12 (Adl2). Virus-specific
RNA was selected from cytoplasmic RNA preparations by hybridization to virus D N A or its
cloned subfragments immobilized on nitrocellulose. The mRNA eluted from the hybridization complex was then translated in a messenger-dependent reticulocyte lysate and the
products identified by one- or two-dimensional gel electrophoresis followed by fluorography.
Translation in vitro of RNAs selected with Ad12 DNA fragments representing each of the
early virus regions
Virus-specific RNA used to direct cell-free protein synthesis was prepared 9 h
post-infection from KB cells with Adl2. In most experiments the cells were treated with 25
pg/ml cycloheximide from 2 h post-infection to harvest, because it enhances the expression of
at least some early viral mRNAs (Eggerding & Raskas, 1978; Chow et al., 1979). No
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103
Adl2 proteins in infected and transformed cells
(a)
(b)
(e)
(d)
(e)
(f)
q___
E1B 59K
-38K-~
36K~
30K~
E1A, 2 8 K ~
26K~
25K----'-24K~
22K~
E1B 19K
E1B 1 7 K ~
34K E
t
I
Fig. 2. Translation in vitro of Adl2-specific El RNAs prepared from infected KB cells and the
Adl2-transformed hamster cell line HA12/7. Messenger RNA was selected by hybridization to the
Adl2 DNA fragments EcoRI C (b, f), HindlII I (c), and HindlII F (d). These experimentsused RNA
prepared 9 h post-infectionfrom cells treated with 25 gg/ml cyclohexirnidefrom 2 h after infection (a to
e) and from half confluent monolayers of the Adl2-transformed line HA 12/7 (f). The mRNAs thus
selected were translated in the reticulocyte lysate and the products analysed by electrophoresisthrough
a 10 to 15% polyaerylamide gradient gel. Fluorography was done for 3 to 5 days. The results of
cell-freetranslation with no added RNA (e) or RNA from infected cells selected with whole Ad 12 DNA
(a) are shown for comparison.
difference was found in the patterns of polypeptides produced by cell-free translation of virus
RNAs isolated from cells grown in the presence or absence of cycloheximide, although R N A
from cells treated with cycloheximide gave larger amounts of cell-free protein synthesis per
mg of cytoplasmic RNA.
The results of in vitro translation of Ad 12-specific RNAs selected with different restriction
endonuclease fragments of A d l 2 D N A are presented in Fig. 1 and Fig. 2. Virus R N A
transcribed from early region E1 was selected with fragment E c o R I C (0 to 16.5 map units).
The R N A complementary to these sequences directed the synthesis of proteins with apparent
mol. wt. 59K, 38K, 36K, 30K, 28K, 26K, 25K, 24K, 22K and 19K (Fig. 1 d; Fig. 2b).
Several R N A preparations also directed the synthesis of a 10K polypeptide which could be
separated from the labelled material running with the front by different gel systems. Some of
these polypeptides, e.g. those with mol. wt. between 38K and 22K, could be documented
unambiguously only by separation on polyacrylamide gradient gels as shown in Fig. 2. In
addition to the proteins described above, we often detected, after longer exposures of the
fluorograms, a minor polypeptide of 17K among the translation products of El-selected
RNA. Surprisingly, the R N A coding for this protein was found in much larger amounts in
Adl2-transformed cells (Fig. 2f). The absence of the background bands which are seen in
Fig. 1 (b) in several in vitro translations also shown in Fig. 1 and Fig. 2 is probably due to
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104
H. E S C H E
A N D B. S I E G M A N N
-~
Electrophoresis
(a)
(b)
10
lqllp
2!
4
(d)
e~
t~
E
j"
/
J
r..)
J
Fig. 3. Tryptic [35S]methionine-labelled peptide maps of the in vitro synthesized E1A-specific
polypeptides 38K (a), 30K (b), 26K (c) and 24K (d). The protein spots, similar to those shown in Fig.
4, were digested with trypsin, spotted in the lower right-hand corner of thin-layer plates and
electrophoresed from right to left in the first dimension. Peptides shown in (b) were electrophoresed for a
longer time (30 min) in comparison to those shown in (a), (c) and (d) (20 min). Ascending
chromatography was performed in the second dimension from bottom to top. Fluorography was done
for 4 days.
differences in the different, micrococcal nuclease-treated reticulocyte S-30 extracts used. In
order to determine which proteins are encoded by early region E1A and which by region
E1B, RNA was selected with Adl2 DNA fragments representing only parts of region El.
Translation in vitro of RNA selected with the HindlII I fragment (6.8 to 10.5 map units),
which contains exclusively E1B DNA sequences (Sawada & Fujinaga, 1980), resulted in the
synthesis of proteins 59K, 19K and 17K (Fig. 1 e). This finding leads to the conclusion that
these polypeptides are encoded by region E1B. The 19K and 17K proteins were also obtained
with RNA selected by fragment HindlII F (10.5 to 18 map units), suggesting that its RNAs
extend beyond the HindlII cleavage site at 10.5 %. We still have no explanation for why we
could not select with fragment HindlII I RNA, which directs the synthesis of the E1B 59K
protein, although the DNA sequence coding for this polypeptide is located downstream of
that coding for the 19K protein (H. Bos, personal communication). The 10K polypeptide
obtained in several in vitro translations with EcoRI C-selected RNA (Fig. 1 d) was also
detected with RNA selected by hybridization to fragment HindlII F but not to fragment
HindlII I. This protein may represent polypeptide IX of Adl2. The polypeptides of mol. wt.
between 38K and 22K were characterized as E1A-specific proteins by immunoprecipitation
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A d l 2 proteins in infected and transformed cells
105
with monospeeific antibodies, directed against a synthetic peptide which is encoded by a
sequence located on the 3' end of the E1A mRNA (H. Esche et al., unpublished results). To
analyse further the relationship among the E1A-specific polypeptides synthesized in vitro, we
compared the tryptic peptides of these proteins by high voltage electrophoresis (first
dimension) combined with chromatography (second dimension). As shown for the proteins of
38K, 30K, 26K and 24K in Fig. 3, we could demonstrate major common peptides for all
eight E1A polypeptides. We are currently analysing the tryptic peptides of the E1A proteins
labelled in vivo as well, which we immunoprecipitated with the monospecific antibodies
described above. Preliminary tryptic peptide maps of the in vitro synthesized E1B proteins
59K and 19K indicate that they are not related at all (data not shown).
RNA transcribed from early region E2 of Ad 12 was selected by hybridization to fragments
EeoRI F (62.2 to 64.2 map units) and BamHI C (57.9 to 73.1 map units). Translation in
vitro of EeoRI F-selected RNA resulted in the synthesis of one major 61K polypeptide and
some minor proteins of about 42K, 38K and 34K (Fig. 1g). RNA selected with fragment
BamHI C, which comprises parts of region E3 as well as region E2, directed the synthesis of
a 16K polypeptide (Fig. 1 h). This protein was also obtained using RNA selected with
fragment BamHI B (73.1 to 87 map units) which contains exclusively E3 DNA sequences
Fig. 1 i). Therefore, we conclude that the 16K polypeptide is encoded by early region E3.
RNA complementary to region E4 which was selected with the fragments EeoRI A* (approx.
87 to 100 map units) and PstI I (89.1 to 95.3 map units) was translated into proteins of mol.
wt. 20K, 18K, 16K and l l . 5 K (Fig. l j, k). The intensity of labelled products seen in lanes Q')
and (k) is different because the polypeptides shown in lane (j) are from an autoradiogram that
was deliberately over-exposed in order to detect possible additional minor polypeptides. The
observation that the 16K polypeptide synthesized by PstI I-selected RNA occurred in
two-dimensional gels at the same pH range (about pH 8.3) as that encoded by RNA selected
with BamHI C (Fig. 4 e , f ) suggested that this protein is encoded by region E3. Its presence
among the E4-specific translation products indicates that fragments PstI I and also EeoRI A*
contain at least some E3 DNA sequences.
In addition to RNAs encoded by the conventional recognized early regions E1 to E4, we
looked for early Adl2-specific RNA transcribed from DNA sequences encompassing 10.5 to
28.5 map units. It was previously shown for adenovirus type 2 that parts of this region
encode several virus-specific RNAs and proteins at early times after infection (Lewis &
Mathews, 1981). RNA complementary to this region was selected with the fragments EcoRI
D (16.5 to 28.5 map units) and HindIII F (10.5 to 18 map units) and synthesized in vitro a
67K polypeptide always obtained in low amounts (Fig. l f, l). No RNA coding for this
polypeptide was selected with fragment HindIII I.
Analysis of A d12-speeific early proteins in two-dimensional gels
For further characterization we have analysed the virus proteins synthesized in vitro by
electrophoresis on two-dimensional gels (O'Farrell, 1975; O'Farrell et al., 1977). The results
illustrated in Fig. 4 confirm to a large extent our data obtained by one-dimensional gels. In
addition, these results show that most El-coded proteins as well as those of region E4
appeared at acidic pH values between 4.3 and 6.5 (Fig. 4a to e,f). The 61K protein, which
probably represents the single-strand DNA-binding protein of Ad 12, occurred between pH 7
and 8 (Fig. 4d, e). A similar isoelectric point was previously found for the DNA-binding
protein of Ad2 (Harter & Lewis, 1978). The minor E2-specific polypeptides (45K to 34K)
seen in one-dimensional gels appeared also at this pH range which led us to assume that these
polypeptides represented proteolytic degradation products of the 61K protein. As mentioned
in an earlier section, closer inspection of the two-dimensional gels with polypeptides
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106
H. E S C H E A N D B. S I E G M A N N
pH
4.0 5.0 6.3 7.0
(a) ~ - ~
. . . .
7.9
8.3
4.0
8.3
/
E2 6 1 K - x
E1B 5 9 K - - ' ~
~'38/36K-~ "'
E1A) 30/28K-,N__
) 26/25K--~_~ ~
[,.24/22K~ ' i
t
:
t
I i
b
l l
02
3 3
55
~,
~mP'--~
('38/3 6 K - =1~] 3 0 / 2 8 K - ~"~26/25K--~"
1,24/22K
E1B 1 9 K - E1B 17K- / -
(c)
E1B 59K
I ~ |..-.,,,,-ill ~
..~_
~
ram, o,
•
"""
4.0 5.0
v
v
I
q
5 5
31
o4
,o 8
6.1
7.2
4.0
7.2
~'
v
v
!
pb~,,,. •
~2
.
~
1
I
. ~ " I,
7.1
¥
- ,
r3s/36K
t:,A.)30/28K
~'" ~'126/25K
L24/22K
E1B 19K
E1B 17K~ -
"72
4.0
"~"
littqt ~
4 7
8
E4 18K E
19K--~ ~lllt
~
_~ ,,
E1B 17K.-// , ~ , , J t _
- - - !m~ ~
"1 ....
E3 16K - /
~v*
4.0
5.0 6.0
7.1
(b)
v
v ,
v
E1B 5 9 K - - ~
c91
"
__22_"
--
~,/~
6~
5
"44
°11
8
Fig. 4. Fluorograms of two-dimensional gels containing proteins synthesized in vitro by the reticulocyte
ceU-free system programmed with early Ad12-specific RNA. Messenger RNAs were selected by
hybridization to whole Adl2 DNA (a) or the Adl2 DNA fragments EcoRI C (b, c), EcoRl F (d),
BamHI C (e) and PstI I (f). RNA used in these experiments was prepared 9 h post-infection from cells
grown in the presence of 25/lg/ml cycloheximide from 2 h after infection. The products of cell-free
translation were analysed by two-dimensional gel electrophoresis. First dimension: non-equilibrium pH
gradient isoelectric focusing (a, b, d to f ) or equilibrium pH gradient isoelectric focusing (c). Second
dimension: electrophoresis through 15 % polyacrylamide gels. Fluorography was done for 3 to 5 days.
The polypeptides are marked as follows: E2 61K (1), EIB 59K (2), E1A 38/36K (3), E1A 30/28K (4),
E1A 26/25K (5), E1A 24/22K (6), E4 20K (7), E1B 19K (8), E4 18K (9), E1B 17K (ll), E3 16K
(12).
synthesized by R N A from region E3 and E4 (Fig. 4d, e) indicated that only one 16K protein
m a y exist. It is encoded by region E3 and has a basic isoelectric point.
Cell-free translation o f virus R N A isolated f r o m A d l 2 - t r a n s f o r m e d hamster and rat brain
tumour cells
In addition to R N A obtained from lytically infected cells, virus-specific R N A from two in
vitro A d l 2 - t r a n s f o r m e d hamster cell lines ( H A 1 2 / 7 , T637) and one Ad12-induced rat brain
tumour cell line ( R B T 1 2 / 3 ) was translated in vitro. R N A was prepared from cells grown
without any drugs by hybridization to the fragments EcoRI C (region E l ) , B a m H I C (regions
E2 and E3) and PstI M (region E4). El-specific R N A prepared from each o f the three
A d l 2 - t r a n s f o r m e d cell lines directed the synthesis of proteins 59K, 3 8 / 3 6 K , 34K, 3 0 / 2 8 K ,
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A d12 proteins in infected and transformed cells
107
pH
(a')
4.0
V
--
7.0
V
7.9
V
E2 6 1 K - - - - - - ~
#
L
g
4.0
6.9
l
(e)
+
E2 61K.
i
E3 16K
(f)
Q
4.0
7.0
7
9-
---
E3 1 6 K - -
8.4
v
E420 -,,
E4 18K
8.3
l
~
~
_
~
.
.
.
.
.
.
.
.
.
.
Fig. 4 (d to f ) cont.
2 6 / 2 5 K , 2 4 / 2 2 K , 19K and 17K (Fig. 2 f ; Fig. 5; Fig. 6 c to e). The protein patterns observed
resemble those which were obtained when early viral R N A prepared from infected cells was
translated in vitro. However, in all A d l 2 - t r a n s f o r m e d cell lines investigated we found an
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108
H. E S C H E
A N D B. S I E G M A N N
(a)
1
E2 6 1 K ~ , , . . . . ~
E1B 5 9 K - - w
2
(b)
3
|
4
1
2
m
~
(c)
3
4
1
2
3
4
_
l
~,~=~
E 34K E1A~ 3 0 / 2 8 K ~ .
(N) 2SKS&2126/25K------~
(E4) 2 4 K ~ 2 4 / 2 2 K ~
E420Ki
~
----E1B
"
E1B
E3
dlll~ ~-,-~ ~ .
Fig 5. Proteins synthesized in vitro by the reticulocyte cell-free system programmed with virus-specific
RNA prepared from the Adl2-transformed hamster cell lines HA12/7 (a) and T637 (b) and the
Adl2-induced rat brain tumour line RBTI2/3 (c). Messenger RNA was selected from cells grown as
monolayers to half confluence by hybridization to the Ad 12 DNA fragments EcoRI C (region E 1, lane
1), B a m H I C (regions E2 and E3, lane 2) and PstI M (region E4, lane 3). The products of cell-free
translation were analysed by electrophoresis through 15 % polyacrylamide gels which were dried and
exposed for fluorography for 2 to 6 days. The results of in vitro translations with no added RNA or
RNA selected with whole Adl2 DNA are shown for comparison (lane 4).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
E1B 59K
"38K--~__ ~
36K~
30K~
E1A~ 2 8 K - - ' - - ~
26K~
25K ~
24K--~
~22K~"
E1B 19K
E1B 1 7 K ~
m
v~e_ ~
--34K E
tm
p
W
Fig. 6. Translation in vitro of El-specific Adl2 RNA prepared from the Adl2-transformed hamster
cell lines HA12/7 (c) and T637 (d), and the Adl2-induced rat brain tumour line RBT12/3 (e). RNA
was prepared from cells grown as monolayers by selection/hybridization to the Adl2 DNA fragment
EcoRI C. The products of cell-free translation of the selected RNAs were analysed by electrophoresis
through 12% polyacrylamide gels followed by fluorography (3 days). Results of in vitro translations
with no added RNA (g) and with RNA prepared from infected cells by hybridization to the fragments
EcoRI C ( a , f ) and HindlII I (b) are shown for comparison.
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A d 1 2 proteins in infected and transformed cells
109
RNA species which directed the synthesis of a 34K polypeptide not detected in Ad 12-infected
cells. This 34K protein shares common peptides with E1A-specific proteins (data not shown).
We are just analysing the RNA coding for this protein. Region E1B RNA which codes for
the 17K polypeptide was found in the Ad 12-transformed cells in significantly larger amounts
in comparison with cells infected with Adl2. RNA transcribed from regions E2 and E3 was
detected in lines T637 and RBT12/3 only, and directed the synthesis of the 61K and 16K
proteins. Unusually high amounts of E2- and E3-specific RNA were observed in the
Adl2-induced rat brain tumour line RBT12/3 which synthesized in vitro additional proteins
in variable amounts. The absence of any functional RNA expressed at least from region E3
in HA ! 2/7 cells is in agreement with previous mapping data of RNA prepared from these
cells (Ortin et al., 1976; Schirm & Doerfler, 1981). RNA complementary to early region E4
was detected in all Adl2-transformed cell lines investigated, and was translated into proteins
of mol. wt. 25K, 24K and 20K. Some RNA preparations also directed the synthesis of an
18K and a 14.5K polypeptide. Messenger RNA for the 25K and 24K proteins, which are the
most prominent bands in the fluorograms, was not found in any RNA preparation from
infected cells. In general, region E4 is expressed very poorly in these Adl2-transformed cells.
In none of the transformed cell lines used could m R N A for the 67K protein be detected
encoded by DNA sequences between 11 and 28 map units, even upon longer exposures of the
fluorograms (data not shown).
DISCUSSION
This report describes the identification and mapping of Adl2 early proteins by in vitro
translation of Ad12-specific RNA prepared from infected and Ad12-transformed cells.
Messenger RNA was selected from cytoplasmic RNA preparations by hybridization to
different virus DNA sequences.
In the course of this study, we used RNA preparations from cells grown after infection in
the presence or absence of the drug cycloheximide. Except for the observation that
cycloheximide enhances the synthesis of many early RNAs (Eggerding & Raskas, 1978), we
found virtually no difference in the patterns of polypeptides produced by cell-free translation
of RNA from cells untreated or treated with cycloheximide.
The data from a large number of translation experiments with RNA from infected cells,
similar to that of Fig. 1, are summarized in Fig. 7 (a). A surprising result is that RNA specific
for region El, and particularly for region E1A, is translated into such a large number of
proteins (see Fig. 1, 2 and 3): 59K, 38K, 36K, 30K, 28K, 26K, 25K, 24K, 22K, 19K and
17K. As the sum of their mol. wt. far exceeds the maximum coding capacity of region El,
these proteins cannot represent only products of separate genes. The coding area of the
proteins with mol. wt. from 38K to 22K is located between 1 and 5 map units (region E1A),
since their RNAs could be certainly selected with the EcoRI C fragment but not with
fragment HindlII I. Peptide mapping showed that these E1A proteins are structurally related
and, therefore, derived at least in part from common DNA sequences. This finding is also
supported by the observation that all E1A proteins synthesized in vitro can be
immunoprecipitated with monospecific antibodies directed against a synthetic peptide of 10
amino acids, which is encoded on the 3' end of E1A-specific mRNAs (H. Esche et al.,
unpublished results).
Recently, it has been shown by Sawada & Fujinaga (1980) that at least four RNA species
with partially common sequences are transcribed from region E1A. The large number of
proteins may thus be explained by the existence of overlapping RNAs as well as by premature
termination and degradation of R N A s or proteins. The latter interpretation, however, seems
to be unlikely, as almost all E1A proteins observed after in vitro translation of E1A-specific
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H. E S C H E AND B. S I E G M A N N
110
38K 59K
36K 19K
30K 17K
28K
26K
25K
24K (10K)
22K
IX
(a)
0
41K
(b) 23-39K
15.17K
10
I
20
- ( 67K ) . . . .
17K
19K
60K
I
30
I
40
I
50
1( E2 I~0 I
61K
E3
80
20K
18K
ll-5K
61K
14.5K (1)
13.5K
61K
(2)
Fig. 7. Adenovirus proteins synthesized in the absence of virus DNA replication. The conventional
early gene blocks (El to E4) are shown as arrows; the direction of the arrow gives the polarity of
transcription in each block. Polypeptides translated in vitro from RNA deriving from each block are
listed above or below their points of origin. The circled numerals (1 to 3) mark the loci of the elements of
the three-part leader associated with late mRNAs. (a) Our results; (b) results from (1) Jochemsen et aL
(1980) and (2) Rosenwirth et al. (1975).
R N A can be immunoprecipitated from in vivo labelled extracts of infected cells with the
monospecific antibodies described above (H. Esche et al., unpublished results). A higher
number of E 1A polypeptides than E 1A-specific RNAs was also reported for adenovirus types
2 and 5. Both viruses transcribe from early region E1A three overlapping and differently
spliced R N A s (Chow et al., 1979). At least two of these RNAs each code for two related
polypeptides of different mol. wt. (Esche et al., 1980). The different sizes of the two proteins
derived from one R N A species might be accomplished by a post-translational modification,
such as phosphorylation or proteolytic removal of a portion of at least one of the two
molecules.
'~
I n vitro translation of R N A selected with fragment H i n d I I I I resulted in the synthesis of
the E1B polypeptides 59K, 19K and 17K. The finding that the E1B m R N A s coding for the
19K and 17K proteins could be selected also with fragment H i n d I I I F indicates that the 3'
ends of these RNAs map to the right of the H i n d I I I cleavage site at 10.9 map units. These
data which confirm similar observations by Jochemsen et al. (1980) would imply that
transcription of early region E I B during lyric infection extends further to the right than has
been reported by Ortin et al. (1976) and Smiley & Mak (1978). The fact that we failed to
select m R N A which codes for the E1B 59K polypeptide, although D N A sequence data of the
A d l 2 E1B region indicate that the coding region for the 59K protein is located downstream
of the region coding for the 19K polypeptide (H. Bos, personal communication), remains to
be explained. I n vitro translation of H i n d I I I F-selected RNA, prepared early as well as late
after infection in a fractionated cell-free system prepared from rabbit reticulocytes, resulted in
some experiments, in addition to the proteins 19K and 17K, in the synthesis of a 10K
polypeptide (data not shown). This protein which may probably be identical with the 10K
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A d l 2 proteins in infected and transformed cells
111
polypeptide obtained also with EcoRI C-selected mRNA (Fig. 1d) may represent polypeptide
IX of Adl2.
RNA specific for early region E2a directed the synthesis of mainly one protein of tool. wt.
61K probably representing the single-strand DNA-binding protein of Adl2 (Rosenwirth et
al., 1975). The weak protein bands of 45K to 34K which appeared at approx, the same pH
(about 7.5) as the 61K protein in two-dimensional gels (Fig. 3) may represent proteolytic
degradation of the 61K polypeptide. To early viral region E3 we could assign a protein of
16K. This protein we obtained with RNA selected among other fragments with BamHI C but
also PstI I, suggesting that region E3 extends at least from 73 to 90 map units. Early region
E4 codes for polypeptides of mol. wt. 20K, 18K and 11.5K. Cycloheximide strongly
enhances the concentration of early region E4-speciflc RNAs, which is in agreement with the
results ofEggerding & Raskas (1978) and Chow et al. (1979).
In addition to the proteins encoded by the conventional early regions El, E2a, E3 and E4,
we have identified a 67K polypeptide whose mRNA maps between 11 and 28 map units. It
may represent the precursor of the polypeptide covalently attached to the 5' ends of Adl2
virion DNA in analogy to the Ad2 87K protein expressed from this region (Stillman et al.,
1981). For comparison of our protein data with those from other groups, we have listed in
Fig. 7 (b) early Ad 12-specific proteins which have been found by other laboratories.
Translation of Ad 12-specific RNA prepared from the Ad 12-transformed hamster cell lines
T637 and HA12/7, as well as the Adl2-induced rat brain tumour cell line RBT12/3, showed
that early regions E1 and E4 are expressed in these cell lines. We have found functional
cytoplasmic RNA specific for regions E2 and E3 in lines T637 and RBT12/3 but not in the
hamster line HA12/7. The expression of early region E1 in all three Adl2-transformed cell
lines reflects the requirement of the products of this region for initiation and maintenance of
cellular transformation. The proteins obtained with E 1 RNA from the transformed cells have
very similar if not identical tool. wt. as those produced by RNA from infected cells (Fig. 2, 5,
6). An additional protein of 34K, however, was found only with RNA prepared from the
Ad12-transformed cells and showed common tryptic peptides with EIA polypeptides. One
possible explanation for the synthesis of this 34K polypeptide by RNA from the transformed
cells might be that it is encoded by an RNA species which is spliced differently in transformed
cells in comparison to RNAs in infected cells. It has been shown that the processing of
primary transcripts to cytoplasmic mRNAs can follow several alternative pathways with
different intermediates (Chow et al., 1979). Such small variations in virus-specific RNA
populations in the transformed cells may be caused by (small) differences in growth
conditions of these cells. Translation of El-specific RNA further indicated that the E1B 17K
mRNA transcribed poorly in Ad12-infected cells is present in much larger amounts in the
Ad 12-transformed cells.
The finding that not only the transforming region but also early region E4 is expressed in
all the Adl2-transformed cell lines used, which has been described for other Adl2- and
Ad2-transformed cell lines also (Jochemsen et al., 1980; H. Esche, unpublished results),
raises the question as to whether the function of some of the E4 gene products can exert any
influence on the transforming process or .the phenotype of the transformed cells. The protein
patterns observed with E4-specific RNA prepared from the transformed cells showed, in
addition to proteins obtained with RNA from infected cells, two proteins of 25K and 24K.
We are currently analysing these proteins in more detail.
RNAs transcribed from early regions E2 and E3 in cell lines T637 and RBTI2/3 directed
mainly the synthesis of the polypeptides found using RNA from infected cells (E2 61K, E3
16K), which are apparently not required for transformation. That we have not obtained
E3-specific proteins with RNA from the Adl2-transformed hamster line HA 12/7 confirms
the observation by Ortin et al. (1976) and Schirm & Doerfler (1981) that region E3 is not
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112
H. ESCHE
AND
B. SIEGMANN
transcribed in these cells. RNA from region E2, however, was shown to be expressed and
transported into the cytoplasm of HA12/7 cells (Ortin et al., 1976). Why this RNA cannot
be translated in vitro has still to be explained.
In none of the transformed cell lines investigated has virus RNA been found transcribed
from DNA sequences between 11 and 28 map units. Functional RNA from late viral regions
has not been detected either (Schirm & Doerfler, 1981).
This research was supported by the Deutsche Forschungsgemeinschaft through SFB 74.
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