J. gen. Virol. (1989), 70, 1913-1919.

Printedin Great Britain

1913

Key words: HPV-16 genome/cervical carcinoma cell line/flanking sequences

Structure and Expression of an Integrated Human Papillomavirus Type 16

Genome Amplified in a Cervical Carcinoma Cell Line
By H I R O S H I S H I R A S A W A , 1 Y O S H I M I T O M I T A , 1 A K I R A F U S E , 1
TOMOKO YAMAMOTO, 1 HIDEKI TANZAWA, 1 SOUEI SEKIYA, 2
H I R O Y O S H I T A K A M I Z A W A 2 AND B U N S I T I S I M I Z U 1.
1Department o f Microbiology and 2Department o f Obstetrics and Gynecology, School o f
Medicine, Chiba University, Chiba 280, Japan
(Accepted 17 February 1989)

SUMMARY
A cellular sequence containing the integrated human papillomavirus type 16 genome
in a cervical carcinoma cell line QG-U was cloned and analysed. The transcriptionally
active viral genome disrupted at the E2 and L2 open reading frames was amplified with
its flanking sequences.
Certain types of human papillomavirus (HPV) are associated with precancerous cervical
intraepithelial neoplasias and carcinomas of the uterine cervix (Diirst et al., 1983; Tomita et al.,
1986; Di Luca et al., 1986; Shirasawa et al., 1986). In particular, HPV type t6 is the most
prevalent type and its genomic DNA has been shown to have the ability to transform rodent
cells and immortalize human keratinocytes (Yasumoto et al., 1986; Tsunokawa et al., 1986;
Dtirst et al., 1987; Pirisi et al., 1987). In cervical carcinoma tissues, cell lines and in some
precancerous lesions, HPV-16 DNA is most often integrated into the host DNA although
extrachromosomal viral DNA can persist concurrently (D/irst et al., 1985; Lehn et al., 1985,
1988; Pater & Pater, 1985; Yee et al., 1985; Di Luca et al., 1986; Shirasawa et al., 1986). The
integrated HPV-16 genomes have been shown to be disrupted usually at the E1 and E2 region
retaining the non-coding region and the E6/E7 region which is transcriptionally active (Lehn et
al., 1985; Smotkin & Wettstein, 1986; Shirasawa et al., 1987, 1988; Awady et al., 1987; Baker et
al., 1987; Schneider-Maunoury et al., 1987). These observations support the hypothesis that the
integration of the viral genome plays some role in the development and maintenance of the
malignancy.
To know the precise structure of the integration site, the cellular nucleotide sequence
containing the integrated HPV-16 genome in the cervical carcinoma cell line QG-U was cloned
and analysed. Here we show the precise structure of the integration site, the presence of virushost fused mRNAs and direct evidence of viral genome amplification with its flanking
sequences.
QG-U is a cell line derived from a cervical squamous cell carcinoma of a Japanese patient and
harbours exclusively integrated HPV-16 genomic DNA which is transcriptionally active as
reported previously (Shirasawa et aL, 1987). Southern blot analysis of the QG-U DNA with
restriction enzymes BamHI, HindlII, EcoRI and PstI (single-cut, no-cut, two-cut and six-cut
enzymes for HPV-16 DNA, respectively) using HPV-16 DNA as a probe showed that the viral
genome is monomeric and integrated at a single site (Shirasawa et al., 1987) (Fig. 1). The viral
genome was judged to be interrupted at the 1.1 kb PstI fragment region which contains the 3'
portion of the E2 open reading frame (ORF) and the 5' portion of the L 2 0 R F , as indicated by
the absence of the 1.1 kb PstI fragment (Fig. 1, lane 4). Although the HindlII cleavage yielded a
single fragment containing the viral genome (Fig. 1, lane 2), the fragment was 22 kb in size and
too large to introduce into the 3~phage vector. Therefore, we chose to introduce the two BamHI
fragments hybridizing to HPV-16 DNA (11 kb and 7 kb as shown in Fig. 1, lane 1) into the
BamHI site of the/l phage L47-1.
0000-8821 1989 SGM

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19.3~
7.7~
6.2~
4-3~
3.5-2.7~
1.9-1.5~
1-1~
0.9-0.6~

it

Fig. 1. Southern blot analysis of the DNA from the cervical

carcinoma cell line QG-U. The DNA (5 ~tg) from the QG-U
ceils was digested with one of the four restriction enzymes,
separated on a 0.8~ agarose gel and subjected to Southern blot
hybridization with HPV-16 DNA fragment probes (lane 1,
BamHI; lane 2, HindlII; lane 3, EcoRI; lane 4, PstI). The
symbols O and indicate the 11 kb and 7kb BamHI
fragments, respectively. The bars represent the positions of the
size markers, 2 phage DNA digested with EcoT14I or q~X174
DNA digested with HaelII; the numbers represent the sizes of
the fragments in kb.

The D N A extracted from t)G-U cells was treated with B a m H I and fractionated on a 0.5~
agarose gel. The gel was sliced and individual D N A fractions were eluted and recovered by
D N A C E L L (Daiichi Pure Chemicals) as recommended by the manufacturer. The D N A
fractions that hybridized to HPV-16 D N A were introduced into 2 L47-1 D N A at the BamHI
site. The ligated DNAs were packaged in vitro with Gigapack Gold (Stratagene) for the
construction of enriched partial genomic libraries. Approximately 5 x 10s recombinant phages
for the 7 kb inserts and 1 106 recombinant phages for the 11 kb inserts were plated on a P2
lysogen, WL95, as host and screened by plaque hybridization using HPV-16 D N A as a probe.
One positive clone (UD913) for the 7 kb fragment and eight positive clones for the 11 kb
fragment were obtained. Four clones (UU111, UU211, UU311, UU411) out of the eight clones
for the 11 kb fragment were analysed by digestion with restriction enzymes and proved to be
identical. The UD913 and UU411 inserts were subcloned into the B a m H I site of pUC19 and
used for further analyses.
The restriction map of UD913 and UU411 is depicted in Fig. 2(a). The recognition sites for
several restriction enzymes were the same as for the prototype HPV-16 except for the HincI1 site
in the non-coding region (Fig. 2a, Hc*). Neither large duplications nor internal deletions were
detected by restriction enzyme analysis. This analysis indicated that the 5' and 3' virus-host
junctions are located at the 5' end of the L 2 0 R F and the 3' end of the E 2 0 R F , respectively. The
E4 ORF was intact and the E5 ORF was entirely deleted. To identify more precisely the site of
the virus-host junctions, the nucleotide sequences around the junctions were determined by the
dideoxynucleotide chain-termination sequencing method (Sanger et al., 1980) using an M13specific oligonucleotide primer after introducing the various D N A fragments into M13mpl8
and M13mpl9. Sequence analyses revealed that the virus-host junctions reside at nucleotide
positions 3741 ( E 2 0 R F ) and 4416 (L20RF). Part, 674 bp, of the viral genome (nucleotides 3742
to 4415) containing the putative early polyadenylation signal, was deleted. The nucleotide
sequences of the virus-host junctions are shown in Fig. 3. Around the 5' junction, no significant
homology was found between the host and the viral sequences, but an AT-rich region was
present in the flanking sequence (Fig. 3a). Around the 3' junction, a TAAAA/TTTTA 5 bp
inverted repeat was present (Fig. 3b). No large fused ORFs of viral or cellular sequences were

1915

Short communication
(a)
i[

UU411

UD913
X,

5'

So

E Hc*

P E

3'

B
I

4416"~ .........................................................................
.<
HPV-16 genome
UUh'.

;"~'3741 :~
" UDh

..

HEII
N

"

DIN!

"

....

:E5:

(b)

-B

EP

,,,

:-'~-- Deleted cellular region

Bg i
PH

II

II

,,,

...~: E
Bg! P
"

H:B
II

Allelic cellular sequence

Fig. 2. Restriction maps of the cloned cellular sequences containing the HPV-16 genome and of the
aUelic cellular sequence in the QG-U cells. (a) The restriction map of the clones UU411 and UD913.
The symbol V denotes virus-host junctions. The hatched bar and shaded bar represent the HPV-16
genome and the deleted cellular region at the integration site, respectively. The ORFs of HPV-16 are
indicated below the map. (b) The restriction map of the allelic cellular sequence deduced from Southern
blot analysis with the flanking sequences as probes. The symbols represent the cleavage sites of the
restriction enzymes (B, B a m H I ; Bg, BgIII ; E, EcoRI ; H, HindIII ; Hc, HinclI ; K, KpnI ; P, PstI ; S, SphI ;
Sp, SpeI; X, XhoI).

(a)
5' Junction

CATCAGTACC

ATGGAACCCT

ACTCAGCAAT

AAAAAGAAAA

AACTATTGAT

AAATGCAACA

ACTTAGATGA

ACATCAAGGA

AATTATACTG

AGTGAAAGAA

GCCAATCCCC

AAAAGATACA

TATGGCATAA

TCAATTATTA

TTATTAATTT

CATGAATAAA

ATTATAGAGA

TGAACAgaac

atactgcagt
"-

gtcgtctaca

tggcattgga

caggacataa

tgtaaaacat

GAATATATAT

ATTTTTAACC

TTCACCCCCA

TCCCCTCAGG

GAATTCCTAA

TGGTGGGAAT

CACCAGTTCA

&TCCACCGGC

CAGCTTTGCG

GATGAGAACA

CAGACAGACC

CAGCCACTCT

GTTGAGTTTT

GTGTCTGCTG

***

(b)
3' Junction

~4416
agggtcgggt
"- L 2 0 R F
"~

~ 3741
aaaGGGCTGG

E20RF

Fig. 3. Nucleotide sequences around the 5'(a) and 3'(b) virus-host junctions of the integr~tted HPV-16
genome in the QG-U cell. The upper case and lower case letters represent the cellular and viral
sequences, respectively. The interrupted L2 and E 2 0 R F s are represented by arrows (~ ~). The stop
codons in each frame are indicated by asterisks (*). The underlined sequences represent the AT-rich
region found in the 5" flanking sequence (a) and the inverted repeats around the 3" junction (b).

g e n e r a t e d at e i t h e r the 5' or 3' viral t e r m i n u s as a result of integration. T h e E 2 0 R F

was
t e r m i n a t e d 55 nucleotides d o w n s t r e a m f r o m the j u n c t i o n as s h o w n in Fig. 3(b).
W e d e t e r m i n e d 1449 bp of the 3' flanking sequence, 568 bp of the 5' flanking sequence and
a p p r o x i m a t e l y 1.6 kb of the viral s e q u e n c e including the n o n - c o d i n g region, and the E2, L2, E6
and E 7 0 R F s . T h e r e was no large O R F in the flanking host sequences. A h o m o l o g y search in
G e n b a n k r e v e a l e d t h a t the 3' flanking s e q u e n c e has no h o m o l o g y to k n o w n sequences. H o w e v e r ,
the 5' flanking sequence, 357 bp u p s t r e a m f r o m the j u n c t i o n , c o n t a i n s 190 bp w h i c h show h i g h

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1916

Table 1. Nucleotide changes in the sequenced portion* of the HPV-16 genome integrated in

Q6-U
Nucleotide
position

ORF

Nucleotide
" change

647
846
3684
7519

E7
E7
E2
Non-coding
region

A-*G
T~C
C~A
G~A

Consequence
Amino acid change (Asp--,Ser)
Silent (Ser)
Amino acid change (Thr~Lys)

* Sequenced portions are the region from nucleotide positions 58 to 1060, 3619 to 3741, 4416 to 4570 and 7460 to
7850.
homology to part of the 350 bp 'O' family repeat. Polyadenylation signals, AATAAA, were
found at 855 bp and 864 bp downstream from the 3'junction; these might be used by the virushost fused mRNAs. In the sequenced portion of the viral genome, four nucleotide changes were
found in comparison with the published prototype HPV-16 sequence (Seedorf et al., 1985) as
shown in Table 1. One of the changes in the E 7 0 R F at the nucleotide position 846 was the same
as that reported by Choo et al. (1988).
To elucidate how the integration of the HPV genome occurred, we analysed QG-U D N A by
Southern blotting using the flanking cellular sequences, U U h (BgllI-SpeI fragment of UU411)
and UDh (EcoRI-HindlII fragment of UD913) which are indicated in Fig. 2(a). The 513 bp
U U h contains 153 bp of viral sequence. The cellular D N A was digested with the indicated
restriction enzymes and analysed. The QG-U D N A showed heterozygosity, indicating that the
allelic cellular sequence unaltered by HPV D N A integration is present (Fig. 4). The existence of
the same allelic cellular sequence in normal human fibroblasts was revealed by Southern blot
analysis with several restriction enzymes (data not shown). These analyses allowed us to map the
intact allelic cellular sequence as depicted in Fig. 2 (b). From these results it appears that 5.6 kb
of the cellular sequence was deleted at the integration site. In addition, the hybridization signals
of the fragments from the allelic cellular sequence were weaker than those of the fragments from
the viral D N A and its flanking sequences, indicating that the viral genome is amplified with its
flanking sequences. The magnitude of this amplification was estimated by cutting out the 32p_
labelled portion of the filter corresponding to the fragments a and b shown in the Fig. 4 (b) and
counting in liquid scintillant. Fragment b, which consists of both the 3' flanking sequence and
the unaltered sequence, was four times as radioactive as fragment a, revealing that the viral
genome and its flanking sequences are present as three copies per diploid genome.
As a result of the integration, the viral genome lacks the putative early polyadenylation signal
and this seemingly causes the viral transcripts to be a chimeric m R N A of virus and flanking
cellular sequences as reported previously (Shirasawa et al., 1987, 1988). To confirm this and to
see whether the flanking sequence is transcribed in normal cells, total R N A was extracted from
QG-U cells and from a primary culture of human fibroblasts, and subjected to Northern blot
analysis using the 3' flanking sequence UDh as a probe. As shown in Fig. 5, there was no
transcript hybridizing to the host D N A sequence in normal human fibroblasts. The 3.6 kb and
2.0 kb transcripts which hybridized to the E6/E7 region also hybridized to the UDh, suggesting
that these m R N A s were generated by readthrough into the flanking sequence. The other viral
transcripts did not hybridize to UDh, suggesting that this region is spliced out in these
transcripts which terminate further downstream.
The alteration of the structure in viral transcripts by readthrough into the flanking sequence
may have some biological meaning (Shirasawa et al., 1988), for instance stabilization o f m R N A .
Indeed, an increase in the half-life of the viral transcripts in HeLa cells has been observed
(Schwarz et al., 1985; Schneider-G/idicke & Schwarz, 1986; Kleiner et al., 1986). The
transcriptional regulation of the viral genome may also be altered. In CaSki and QG-H cells
which have tandem repeat HPV-16 copies, the viral transcripts do not utilize the viral
polyadenylation signals (Smotkin & Wettstein, 1986; Shirasawa et al., 1988). The deregulation
of viral transcription by integration as well as by cellular events might be necessary for

Short communication

1917
=

.v

(a)

(b)

11

1o

Fig. 4. Southern blot analysis of QG-U DNA using the flanking cellular sequences as probes, The
DNA (5 ~tg)extracted from QG-U cells was digested with the restriction enzymes indicated, separated
on a 0.8~ agarose gel and subjected to Southern blot hybridization with the 5' flanking cellular
sequence UUh (a) or the 3' flanking cellular sequence UDh (b). The radioactivity of the fragments
indicated by the letters a and b was compared. Arrowheads indicate the fragments from the allelic
cellular sequence. The bars represent either the positions of ,~ phage DNA digested with EcoT14I or
~bX174 DNA digested with HaelII. The numbers represent the sizes of the fragments in kb.

carcinogenesis. This integration seems to need no specific site in either viral or cellular
sequences. Our d a t a and previously reported junction sequences ( A w a d y et al., 1987; Baker et
al., 1987; Schneider-Maunoury et al., 1987; Choo et al., 1988) indicate that, like that of other
viruses, integration o f HPV-16 D N A is mediated by a mechanism o f illegitimate re~,ombination
with very short homologous stretches (Ruley & Fried, 1983).
The effect of the cellular sequence on the viral genome must also be taken into conskteration.
The 5' flanking sequences including the incomplete ' O ' family repeat could perhaps modulate
viral transcription. The ' O ' family repeat is thought to be long terminal repeat-like sequence of a
transposon-like element (Paulson et al., 1985).
The process of carcinogenesis m a y be triggered by the deletion or inactivation of cellular
genes such as tumour suppressor genes. In Q G - U cells, approximately 5.6 kb o f the cellular
sequence was deleted at the integration site. The deleted cellular sequence, however, seems to be

1918
(a)

Short communication
(b)

....

.... ~ . . ~

Fig. 5. Northern blot analysis of RNA from QG-U cells and

normal human fibroblasts. The total RNAs (10 ~tg) from QGU cells (lanes 1) and normal human fibroblasts (lanes 2) were
separated on a 0.8% agarose gel and subjected to Northern
blot hybridization with the HPV-16 E6/E7 region (EcoRIPstI fragment, nucleotides 7453 to 875) (a) and the 3' flanking
cellular sequence UDh (EcoRI-HindlII fragment) (b). The
bars indicate the transcripts and the accompanying numbers
represent their sizes in kb.

inactive because the flanking sequence is not transcribed in normal human fibroblast cells.
The amplification of the viral genome could be an early event, as the integration can occur in
the precancerous stage (Di Luca et al., 1986; Shirasawa et al., 1986; Schneider-Maunoury et al.,
1987; Lehn et al., 1988). In fact, this phenomenon is not unusual, because the integrated
monomeric viral genomes often exist as several copies even if the integration site is estimated to
be single. This event might enhance cell proliferation. The amplification of the transcriptionally
active HPV-16 genome suggests the involvement of the HPV-16 genomes in the development
and maintenance of the malignancy.
We thank Dr H. zur Hausen and Dr L. Gissmann, Heidelberg, F.R.G., for their kind gift of cloned HPV-16

DNA. The DNA analysis by the IDEAS program on the VAX computer was done with the help of Dr M.
Yamamoto and Dr A. Ito, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
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(Received 5 December 1988)