Download Mechanism of translation of the bicistronic mRNA encoding human

Journal of General Virology (1994), 75, 2663-2670. Printedin Great Britain
2663
Mechanism of translation of the bicistronic mRNA encoding human
papillomavirus type 16 E6-E7 genes
Theresa M. C. Tan,* Bernd Gloss,~ Hans-Ulrich Bernard and Robert C. Y. Ting
Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511,
Republic of Singapore
The transforming genes E6 and E7 of human papillomavirus (HPV) type 16 and other HPV types are
expressed from a bicistronic mRNA with a characteristic
spacing of 3 to 6 bp between the termination codon of
E6 and the initiation codon of E7. Plasmid pSP64E6E7
which contains the reading frames of both E6 and E7
was constructed in order to study the expression of both
proteins in a coupled transcription/rabbit reticulocyte
translation system. Both E6 and E7 proteins were
expressed simultaneously. This translation could be
interfered with by antisense oligonucleotides corresponding to various regions of the transcript. Antisense
oligonucleotides targeted at sequences flanking either
side of the translation initiation codon of the E6 open
reading frame were effective in inhibiting the synthesis of
both proteins, whereas oligonucleotides complementary
to the coding regions downstream of the first start codon
showed either a considerably reduced effect or none at
all. In particular, there was limited inhibition of E7
translation by antisense oligonucleotides flanking the
translation start region of the E7 gene. In the presence of
RNase H, it was possible to selectively inhibit the
synthesis of either E6 or E7 by several gene-internal
antisense oligonucleotides. We conclude that HPV16
E6-E7 bicistronic mRNA is fully functional and that
both proteins are translated with equal efficiency via the
scanning mechanisms with reinitiation at the second
open reading frame. In addition, both AE6 and AE7 may
have therapeutical potential as they are capable of
inhibiting the proliferation of CaSki cells which contain
the HPV16 genome.
Introduction
encoded in the E6 and E7 genes whose products interfere
with the functions of the p53 and the Rb tumour
suppressor genes (Hudson et al., 1990; Mtinger et al.,
1989; Hawley-Nelson et al., 1989; Werness et al., 1990;
Dyson et al., 1989). The fact that the E6 and E7 proteins
are consistently expressed in HPV-positive cervical
cancers and in cell lines derived from this cancer (Schwarz
et al., 1985; Schneider-G/idicke & Schwarz, 1986;
Smotkin & Wettstein, 1986; Androphy et al., 1987;
Bedell et al., 1989; Cullen et al., 1991) supports the
hypothesis that expression of E6 and E7 plays a role in
cervical carcinogenesis. The HPV16 E6 and E7 proteins
are transcribed from the same promoter, P97, in the form
of a bicistronic message (Schneider-G/idicke & Schwarz,
1986; Smotkin & Wettstein, 1986).
This study examines whether the E6-E7 message
allows E7 to be translated efficiently. The use of antisense
oligonucleotides to elucidate the mechanism for translation of this bicistronic message will be described. The
implications of the results for understanding the mechanism of translation of this bicistronic message and for
the design of specific and effective antisense oligonucleotides will be discussed.
Antisense oligonucleotides are short segments of single
stranded RNA or DNA with nucleotide sequences
complementary to a specific gene or its mRNA. When
targeted at mRNA or its precursor (pre-mRNA),
antisense oligonucleotides hybridize and form duplexes
which prevent the translation of the message into the
encoded protein (Paterson et al., 1977; Izant &
Weintraub, 1985; Melton, 1985; Blake et al., 1985). A
refined understanding of this inhibitory mechanism could
lead to using it as a tool for interfering with pathogenic
processes. To study this mechanism we used the
transforming genes of the human papillomavirus.
Papillomaviruses have a circular DNA genome of
approximately 8 kb. To date, more than 60 human
papillomavirus types (HPV) have been identified. Among
these a subgroup, including HPV16 and HPV18, is
frequently detected in cervical cancer (DeVilliers, 1989).
The transforming properties of HPV16 and HPV18 are
-~ Presentaddress:Schoolof Medicine,Universityof California,San
Diego, 9500 GilmanDrive, La Jolla, California92093, U.S.A.
0001 2654 © 1994SGM
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T. M. C. Tan and others
2664
Methods
Synthesis of oligonucleotides. Oligonucleotides were synthesized by
B. Li at the Institute of Molecular and Cell Biology or custom-made by
New England Biolabs. The oligonucleotides were purified by HPLC.
All oligonucleotides were dried and resuspended at a concentration of
~
~f
'=PeTS
I
r,.
~
I~SP6 TS
~I~P97
~
o
Region A /~TTTAGGTGACACAI"rAGAATACAAGCTTAACTGCA~TGTn'CAGGACCCACAG
sPrPromotor
HindIII
Met PheGtn Asp Pro Gin
ASP6
AE6
Region B CAGCTGTAATCATGCATGGAGATACACCT
Gin Leu Ter
Met His Gly Asp Thr Pro
q
b
AE7
Region C AAACCA~AATCTACCATGGCTGATCCTGCAG
Lys Pro Ter
PstI
Oligonucleotides
ASP6
AE6
A
B
AE7
c
D
Control
Sequences (5-3")
Position targeted
AGTTAAGC'FrGTATI'C
SP6 TS
GTGGGTCCTGAAACAT
104-119
TATTGCTGTTCTAATG
363-378
GATGATCTGCAACAAG
516-531
GTGTATCTCCATGCAT
562-577
TGGTTTCTGAGAACAG
840-855
CTGCTTGTCCAGCTGG
682-697 (455-469)
TTGGCCGCTGCCATCC ....
Fig. 1. Structure of the plasmid pSP64E6E7 and positions of antisense
oligonucleotides. The numbering of HPV16E6/E7 follows that
published by Seedorf et al., 1985. Sequences in oligonucleotide D that
are complementary to E6 are underlined. The ORFs of HPV16 E6 and
E7 are cloned using the HindlII and PstI sites of the pSP64 plasmid.
The SP6 transcription start (SP6 TS) is 12 bases upstream of the HPV
16 sequences starting at P97 and ending at the PstI restriction site.
Plasmid pSP64E6 is similar to pSP64E6E7 but ends with the bases TC
after the E6 termination codon followed by the PstI site. Plasmid
pSP64E7 is also similar to pSP64E6E7 but starts with the sequence
TGTAATC after the HindlII site followed by the ATG of E7.
1 mM in water. The sequence of the oligonucleotides and their expected
positions upon hybridization are shown in Fig. 1. Phosphorothioate
analogs were custom-made by Oligos Etc and resuspended at a
concentration of 2 mM in water.
Plasmids. Segments of the HPV-16 genome that either encoded the
E6 protein, E7 protein or both proteins together as a bicistronic
sequence were obtained from a HPV-16 clone using the PCR. The
forward PCR primer contained a HindIII restriction site whereas the
reverse primer had a PstI site (Table 1). After cleavage with these
enzymes, each of the three PCR products were ligated into the plasmid
pSP64. The recombinant vectors will be referred to as pSP64E6,
pSP64E7 and pSP64E6E7 (Fig. 1).
In vitro protein translation. The TNT SP6 coupled reticulocyte lysate
system (Promega) was used for the coupled transcription-translation
reactions. The pre-mix contained 52 ~tl lysate, 4 lal reaction buffer, 4 lal
SP6 polymerase, 2 ~tl amino acid mixture without cysteine (all the
above reagents were supplied with the system), 4 lal recombinant
RNasin ribonuclease inhibitor (40 U/~I; Promega), 6 lal of H20 and 8 lal
of 35S-labelled L-cysteine containing 74 pmol (NEN, DuPont). The
specific activities of the four batches of zsS-labelled L-cysteine ranged
from 1150 to 1220Ci/mmol. Pre-mix (8 lal) was added to 1 lag of
recombinant vector DNA and the reaction volume was adjusted to
10 lal before incubation at 37 °C for 120 min.
Hybrid arrested in vitro translation. The 10 lal reaction mixture
containing 1 lag of pSP64E6E7 was similar to that described above
except for the presence of the antisense oligonucleotides at a final
concentration of 50, 100 or 200 taM. Translation of pSP64E7 was also
performed in the presence of 50 and 100 tam of antisense oligonucleotides.
Inhibition of translation by RNase H and antisense oligonucleotides.
The 10 p.l reaction mixture containing 1 lag of pSP64E6E7 was similar
to that described above except for the presence of 25 taMof the antisense
oligonucleotide and 1.5 U of RNase H (Promega). For reactions
conducted in the presence of 25 tam of AE6 or the control oligonucleotide, 0-25 lag of a luciferase expression plasmid (provided as a
positive control in the TNT SP6 coupled reticulocyte lysate system) was
added as a control.
Denaturing gel analysis of translation products. After 120 min of
incubation at 37 °C, 2 ~tl samples were removed from each reaction
mixture for analysis on 12.5 % SDS-PAGE. The electrophoresis was
stopped when the bromophenol blue dye had run off the bottom of the
gel. The gel was fixed in acetic acid :isopropylalcohol:water (10: 25 : 65)
for 20 min, soaked in Amplify (Amersham) for 10 min followed by
immersion in 5 % glycerol for 20 rain before being dried and then
fluorographed overnight at 70 °C. The bands were quantified with t h e
Table 1. Primers used for obtaining the E6, E7 and E6-E7 open reading
frames*
Open reading
frames/
(recombinant
vector)
E6(pSP64E6)
E7(pSP64E7)
E6/E7
(pSP64E6E7)
PCR primers
Forward primer a 5" GGAAGCTTAACTGCAATGTTTCAGGACCC 3'
Reverse primer b 5' GGCTGCAGGATTACAGCTGGGTT 3'
Forward primer c 5' GGAAGCTTTGTAATCATGCATGGAGATAC 3'
Reverse primer d 5' GGCTGCAGGATCAGCCATGGTAGATTATG 3'
Forward primer a and reverse primer d
* The underlined bases in the forward primers show the Hindlll restriction site whereas those on
reverse primers show the PstI site. Bases in bold indicate either the start codon (ATG) or the
termination codon (TTA).
the
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Translation o f H P V 1 6 E 6 - E 7 m R N A
image analysing system Visage 110 (Biolmage) using the Sunview
program.
Cell proliferation as determined by tetrazolium reduction. An
established cervical epithelial tumour cell line, CaSki, containing
HPV16 was used in this study. The cells were grown in MEM
supplemented with 10% heat-inactivated fetal bovine serum,
100 units/ml penicillin, 100 Ixg/ml streptomycin and 2 mM-glutamine.
Another cervicaltumour cell line, C-33A, lacking the HPV genome was
used as a control cell line. The C-33A cells were grown in medium
similar to that used for CaSki cells with the addition of 0.8 mg/1 of
MEM non-essential amino acids and 1 mu-pyruvate. Cells (5 x 10a)
were seeded in each well of the 96-weU plate (Nunc) and allowed to
recover for 48 h prior to the treatment with phosphorothioate
oligonucleotides.Fresh medium containing 5 ~tg/mlof the transfection
reagent, DOTAP (Boehringer-Mannheim) and 20 laM of phosphorothioate analogs was then added to the cellsand the cellswere treated for
48 h with a change of medium after 24 h. Twenty lal of freshly prepared
MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium]PMS (phenazine methosulphate) was
then added to each well. (MTS and PMS were supplied as solutions of
2 mg/ml and 0.92 mg/ml respectively in the CellTitre 96 AQueous
Non-radioactive cell proliferation assay kit from Promega). After
incubation at 37 °C for 3 h, the A490was read using an ELISA plate
reader. Assays were performed in triplicate and the results were
expressed as percentage inhibition of the untreated culture. This was
determined by finding the difference in A490 values between the
untreated and oligonucleotide-treated cultures, and subsequently by
calculating the ratio between this and the A490value of the untreated
culture as a percentage.
Results
Synthesis of E6 and E7
O u r initial a i m was to identify the H P V 1 6 proteins E6
a n d E7 after expression in a c o u p l e d in vitro
t r a n s c r i p t i o n - r a b b i t reticulocyte t r a n s l a t i o n system, a n d
to m o n i t o r their expression f r o m a bicistronic m R N A .
T h e plasmids pSP64E6 a n d pSP64E7 were used to
confirm the identities o f the E6 a n d E7 t r a n s l a t i o n
p r o d u c t s a n d pSP64E6E7 was c o n s t r u c t e d as a source
for the bicistronic m R N A (Fig. 1). T h e p r o d u c t s were
analysed by S D S - P A G E a n d f l u o r o g r a p h y (Fig. 2). The
E6 p r o t e i n m i g r a t e d as predicted as a 17K protein. By
contrast, the H P V 1 6 E7 p r o t e i n m i g r a t e d slowly to a n
a b n o r m a l extent; with a Mr of 19K instead o f the expected
12K value. This a b e r r a n t m i g r a t i o n b e h a v i o u r o f H P V 16
E7 has been previously described (Mfinger et al., 1991 ;
Scheffner et al., 1992). The bicistronic m R N A t r a n scribed f r o m pSP64E6E7 resulted in the synthesis o f b o t h
proteins. The intensity of the E7 signal was d e t e r m i n e d
to be 51.9 % +3"7 % ( N = 4) o f the E6 signal.
Effects of antisense oligonucleotides on E6 and E7
translation
T o study the effects o f interference b y antisense oligonucleotides o n the t r a n s l a t i o n o f E6 a n d E7 f r o m the
bicistronic E 6 - E 7 m R N A , p r o t e i n synthesis was carried
2665
o u t in a c o m b i n e d t r a n s c r i p t i o n - t r a n s l a t i o n system with
pSP64E6E7 as described above b u t with antisense
oligonucleotides a d d e d at the start o f the reaction.
Oligonucleotides A E 6 a n d ASP6, which were c o m p l e m e n t a r y to sequences flanking the E6 start c o d o n or
o v e r l a p p i n g the start of the SP6 t r a n s c r i p t respectively,
were effective in i n h i b i t i n g b o t h E6 a n d E7 p r o t e i n
synthesis. I n h i b i t i o n levels were greater t h a n 90 % with
1
2
3
4
-
32
-
27
-
21-5
18
E71~
E6P
-
14
Fig. 2. In vitro translation of HPV16 E6 and E7 in a combined
transcription-rabbit reticulocytetranslation system. Lanes 1and 2 show
the synthesis of E7 from pSP64E7 and E6 from pSP64E6, respectively.
Lane 3 shows the negative control without any plasmid. Lane 4 shows
simultaneous synthesis of E6 and E7 from pSP64E6E7. The positions
of Mr markers are indicated on the right and the positions of E6 and
E7 are indicated by arrowheads on the left.
T a b l e 2. Inhibition o f H P V 1 6 E6 and E7 translation by
antisense oligonucleotides
Antisense
oligonucleotides/
concentrations (pM)
A
50
100
200
B
50
100
200
C
50
100
200
D
50
100
200
ASP6 50
100
200
AE6 50
100
200
AE7 50
100
200
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Percent inhibition
of translation
of E6
38"7
37'8
60"5
1"7
5"9
5"0
0"0
0"0
0"0
7"6
0"0
0-0
27-0
90"8
92-2
80.5
96"9
100"0
5"6
19-5
65"0
Percent inhibition
of translation
of E7
0"0
0"0
28"2
0"0
0"0
10"6
0"0
0"0
0"0
0"0
0'0
0'0
52"1
94'0
92'2
86"2
97"2
100"0
25-0
39'5
66"3
T. M. C. Tan and others
2666
1
2
3
4
5
6
1
7
2+
3+
4
5+
6
7
- 49
L~
- 32
97
69
49
27
E71~
21.5
- 18
32
14
27
Fig. 3. Inhibition of E7 translation by antisense oligonucleotides AE6
and AE7. Lane 1 is a negative control without any plasmid. Lanes 2
and 7 show the translation of E7 from pSP64E7. Lane 3 has 100 laMof
AE7 and lane 4 has 50 laMof AE7. Lane 5 has 100 laMof AE6 and lane
6 has 50 gM of AE6. The positions of Mr markers are indicated on the
fight and the position of E7 is indicated by the arrowhead on the left.
1
2
3
4+
5
6+
7
8+
9
E7
E61~
10+
14
32
-
27
21.5
-18
-
-14
Fig. 4. Selective inhibition of E6/E7 translation by antisense oligonucleotides A, B, C and D in the presence of RNase H. All lanes with
(+) indicate the addition of RNase H. Lane 1 shows the normal
translation of E6 and E7. Lane 2 is a negative control without any
plasmid. Lanes 3 and 4 have oligonucleotide A, lanes 5 and 6
oligonucleotide B, lanes 7 and 8 oligonucleotide C and lanes 9 and 10
oligonucleotideD. All oligonucleotidesare at a concentration of 25 laN.
In the presence of RNase H, oligonucleotides A and B selectively
inhibit E6 synthesis whereas D inhibits E7 and to a lesser extent E6 as
well. The positions of Mr markers are indicated on the fight and the
positions of E6 and E7 are indicated by arrowheads on the left.
100 or 200 gM of either oligonucleotide for E6, as well as
for the d o w n s t r e a m E7 gene (Table 2). I n the case o f the
oligonucleotide AE7, which is c o m p l e m e n t a r y to
sequences flanking the E7 i n i t i a t i o n c o d o n , only approx.
40 % a n d 65 % i n h i b i t i o n levels were observed at 100 a n d
200 gM-AE7. A E 7 also h a d some effect o n the expression
of the E6 gene especially w h e n a d d e d in a m o u n t s of
200 JaM. This non-specific i n h i b i t i o n is due to the partial
identity b e t w e e n oligonucleotides A E 6 a n d A E 7 (nine
o u t of 16 bp). Both 100 JaM-AE6 a n d -AE7 did n o t affect
the t r a n s c r i p t i o n o f pSP64E6E7 (data n o t shown).
Oligonucleotides A, B, C a n d D, which were c o m p l e m e n t a r y to sequences w i t h i n the E6 or the E7 gene or
Fig. 5. Inhibition of E6/E7 translation by antisense oligonudeotides
AE6 and AE7 in the presence of RNase H. A control protein, luciferase
(62K) was simultaneously translated. The translation reactions were
similar to those described in Methods with the exception of the
addition of 0.25 lag of the luciferase plasmid (provided as a positive
control with the Promega kit). Lanes with (+) indicate the addition of
RNase H. Lane 1 is without any plasmid. Lane 2 shows the translation
of L, E6 and E7 in the presence of RNase H. The synthesis of all three
proteins is slightly affected by the presence of RNase H. Lanes 3 and
4 have an unrelated control oligonncleotide (5' TTGGCCGCTGCCATCC 3') and lanes 5 and 6 have oligonucleotide AE6. Lane 7 shows
the normal translation of all three proteins. The concentration of all
oligonucleotideswas 25 laM.The positions of Mr markers are indicated
on the right and the positions of luciferase (L), E6 and E7 are indicated
by arrowheads on the left.
to the sequences at the 3' t e r m i n u s of the genes, h a d little
or n o effect o n E6 or E7 t r a n s l a t i o n (Table 2).
I n addition, the effect o f A E 6 a n d A E 7 o n the
t r a n s l a t i o n o f E7 from pSP64E7 was also examined. A E 6
did n o t i n h i b i t the synthesis o f E7 w h e n added at the
50 ~tM level b u t there was some i n h i b i t i o n at the 100 jam
level which was due to the p a r t i a l identity b e t w e e n A E 6
a n d AE7. I n contrast, A E 7 affected E7 synthesis m o r e
significantly w h e n a d d e d i n 50 gM a m o u n t s a n d there was
almost complete i n h i b i t i o n w h e n a d d e d at 100gM
a m o u n t s (Fig. 3).
RNase H and antisense inhibition of E6 and E7
translation
E x o g e n o u s R N a s e H cleaves R N A - D N A hybrids a n d it
has been d e m o n s t r a t e d that its a d d i t i o n to in vitro
t r a n s l a t i n g systems c a n increase the extent of h y b r i d arrest t r a n s l a t i o n o f m R N A s ( M i n s h u l l & H u n t , 1986).
I n this study, we e x a m i n e d the effects of R N a s e H o n the
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Translation of HPV16 E6-E7 m R N A
70
T
60
synthesis of luciferase and E7 (Fig. 5). The unrelated
control oligonucleotide also had no effect on the
expression of the three proteins.
Effect of the antisense oligonucleotides on cell
proliferation
50
20
Treatment of the CaSki cell cultures for 48 h with either
AE6 or AE7 phosphorothioate analogs, resulted in the
inhibition of proliferation of CaSki cells by 38"6 % and
52.6 %, respectively. In contrast, treatment of C-33A cell
cultures only led to slight inhibition levels of 6.6 % and
18-2 % by AE6 and AE7, respectively. Treatment of both
cell lines with the unrelated control oligonucleotide also
resulted in minimal inhibition.
10
Discussion
40
e~
o
2667
30
,.Q
.=
0
Control
AE6
AE7
Fig. 6. Inhibition of cell proliferation by AE6 and AE7. Treatment of
CaSki cells by phosphorothioate analogs of AE6 and AE7 led to
inhibition of cell growth ( , ) whereas little inhibition was observedfor
similarlytreated C-33Acells(D). There was also little inhibition of cell
growth for both cell lines by the unrelated control oligonucleotide
(control). The results are expressed as mean (+S.D.) percentage
inhibition of growth.
expression of E6 and E7 proteins from the bicistronic
mRNA. Fig. 4 illustrates the effects of RNase H in
combination with 25 gM o f oligonucleotides A, B, C or
D. In the absence of RNase H, these oligonucleotides
had little or no effect (Table 2). However, together with
RNase H, oligonucleotides A and B could selectively
inhibit the synthesis of E6 (Fig. 4). Oligonucleotide C in
the absence or presence of RNase H had little effect,
whereas oligonucleotide D caused the complete inhibition of E7 synthesis in the presence of RNase H and
also a significant inhibition (77"6 %) o f E6 synthesis. The
inhibition in E6 expression is due to the 75 % identity
between the oligonucleotide and the E6 coding region.
As a means o f examining the specificity of the RNase
H and antisense oligonucteotide combined inhibitory
effects, a series of experiments using a luciferase
expression plasmid as the control were carried out. Fig. 5
(lane 2) shows the synthesis of E6, E7 and the 62K
luciferase protein in the presence of RNase H. The
synthesis of all three proteins was slightly decreased in
the presence of RNase H and this is because of the effects
of minimal unspecific degradation o f the luciferase and
bicistronic E6-E7 RNAs. When 25 gM of oligonucleotide
AE6 and RNase H were used it was possible to selectively
inhibit E6 synthesis without significant effect on the
Our research examined the efficiency of translation of
both proteins from a bicistronic viral m R N A , and the
possibility o f interfering differentially with the expression
of both genes by antisense oligonucleotides. In cervical
cancers and cervical carcinoma-derived cell lines containing HPV16, mRNAs encoding E6 and E7 proteins
are transcribed from the same promoter, P97 (SchneiderG~idicke & Schwarz, 1986; Smotkin & Wettstein, 1986),
in the form of a bicistronic mRNA. The unspliced E6-E7
m R N A encodes the full-length E6 as well as E7 proteins.
In addition, two spliced transcripts E6*I-E7 and
E6*II-E7 whose biological functions are unknown are
usually detected (Shirasawa et al., 1991). It has been
proposed that it is a function of these spliced transcripts
to stimulate E7 translation (Schneider-Gfidicke &
Schwarz, 1986; Smotkin et al., 1989). The unspliced
transcripts of all HPV types that infect genital epithelia,
have a short spacing region (5 bp in the case of HPV16)
between the termination codon of E6 and the initiation
codon of E7. As it is not possible to examine the
translation of the bicistronic HPV16 E6-E7 in cultures
using cell lines containing the HPV16 genome since
splicing events occur, we examined the translation in
vitro using an eukaryotic-derived rabbit reticulocyte
system which should mimic the intracellular conditions
that are present for translation of HPV mRNAs.
Most mRNAs of eukaryotes and their viruses are
monocistronic but in the case of those m R N A s which are
bicistronic, the ribosomes can reinitiate translation at the
next A U G codon downstream (Kozak, 1984; Liu et al.,
1984; Peabody et al., 1986). The efficiency of reinitiation
in such cases is usually low (Kozak, 1984; Liu et al.,
1984) but this progressively improves as the intercistronic sequence lengthens (Kozak, 1987). This reinitiation only occurs when the upstream open reading
frame (ORF) is a 'minicistron' and encodes for a small
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2668
T. M . C. Tan and others
peptide. For plant and animal virus mRNAs that are
structurally bicistronic, encoding two full-length proteins,
only the 5'-proximal end ORF is usually translated, i.e.
they are functionally monocistronic (Kozak, 1986). This
study demonstrates that despite the close proximity of
the ORFs of E6 and E7 and the fact that the message
codes for two full-length proteins, the translation of the
protein E7 is as efficient as that of E6 in vitro. This
quantification was done by comparing the E7 and E6
signals in radiolabelling experiments using [35S]cysteine.
The E7 signal was expected to be half that of E6 as
there are seven cysteine residues in E7 as compared with
the 14 cysteine residues in E6. In our experiments the E7
signals had an intensity which was 51% of that of E6,
leading us to conclude that both proteins are translated
with almost equal efficiency. Similar effective expression
of both proteins was also observed when E6 and E7 were
translated in a rabbit reticulocyte lysate system using
RNAs transcribed from pSP64E6E7 (data not shown).
The efficient translation of the E6 and E7 proteins of
other HPVs has also been reported (Roggenbuck et al.,
1991).
For antisense inhibition studies, a series of antisense
oligonucleotides was constructed. The AE6 and AE7
oligonucleotides were constructed to include sequences
flanking the start codons of E6 and E7 respectively as
these regions are probably most sensitive to antisense
inhibition (Gupta, 1987; Maher & Dolnick, 1987). As
such, there is partial identity between AE6 and AE7
(nine out of 16 bp). However, at concentrations of 50 ~tM
or less, AE6 could selectively bind to sequences flanking
the E6 start codon and did not inhibit the translation of
E7 from pSP64E7 which encodes for E7 alone. In
contrast, 50 gM of AE6 inhibited the synthesis of both E6
and E7 from pSP64E6E7 which encodes the bicistronic
E6-E7 message whereas AE7 did not affect this synthesis.
At high concentrations, especially at 200 gM levels, there
were some unspecific effects from both oligonucleotides.
Our data on antisense inhibition also indicate that
there is efficient translation of E6 and E7 from the
bicistronic message. Oligonucleotides ASP6 and AE6
targeted at sequences around the E6 initiation codon can
inhibit translation of both proteins efficiently at 100 and
50 gM levels respectively. This observation fully supports
the scanning process model. In its simplest form, this
model (Kozak, 1989) postulates that a 40S ribosomal
subunit and an imperfectly defined set of initiation
factors (Pain, 1986) enters at the 5' end of the mRNA
and migrates along the mRNA until it reaches the first
start codon, whereupon a 60S ribosomal subunit joins it
and the first peptide bond is formed. In line with this
model, the inhibition of expression of E6 by ASP6 is
probably due to the interference with the binding of the
40S ribosomal subunit and the initiation factors or their
passage to the start codon, whereas AE6 probably
interferes with the binding of the 60S subunit.
Oligonucleotides B and AE7 that are targeted at
sequences flanking the E7 initiation codon, however, are
less effective in reducing E7 translation that arises from
the bicistronic message, although partial inhibition was
observed at 200 [.tM levels of these oligonucleotides.
These observations together with the inhibition by ASP6
and AE6, strengthen the hypothesis that E6 and E7 are
translated from a bicistronic sequence and that the
ribosomes reinitiate translation efficiently after termination of E6 translation. The hindrance caused by the
antisense oligonucleotides B and AE7 is not sufficient to
dislodge the ribosomes and prevent E7 synthesis.
Alternatively, a mechanism whereby the ribosomes bind
mRNA internally at the initiation A U G of E7 could be
operating. Our data do not support this possibility since
oligonucleotide AE7 would then have blocked translation of E7 more effectively. In addition, the ratio of the
E7:E6 [35S]cysteine signal would not be 51% if E7 was
translated by the internal ribosome-binding mechanism
since one would not expect equal molecules of both
proteins to be translated. The coding region-specific
antisense oligonucleotides also did not prevent protein
synthesis. This again is probably due to the fact that the
translating ribosome can dislodge the oligonucleotide
from the RNA template (Sankar et al., 1989).
RNase H was used to examine whether the genes 1
internal binding antisense oligonucleotides which did not
lead to a significant inhibition of E6 and E7 expression
were interacting specifically with the targeted regions
under the experimental conditions used. We observed
that RNase H has a dramatic effect on translation
inhibition even in the case of oligonucleotides other than
AE6 and ASP6. Also, the concentration of oligonucleotide AE6, which is effective without RNase H at
a concentration of 50 gM, can be reduced to 25 ~tMin the
presence of RNase H. The interaction of the oligonucleotide AE6 with the mRNA was observed to be
highly specific since the translation of an unrelated
protein, luciferase, was not affected at all. Taken
together, these results are in agreement with published
data demonstrating that oligonucleotides directed against
gene-internal sequences form heteroduplexes with mRNA
efficiently. These heteroduplexes can be detected due to
their destruction by RNase H, but they are unstable
during the translation process (Minshull & Hunt, 1986;
Walder & Walder, 1988; Gagnor et al., 1987).
Therefore, for effective interference of translation
from such bicistronic messages, one should employ
antisense oligonucleotides that are complementary to
regions at the 5' end or at the initiation codon of the first
open reading frame. Hence, antisense inhibition of
translation from such a bicistronic message closely
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Translation of H P V 1 6 E 6 - E 7 m R N A
resembles that of monocistronic messages in that regions
flanking the first initiation codon at the 5' end are most
sensitive to antisense oligonucleotide inhibition of translation (Sankar et al., 1989; Maher & Dolnick, 1987;
Gupta, 1987).
In vivo, a large fraction of the E6-E7 transcript is
spliced to produce two shorter transcripts, E6*I-E7 and
E6*II-E7. Since these two shorter transcripts have the
same 5' oligonucleotide sequence in common with the
full-length E6-E7 transcript, one would assume that the
effects of the AE6 oligonucleotide would be on all three
transcripts. To monitor this possibility, we studied the
growth inhibitory effects of AE6 and AE7 on CaSki and
C-33A cells. It is well documented that the expression of
the HPV E6 and E7 genes is directly linked to the
proliferative capacity of cervical cancer cells (von Kenbel
Doeberitz et al., 1988, 1990; Watanabe et al., 1993).
Interference with the expression of these proteins in
CaSki cells by the phosphorothioate analog of AE6 is
sufficient to inhibit cell proliferation. The AE7 oligonucleotide was also effective in inhibiting cell proliferation. This inhibition of cell proliferation is specific
since both antisense oligonucleotides did not affect
C-33A cells that do not harbour HPV genome. These
results suggest that these oligonucleotides may be useful
therapeutically.
In conclusion, our research provides evidence for the
mechanism of translation of a bicistronic mRNA of a
human virus. The use of antisense oligonucleotides as a
means of dissecting the mechanism of translation
provides an useful method for analysing protein synthesis. This type of analysis is an important prerequisite
for studies conducted in vivo and to predict optimal
targets in research that aims to use antisense oligonucleotides as pharmaceutical substances.
References
ANDROPHY, E.J., HUBBERT, N. L., SCHILLER,J.T. & LowY, D. R.
(1987). Identification of the HPV-16 E6 protein from transformed
mouse cells and human cervical carcinoma cell lines. EMBO Journal
6, 989 992.
BEDELL, i . A . , JONES, K.H., GROSSMAN, S.R. • LAIM1NS, L.A.
(1989). Identification of human papillomavirus type 18 transforming
genes in immortalized and primary cells. Journal of Virology 63,
1247-1255.
BLAKE, K. R., MURAKAMI,A. & MILLER, e. S. (1985). Inhibition of
rabbit globin mRNA translation by sequence-specific oligodeoxynucleotides. Biochemistry 24, 613~6138.
CULLEN, A.P., REID, R., CAMPION, M. & LORINCZ, A.T. (1991).
Analysis of the physical state of the different human papillomavirus
DNAs in intraepithelial and invasive cervical neoplasm. Journal of
Virology 65, 606612.
DEVILLIERS,E. M. (1989). Heterogeneity of the human papillomavirus
group. Journal of Virology 63, 4898-4903.
DYSON, N., HOWLEY,P. M., M1)NGER, K. & HARLOW, L. (1989). The
human papilloma virus-16 E7 oncoprotein is able to bind to the
retinoblastoma gene product. Science 243, 934-937.
GAGNOR,C., BERTRAND,J-R., THENET,S., LEMAiTRE,M., MORVAN,F.,
RAYNER, B., MALVY, C., LEBLEU,B., IMBACH,J. L. & PAOLETTI,C.
2669
(1987). ~-DNA VI: comparative study of ~t- and fl-anomeric
oligonucleotides in hybridization to mRNA and in cell-free translation inhibition. Nucleic Acids Research 15, 10419-10436.
GUPTA, K. C. (1987). Antisense oligonucleotides provide insight into
mechanism of translation initiation of two sendai virus mRNAs.
Journal of Biological Chemistry 262, 7492-7496.
HAWLEY-NELSON,P., VOUSDEN,K. H., HUBBERT,N. L., LOWY, D. R.
& SCHILLER,J. T. (1989). HPV16 E6 and E7 proteins cooperate to
immortalize human foreskin keratinocytes. EMBO Journal 8,
3905-3910.
HUDSON, J.B., BEDELL, M.A., MCCANCE, D.J. & LAIMINS, L.A.
(1990). Immortalization and altered differentiation of human
keratinocytes in vitro by the E6 and E7 open reading frames of
human papillomavirus type 18. Journal of Virology 64, 519-526.
IZANT, J. G. & WEINTRAUB,H. (1985). Constitutive and conditional
suppression of exogenous and endogenous genes by anti-sense RNA.
Science 229, 345-352.
KOZAK, M. (1984). Selection of initiation sites by eucaryotic ribosomes:
effect of inserting AUG triplets upstream from the coding sequence
for preinsulin. Nucleic Acids Research 12, 3873-3893.
KOZAK, M. (1986). Regulation of protein synthesis in virus infected
animals cells. Advances in Virus Research 31, 229-292.
KOZAK, M. (1987). Effects of intercistronic length on the efficiency of
reinitiation by eucaryotic ribosomes. Molecular and Cellular Biology
7, 3438-3445.
KOZAK, M. (1989). The scanning model for translation- an update.
Journal of Cell Biology 108, 229 241.
LIu, C.C., SIMONSEN,C. 8/. LEVINSON, A.D. (1984). Initiation of
translation at internal AUG- codons in mammalian cells. Nature,
London 309, 82 85.
MAILER,L. J., III & DOLNICK,B. J. (1987). Specific hybridization arrest
of dihydrofolate reductase mRNA in vitro using anti-sense RNA or
anti-sense oligonucleotides. Archives of Biochemistry and Biophysics
253, 214-220.
MELTON, D.A. (1985). Injected anti-sense RNAs specifically block
messenger RNA translation in vivo. Proceedings of the National
Academy of Sciences, U.S.A. 82, 144-148.
MINSHULL,J. & HUNT, T. (1986). The use of single-stranded DNA and
RNase H to promote quantitative 'hybrid arrest of translation' of
m R N A / D N A hybrids in reticulocyte lysate cell-free translations.
Nucleic Acids Research 14, 6433-6451.
Mf~GER, K., PHELPS,W. C., BUBB,V., HOWLEY,P. M. & SCHLEGEL,R.
(1989). The E6 and E7 genes of the human papillomavirus type 16
together are necessary and sufficient for transformation of primary
human keratinocytes. Journal of Virology 63, 4417-4421.
MONGER, K., YEE, C.C., PHELPS, W. C., PIETENPOL, J.A., MOSES,
H.L. & HOWLEY, P.M. (1991). Biochemical and biological differences between E7 oncoproteins of the high- and low-risk human
papillomavirus types are determined by amino-terminal sequences.
Journal of Virology 65, 3943-3948.
PAIN, V. M. (1986). Initiation of protein synthesis in mammalian cells.
Biochemical Journal 235, 625-637.
PATERSON,B. M., ROBERTS,B. E. & RUFF, E. L. (t977). Structural gene
identification and mapping by DNA-mRNA hybrid-arrested cellfree translation. Proceedings of the National Academy of Sciences,
U.S.A. 74, 437(V4374.
PEABODY,D. S., SUBRAMANI,S. & BERG, P. (1986). Effects of upstream
reading frames on translation efficiency in SV40 recombinants.
Molecular and Cellular Biology 6, 2704-2711.
ROGGENBUCK,B., LARSEN,P. M., FEY, S. J., BARTSCI-I,D., GISSMANN,
L. & SCHWARZ,E. (1991). Human papillomavirus type 18 E6*, E6
and E7 protein synthesis in cell-free translation systems and
comparison of E6 and E7 in vitro translation products to proteins
immunoprecipitated from human epithelial cells. Journal of Virology
65, 5068-5072.
SANKAR, S., CHEAH, K.C. & PORTER, A.G. (1989). Antisense
oligonucleotide inhibition of encephalomyocarditis virus RNA
translation. European Journal of Biochemistry 184, 39-45.
SCHEFFNER, M., MONGER, K., HUIBREGTSE,J. M. & HOWLEY, P. M.
(1992). Targeted degradation of the retinoblastoma protein by
human papillomavirus E7 E6 fusion proteins. EMBO Journal 11,
2425-2431.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 09:22:45
2670
T. M . C. Tan and others
SCHNEIDER=G,g~DICKE,A. & SCHWARZ,E. (1986). Different human
cervical carcinoma cell lines show similar transcription patterns of
human papillomavirus type 18 early genes. EMBO Journal 5,
2285-2292.
SCHWARZ, E., FREESE,U- K., GISSMANN,L., MAYER,W., ROGGENaUCK,
B., STe,~MLAU, A. & ZLrR HAUSEN, H. (1985). Structure and
transcription of human papillomavirus sequences in cervical carcinoma cells. Nature, London 314, 111-114.
SEEDORF, K., KR~M)4ER, G., DORST, M., Sb'HAL S. & RbWEKAMP,
W. G. (1985). Human papillomavirus type 16 sequence. Virology 145,
181-185.
SHIRASAWA, H., TANZAWA~H., MATSUNAGA,Z. (~ SIMIZU, B. (1991).
Quantitative detection of spliced E6-E7 transcripts of human
papillomavirus type 16 in cervical premalignant lesions. Virology
184, 795-798.
SMOTKIN, D. (~ WETTSTEIN, F.O. (1986). Transcription of human
papillomavirus type 16 early genes in a cervical cancer and a cancerderived cell line and identification of the E7 protein. Proceedings of
the National Academy of Sciences, U.S.A. 83, 46804684.
SMOTKIN, D., PROKOPH, H. & WETTSTEIN, F. O. (1989). Oncogenic and
nononcogenic human genital papillomaviruses generate the E7
mRNA by different mechanisms. Journal of Virology 63, 1441-1447.
VON KENBEL DOEBERITZ, M., OLTERSDORF, T., SCHWARZ, E. &
GISSMANN, L. (1988). Correlation of modified human papillomavirus
early gene expression with altered cell growth in C4-l cervical cancer
cells. Cancer Research 48, 378(~3786.
VON KENBELDOEBERITZ, M., GISSMANN,L. & ZUR HAUSEN, H. (1990).
Growth-regulating functions of human papillomavirus early gene
products in cervical cancer cells acting dominant over enhanced
epidermal growth factor receptor expression. Cancer Research 50,
3730~3736.
WALDER, R. Y. & WALDER, J. A. (1988). Role of RNase H in hybridarrested translation by antisense oligonucleotides. Proceedings of the
National Academy of Sciences, U.S.A. 85, 5011-5015.
WATANABE,S., KANDA, T. & YOSHIKE, K. (1993). Growth dependence
of human papillomavirus 16 DNA-positive cervical cancer cell lines
and human papillomavirus 16-transformed human and rat cells on
the viral oncoproteins. Japanese Journal of Cancer Research 84,
1043-1049.
WERNESS, B. A., LEVINE, A. J. & HOWLEY, P. M. (1990). Association of
human papillomavirus types 16 and 18 E6 proteins with p53. Science
248, 76-79.
(Received 12 May 1994; Accepted 30 May 1994)
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