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The noncoding region of HPV-6vc contains two distinct transcriptional enhancing elements
VIROLOGY
155,545-556 (1986)
The Noncoding Region of HPV-6vc Contains Two Distinct Transcriptional Enhancing Elements ROBERT F. RANDO,*,’ WAYNE D. LANCASTER,t AND CARLOS LOPEZ*
PAULA HAN,*
*Viral Exanthems and Herpesvirus Branch 7-206,Centers for Disease Control, 1600CliT Atlanta, Georgia 30&U, and TDepartment of Obstetrics and @newlogy, Gewgetown University Medical Center, Washington D.C. 20007
Road,
Received May 12, 1986;accepted September 3, 1986 HPV-6vc eubgenomic fragments were inserted into an enhancer-dependent expression vector for chloramphenicol acetyltransferase (CAT) and assayed for the presence of transcriptional enhancing elements. A transcriptional enhancing element was detected in the noncoding region (NCR) of the HPV-6vc viral genome when the CAT assays were perfomed in viral transformed human kidney cell lines (293 and 324K), in human cervical carcinoma cell lines (HeLa and Siha), and in bovine papillomavirus type 1 (BPV-1) transformed mouse cells (C127-53). The NCR region of the HPV-6b genome was only capable of enhancing transcription of the CAT gene in the HeLa cell line at a level one-third that of the HPV6vc NCR. The HPV-6vc NCR enhancing activity in Cl2753 cells was further stimulated by the addition of sodium butyrate to the growth medium. Localization of the DNA sequences in the HPV-6vc NCR responsible for enhancing transcription revealed two distinct enhancer elements. One element (HPV-6vc position ‘7218-7544) was active in the 293, HeLa, Siha, and C127-53 cells. The second enhancer element (HPV-6vc position ‘7.544-7971) was only capable of stimulating transcription in HeLa, C127-53, and Siha cells. When the HPV NCR-CAT expression vectors were cotransfected with a competitor plasmid (pNCR75) into Cl27-53 or HeLa cells then transcriptional enhancement decreased, indicating competition of cellular factors which affect both segments of the HPV-6vc NCR.
onstrate productive viral infection in tissue culture. To date, much of the analysis of The regulation of viral replication and these processes with papillomaviruses has centered on BPV-1 transformed rodent gene expression for the papovavirus family has been well characterized for the SV4O cells in vitro (Dvoretzky et al, 1980). Stud(Benoist and Chambon, 1981; Gidoni et aL, ies on BPV-l-transformed mouse cells have 1985), polyoma (de Villiers and Schaffner, contributed to the understanding of the 1981) and the bovine papillomavirus type physical state of the viral genome in la1 (BPV-1) (Lusky et aL, 1983; Spalholz et tently infected cells (Law et al, 1981), the al, 1985) viruses. In these viruses, a com- viral DNA sequences required for mainteplex pattern of regulation is being eluci- nance of the viral genome in infected cells (Lusky and Botchan, 1984), and the mechdated which includes c+ and trans-acting viral functions as well as truns-acting cel- anism by which the genome is replicated (Waldeck et a& 1984). Genetic analysis of lular components. However, investigations of viral replication and regulation of gene the BPV-1 genome has defined specific recontrol in human papillomaviruses have gions of the viral genome which are needed been hampered by the inability to dem- expression of the transformed phenotype (Androphy et al, 1985; Groff and Lancaster, 1986; Schiller et &, 1986; DeMaio et al, i To whom requests for reprints should be addressed 1986; DiMaio, 1986) and regions which act at Pennsylvania Hospital, Department for the Sick in cis (Lusky et al, 1983) or in tram (Spaland Injured, 8th and Spruce Streets, Philadelphia, PA holz et cd, 1985; Yang et cd, 1985; Howley 19147. INTRODUCTION
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1983), the adenovirus transformed 293 (Aiello et al, 1979) and the murine leukemia virus transformed 143b (Rhim et aL, 1975) cells. Other cell lines used included the mouse fibroblast Cl27 cell line, the BPV-1 transformed mouse fibroblast cell line C127-53and the cervical carcinoma cell lines HeLa, which contains integrated HPV-18 DNA sequences, Siha, which contains integrated HPV-16 DNA sequences and Caski, which contains a high copy number of integrated HPV-16 genomes and a lower copy number of a related HPV genome (Yee et ah, 1985). HeLa, C127,324K, C127-53, and 143b cells were maintained as monolayer cultures in Dulbecco’s modified minimum essential medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), and L-glutamine (540 pg/ml). Caski, Siha, and 293 cells were maintained as monolayer cultures in Eagle’s minimum essential medium supplemented with nonessential amino acids, 7% fetal calf serum, penicillin (100 U/ml), and L-glutamine (500 dml). Plasmid constructs. The recombinant plasmid pML-BPVl containing bovine papillomavirus type one (BPV-1) and the pBR322 derivative pML2d has been described in detail elsewhere (Sarver et al, 1982). The intact recombinant HPV-6 viral genomes (pHPV-6b and pHPV-6vc) were constructed as previously described (Rando et ak, 1986a). The enhancer-dependent expression vector for CAT, pV2lcat (obtained from J. Kriedberg), contains a 2488bp derivative of pBR322 that is minus the poison sequences, and the SV40 origin of replication, early gene promotor, T antigen splice and polyadenylation sequences. The CAT gene is located 3’ to the SV40 early promoter, and there is a unique SacI recognition site 5’ to the early promoter (Fig. 1). The CAT expression plasmid pSV2cat containing the SV40 72-bp repeat enhancer element has been described previously (Gorman et al, 1982). In order to isolate the HPV-6b NCR the recombinant HPV-6b genome, pHPV-Gb, MATERIALS AND METHODS was cleaved with AvaI (position 6660) and Cell lines. Cell lines used in this study HpaII (position 7860), the overhanging consisted of the simian virus 40 (SV40) ends were repaired, and ligated to Hind111 transformed 324K (Tattersall and Bratton, linkers. The recombinant molecules were
et al, 1985) as transcriptional enhancer elements. Human papillomavirus (HPV) type 6vc was isolated from a highly aggressive vulvar verrucous carcinoma (Rando et al, 1986b) which arose within a condyloma. The HPV-6vc genome was characterized (Rando et al, 1986a) and compared to the well-characterized HPV-6b genome (Schwarz et a.!., 1983) which was cloned from a benign anogenital condyloma. The two HPV-6 DNAs showed a high degree of homology except for sequences in the noncoding region (NCR) of the viral genomes. The major difference between the two genomes was a 74-bp insert in the HPV-6vc genome which caused the formation of a triple 23-bp repeat instead of a 24-bp double repeat in the HPV-6b genome. Recently, Boshart and zur Hausen (1986) reported that the HPV-6b DNA sequence may be lacking sequences which map within the viral NCR due to a cloning artifact and that other HPV-6 subtypes contain DNA sequence alterations in their NCRs. Other investigators have also observed duplications in the NCR of papillomavirus genomes (Giri et al, 1985, and Cole and Streeck, 1986). The following experiments were undertaken to characterize HPV-6 enhancer elements, located in the NCR of the viral genome, using an enhancer-dependent expression vector for chloramphenicol acetyltransferase (CAT). The NCRs of HPV-6vc and HPV-6b were used in these experiments to determine if the DNA sequence differences between these two genomes affected enhancer activity. HPV-6 NCR enhancer activity was detected in specific tissue culture cell lines and evidence for cellular factors that might regulate this enhancer activity was obtained. HPV-6vc enhancer activity was always stronger compared to HPV-6b and multiple viral enhancer sequences were identified in the HPV-6vc NCR.
HPV-6
TRANSCRIPTIONAL
digested with Hind111 and the DNA fragments were fractionated by agarose gel electrophoresis according to standard laboratory procedures (Maniatis et cd, 1982). The DNA fragment corresponding to 1.2 kb was excised from the agarose gel and ligated into the unique Hind111 site on pBR322. The 1.2-kb HPV-6b fragment was amplified in the pBR322 recombinant pNCR12 which was then restricted with Hind111 and HgaI (HPV-6b nucleotide position 7167). The resultant overhanging ends were repaired and attached to Sac1 linkers, The 0.68kb band was excised from an agarose gel and ligated into the unique Sac1 site of pV2lcat (Fig. 1). Sac1 linkers were added to the HPV-6vc 0.75 and 1.3-kb DNA fragments generated by digestion of pHPV-6vc with HpaII and to the 2.1-kb DNA fragment generated by digestion of pHPV-6vc with Hind111 (position 2310) and iVoo1(position 4421). These fragments were then ligated into the Sac1 site of pV2lcat (Fig. 1). The 0.75kb DNA fragment corresponding to the HPV-6vc NCR (position 7218-7971) was also modified by the addition of Hind111 linkers and inserted into the Hind111 site of pBR322 (designated pNCR75). DNA transfections were performed by calcium phosphate coprecipitation (Graham and van der Eb, 1973) of various amounts of plasmid DNA (1 to 20 pg) with sonicated calf thymus DNA (adjusted to bring the final amount of transfected DNA to 30 pg) to 5 X lo5 cells in a 60-mm dish. Six hours post-transfection, the cells were treated with 15% glycerol (Frost and Williams, 1978) for 1 min. In the CAT expression assays, cells were given fresh media 24 hr post-transfection. In the BPV-1 transformation assays, Cl27 cells were split (1:12) 24 hr after transfection with pML-BPVl. In indicated experiments, sodium butyrate was added to the growth media to a final concentration of 5 mM after the glycerol shock treatment. Analysis of cellular DNA. Total cellular DNA from pBR322 or pNCR75 transfected HeLa cells was extracted 24,48, and 72 hr after transfection according to published procedures (Law et aZ., 1981). Fifteen micrograms of each cell extract was digested
ENHANCER
ELEMENTS
547
with HindIII, electrophoresed in 0.8% agarose, transferred to a nitrocellulose filter, and analyzed for the presence of transfected plasmid DNA using 32P-labeled pBR322 as a molecular probe (Southern, 1975). CAT assays. CAT assays were performed essentially as described by Gorman et al (1982). Cell extracts were normalized for degree of cell lysis by monitoring the A260,280 ratio before addition of cell extract to the enzyme assay mixture. Enzyme assays were continued for 1 hr before addition of ethyl acetate. The products of the reaction were separated by ascending thin layer chromatography and quantitated by liquid scintillation. RESULTS
Iaknti&ation of transcriptimal enhancer sequences in the genome of HPV-6vc. Two distinct HPV-6 NCRs (Fig. 1) were ligated into an enhancer-dependent CAT gene expression vector, pV2lcat. In the resulting plasmids, inserted DNA fragments were located 5’ to the CAT gene at the unique Sac1site. Thus, an inserted DNA fragment containing an enhancer element should influence the SV40 early promoter-directed transcription of the CAT gene (Fig. 1). Mouse and human cell lines were transfected with the pVZlcat-HPV recombinants. Forty-eight hours after transfection, cells were harvested and assayed for CAT activity. CAT expression was detected in viral transformed 324K or 293 human kidney cell lines and in the human cervical carcinoma cell lines HeLa and Siha when the CAT gene was located downstream from the inserted HPV-6vc NCR (pV21-75). However, when the CAT gene was located downstream from the HPV-6b NCR (pV2168), transcriptional enhancement was only observed in the HeLa cell line (Table 1). The Cl27 cell line was also transfected with pV21-75a and pV21-68a in the presence of 5 mM sodium butyrate (Gorman and Howard, 1983). Sodium butyrate, which stimulates cellular differentiation, was observed to significantly increase CAT gene expression in the HPV-6 NCR recombinant plasmids. In all cell lines tested, the HPV-
_- -...
-
-
bp
bp
15 19
74bp INSERT
A
--
ATGrTrArrGCCACrGCAr 1
1
ACTACATATTGTATA
13
- -_._ ----
15
-.
11 ,zz
-
---
_--
B I
INSERT
1
A
f3
I f E
TArGrACrGrrA~ArGrGrGrArArArGrGrArGrGrGrArArG~ArArGrArGrGrrG~G~ArA~A~ArGrG~ 1
- - _ _._
bp
-----Inserts----
74
‘V-6vc _----
co E = L I
INSERT
.-
- - - .- -
74
.-
.
--
pv21 - 75a
--m-w-
pv21 - 4421
pv21-
m--w--
s-v---
1297
pV21 - 75b
- -
VECTOR
p
I
s1
L
\ (2a
ltsQFs
$* \** I
Mlu-I
HPV INSERT
J
* d
sv40 1 PROMOTOR I
*
1 CAT I
sensa
Sense
Antisense
sema
Antisense
sense
ORIENTATION
FIG. 1. Comparison of the HPV-6b and HPV-6vc NCRs and flanking DNA sequences (HPV-6b nucleotide position 6660-510) and the nucleotide sequences of the HPV-6vc 74-, 15-, and 19-bp inserts (top). The single base changes which occur between the NCRs of HPV-6vc and HPV-6b (A) and the three HPV-6vc inserts are diagrammed (B). Several restriction endonuclease cleavage sites discussed in the text are included with their positions relative to the two genomes (A). Construction of recombinant pV2lcat plasmids containing HPV-6 DNA sequences inserted 5’ to the CAT gene (C). The HPV-6 subgenomic DNA fragments were generated as described under Materials and Methods and ligated into the unique Sac1 site of pV2lcat after addition of Sac1 linker molecules. The shaded areas in pV21-68 and pV21-‘75 indicate the double and triple 23-bp repeat elements found in the NCRs of HPV-6b and HPV-6vc (Rando et al, 1986a).
c
pV21 - 66b
pv21 - 66a
PLASMID
E z
E
XJ
3
LX
!i G =ii 0
2 b
X
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ET AL.
TABLE
1
ASSAY OF CAT EXPRESSION USING HPV-WpVZleat % Chloramphenicol
Plasmid constructs
RECOMBINANT PLASMIDS’
acetylated
from transfected
cell extracts Caski
Cl27
pV2lcat pSV2cat pV21-68a pV21-68b pV21-75a pV21-75b pv21-1297 pV21-4421
-SB
+SB
143b
293
324K
Hela
Siha
-SB
+SB
0.1 10.5 0.1 0.1
0.3 48.5 7.5 -
0.1 0.8 0.1 0.06
0.9 80.3 0.7 1.1
0.17 74.4 0.2 0.08
0.5 86.3 8.3 11.1
0.3 55.7 1.12
0.16 4.2 0.15 -
0.1 9.8 0.13
0.1 0.1 0.1 0.1
40.2 -
0.4 0.08 -
3.2 3.0 1.5 1.3
3.7 1.6 -
34.2 22.1 2.3 1.5
9.2 -
0.15 -
2.3 -
-
a HPV-6 NCR recombinant plasmids (10 pg) were transfected into the indicated cell lines. The cells were harvested and assayed for CAT activity 48 hr after transfection. Each value indicated is the average of three or more experiments. The Cl27 and Caski cell lines were transfected with the indicated plasmids and then grown with (+SB) or without (-SB) 5 mM sodium butyrate in the growth media.
6vc NCR was a stronger enhancer than the analogous region from the HPV-6b genome (Table 1). HPV-6 enhancer activity in BPV-ltransfmed Cl27 cells. Two different transcriptional enhancer elements have been characterized on the BPV-1 genome (Lusky et ah, 1983; Spalholz et ab, 1985). The enhancer element located in the NCR of the BPV-1 genome is activated by a viral tramacting factor (Spalholz et aL, 1985) encoded by the E2 open reading frame (ORF). Since the amino terminal portion of all the sequenced papillomavirus E2 ORFs have a degree of sequence conservation, it was possible that a BPV-1 encoded E2 protein might enhance HPVS transcription. In addition, the Cl27 cell line could be compared directly to BPV-1 transformed Cl27 cells for differences in the activation of these HPV NCR enhancer elements. The genome of BPV-1 was linearized by digestion with BamHI and ligated into the unique BamHI site of pML2d. The recombinant genome was transfected into Cl27 cells and 3 weeks post-transfection, foci of morphologically transformed cells were picked to establish single cell clones. The cell line C127-53 was able to form colonies in soft agar and harbored episomal pMLBPVl molecules (data not shown).
The C12’7-53cell line was used to test the pVBlcat-HPVB recombinant molecules for expression of the CAT gene. The HPV-6vc NCR (pV21-75a) was able to stimulate CAT activity in this cell line, whereas the HPV6b NCR (pV21-68a) was not (Fig. 2). The C127-53 cells were also transfected with these plasmids in the presence of 5 mMsodium butyrate (Gorman and Howard, 1983). Sodium butyrate was able to stimulate pV21-75a enhancer activity four- to fivefold over that of untreated cells (Fig. 2). Although inactive in untreated C127-53 cells, the HPV-6b NCR enhancer was active in C127-53 cells in the presence of sodium butyrate but at one-third the level of pV2175a (Fig. 2). Localization of the HPV-6vc NCR enhancer element. To further localize the HPV6vc DNA sequences capable of enhancing transcription in the pV2lcat system, the recombinant molecules pV21-75 and pV2168 were partially restricted with Sac1 and fully restricted with MluI (Fig. 3). The overhanging ends were repaired and the molecules were religated. The resultant recombinants, containing either the 5’- or 3’-half of the HPV-6 NCR, were tested for their ability to enhance CAT gene expression. The pV21-7544 recombinant contains a triple 23-bp repeat unit (Rando et aL,
HPV-6
TRANSCRIPTIONAL
3 = pV21
ENHANCER
ELEMENTS
551
-68a
4 = pv21.75a
0 12
34 Cl27
1
2 3 Cl27 Butyrate
4
1234 C127-53
1
2 3 C127-53 Butyrate
4
FIG. 2. Transfection of HPV-6 NCR enhancer plasmids into Cl27 and C127-53 cell lines in the presence or absence of 5 m&f sodium butyrate in the growth media. Each cell line was transfected with 10 pg of the indicated plasmid and 25 ng of sonicated calf thymus DNA and assayed for the presence of CAT activity 48 hr after transfection. The CAT assays were performed as described under Materials and Methods and the CAT activity was measured as the percentage of input chloramphenicol that became acetylated. The data values listed are the average of at least three independent experiments.
1986a) and was capable of enhancing CAT activity, in an orientation independent manner, in HeLa, Siha, 293, 324K, and C12’7-53cells and Cl27 cells transfected in the presence of sodium butyrate (Table 2). The pV21-‘7971 recombinants contain an additional 19-bp which are not found on pV21-7860, the corresponding region on the HPV-6b genome (Rando et aL, 1986a). pV21-7971 was able to enhance CAT activity, in an orientation dependent fashion, in HeLa, C127-53, and Siha cells and Cl27 cells transfected in the presence of sodium butyrate (Table 2). The HPV-6b recombinant pV21-7860a produced minimal enhancing activity in C127-53 and HeLa cells. The pV21-7454 (Fig. 3) construct was never isolated without the recombinant having undergone rearrangements or deletions. This observation is consistent with the fact that this region of the HPV-6b ncr is where deletions may have occurred during cloning of the viral genome (Bosart and zur Hausen, 1986). Cmpetitim of transacting factors. In an attempt to identify the presence of cellular factors that effect the HPV-6vc NCR enhancer element in trams, we cotransfected
the pV21-7971a and pV21-7544a plasmids into HeLa and (X27-53 cell lines with various amounts of a competitor plasmid. The competitor plasmid, pNCR75, contains pBR322 with the HPV-6vc NCR inserted into the Hind111 site (see Materials and Methods). The ability of pNCR75 to maintain itself in the tissue culture system as an episome for the duration of the CAT assay (48 hr) was determined by quantitation of the copy number of recombinant plasmid remaining 24,48, and 72 hr after transfection into C127-53 cells (Fig. 4). C127-53 cells were transfected with 5 pg of pBR322 or pNCR75 and 10 pg of carrier DNA and harvested 24, 48, or 72 hr posttransfection. Total DNA was isolated and analyzed for the presence of transfected plasmid by Southern blot transfer hybridization. In the C127-53 cells, episomal pNCR75 was present at approximately 150 copies per cell 24 hr post-transfection, doubling at 48 hr and decreasing to 100 copies per cell by 72 hr after transfection. Competition of pV21-7544a or pV217971a with pNCR75 in C127-53 cells grown in the presence of sodium butyrate demonstrated a decrease in the enhancing
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ET AL.
sense antisense
pv21-7544a pV21-7544b
pv21-7971 pV21-7971
sense antisense
a b
sense
pv21-74540 ‘% pV21-7860a
48 sense
FIG. 3. Subcloning of the HPV-6vc and HPV-6h NCR CAT expression vector plasmids. The recombinant plasmids pV21-‘75a,b and pV21-68a,b were partially digested with Sac1 and fully digested with M!uI (nucleotide position 7544 in HPV-6vc and position 7454 in HPV-6b). The overhanging ends were repaired, religated, and used to transform E coZi HBlOl to ampicillin resistance. The restriction endonuclease sites for Sac1 (S), BumHI (B), EcoRI (R), and Hind111 (H) are indicated.
ability of these two plasmids which was not observed if pUC9 was used as the competitor plasmid (Fig. 4). The level of pV217544a expression was elevated slightly when competed with 1 pmol of pNCR75, but began to fall as the level of competitor increased whereas the level of expression obtained from pV21-7971a began to decrease as the levels of specific competitor increased above 1 pmol (Fig. 4). Competition of these plasmids in HeLa cells resulted in a decrease in the pV21-7971a and pV217544a enhancer activity when they were competed with pNCR75 but showed no change when pUC9 was used as the competitor plasmid (data not shown). DISCUSSION
The region of the HPV-6vc genome between the end of the late open reading
frames (ORFs, L2 and Ll) and the beginning of the early RNA transcripts (E6) contains transcriptional enhancer elements that are similar in genetic organization to the BPV-1 (Spalholz et al, 1985), SV40 (Benoist and Chambon, 1981) and polyoma (de Villiers and Schaffner, 1981) viruses. The identification of a second enhancer element in this region of the viral genome is similar to the observation of two distinct enhancer elements for the Ela gene of adenovirus type 5 (Hearing and Shenk, 1986). Multiple transcriptional enhancer elements may be used by the viral genome in the temporal regulation of the lytic or latent viral life cycle. The use of two well-characterized HPV6 genomes has helped to localize the DNA sequences responsible for transcriptional enhancement in the viral NCR. The pV2175 and pV21-7544 recombinants contain a
HPV-6 TRANSCRIPTIONAL
ENHANCER
ELEMENTS
553
TABLE 2 ASSAYOFCAT EXPRESSION USINGHPV-6 NCR SunFRAGMENT/pV2lcat RECOMBINANT PLASMIDS’ Piasmid constructs
% Chloramphenicol actylated from transfected cell extracts C127-53
Cl27
pV2lcat psv2 pV21-7971a pV21-7971b pV21-7544a pV21-7544b pV21-7860a pv21-1297 pV21-4421
Caski
-SB
+SB
-SB
+SB
293
Heia
Siha
-SB
+SB
0.1 10.5 0.1 0.1 -
0.1 65.1 25.0 22.0 -
0.15 98.0 6.0 0.1 7.7 13.2 0.1 0.1 0.1
0.2 99.0 35.0 3.1 21.0 42.1 3.7 3.9 5.0
0.9 96.0 0.69 10.5 -
0.5 86.3 22.1 3.2 23.5 18.5 2.0 2.3 1.5
0.3 55.7 8.1 8.9 -
0.1 4.2 0.1 0.1 -
0.1 9.8 0.1 0.1 -
‘HPVS NCR subfragment recombinant plasmids (10 pg) were transfected into the indicated cell lines which were harvested and assayed for CAT activity 48 hr after transfection. Each data point is the average of three or more experiments. The C127, C127-53, and Caski cell lines were grown with (+SB) or without (-SB) 5 mMsodium butyrate in the growth media.
triple 23-bp repeat unit followed by a long stretch of alternating purine and pyrimidine residues. The HPV-6b recombinant, pV21-68, contains only two copies of this repeat unit and a shorter stretch of alternating purines and pyrimidines (Schwarz et ak, 1983). The HPV-6vc genome contains a 19-bp segment at nucleotide position ‘7721, located in pV21-75 and pV21-7971, which is not present in the HPV-6b genome. This 19-bp segment (5’-ATGTTTATTGCCACTGCAT-3’) is part of a structure that contains a 7-bp repeat element which starts at position 7705, TATTGCC-13bp-TATTGCC. This repeat element is similar to transcriptional activator elements found in the BPV-1, SV40, and polyoma virus genomes (Lusky et al, 1983). The effect of sodium butyrate on the HPV-6 NCR enhancer elements is consistent with findings for other papovavirus enhancer elements (Gorman and Howard, 1983; Spalholz et ak, 1985). It is interesting that even in the presence of sodium butyrate the HPV-6vc NCR enhancer always produced a higher level of CAT activity than the HPV-6b NCR enhancer. This result suggests that the additional DNA sequences found in the NCR of HPV-6vc maybe part of a larger viral transcriptional control region.
The pV21-7971 construct was able to enhance CAT activity in C127, C127-53, HeLa and Siha cells in the sense orientation (pV21-7971a) but was only able to slightly elevate CAT activity in C127-53 and HeLa cells when tested in the antisense orientation (pV21-7971b) (Table 2). Since CAT activity was still greater than the pV2lcat control plasmid this suggests that pV217971 is capable of enhancing CAT activity but appears to be orientation dependent. The HPV-6b construct pV21-7860a, in the sense orientation, was only able to slightly elevate CAT activity in HeLa cells and C127-53 cells in the presence of sodium butyrate suggesting that the additional 19 bp found in pV21-7971 may play a role in transcriptional enhancement. The presence of a cellular factor(s) which specifically interact with the HPV-6vc NCR enhancer elements was identified in cotransfection experiments using pNCR75 as a competitor plasmid in HeLa and C127-53 cells. In HeLa cells both pV21-7971a and pV21-7544a showed lowered CAT activity with increasing amounts of cotransfected specific competitor plasmid (data not shown). However, pV21-7544a showed increased CAT activity when cotransfected with low levels of the specific competitor plasmid in C127-53 cells but the activity
RAND0
554 m
1
2
ET AL.
3
4
5
6
9.8 6.6 4.3 -
pv21-7971a
pv21
- 7544a
100
0.0
1.0
2.5 pMole
5.0
Competitor
0.0
1.0
X
= pNCR
0
= puc9
2.5 pMole
75
5.0
Competitor
FIG. 4. Southern blot analysis of DNA extracted from C127-53 cells 24 hr (lanes l-2), 48 hr (lane 3), and 72 hr (lane 4) after transfection with pBR322 or pNCR’75 (top). C127-53 cells were transfected with 5 ~g of pBR322 (lane 1) or pNCR75 (lanes 2-4) and 20 pg of carrier DNA and were harvested 24, 48, and 72 hr post-transfection. Total cellular DNA was isolated and analyzed by Southern hybridization (Southern, 1975) for the presence of transfected plasmid DNA using q-labeled pBR.322 as a molecular probe. Each lane contains 15 pg of sample DNA digested with HindIII. Lanes 5 and 6 contain 3 and 30 ng, respectively, of pBR322, digested with Hind111 and mixed with 10 pg of sonicated calf thymus DNA. Competition of trots-acting factors using pNCR75 (bottom). C127-53 cells were cotransfected with 2.5 pmol (10 fig) of pV21-7971a or pV21-7544a and with 1, 2.5 or 5.0 pmol of pNCR75. Sonicated calf thymus DNA was added to the transfection to bring the total amount of DNA in each transfection up to 30 pg. Cell extracts were assayed for CAT activity 48 hr post-transfection as described under Materials and Methods. The CAT activity was measured as the percentage of input chloramphenicol that became acetylated. When pUC 9 was used in the cotransfection experiments instead of pNCR75 then no changes were observed in the enhancing capabilities of pV21-7971a or pV21-7544 in the C127-53 cell line. Each data point is the average of at least three experiments.
decreased upon addition of higher concentrations of competitor plasmid. One possible explanation for this result is that a suppressing factor has been competed from pV21-7544a followed by a gradual titration of an activating factor away from the CAT
plasmid. Alternatively, cellular factors associated with DNA replication may be responsible for this phenomenon if the positioning of the HPVB origin of viral replication is similar to that found with the BPV-1 genome (Waldeck et d, 1984). If this
HPV-6
TRANSCRIPTIONAL
were true then the pV21-7544a recombinant may contain the origin of viral replication. This region of pV21-7544a does contain nucleotides exhibiting the ability to assume the Z-DNA conformation (Rando et ok, 1986a) which would make this region nuclease sensitive (Rosl et al, 1983). This section of the HPV-6vc genome would be more conformationally relaxed and accessible to factors affecting viral replication which might interact with factors affecting transcription. The HPV-6vc enhancer elements are active in the C12’7-53 cells but not in Cl27 cells when CAT expression assays were performed in the absence of sodium butyrate. One of the activating factors in the C127-53 cells, which becomes competed away from the HPV-6vc NCR enhancer elements upon cotransfection with competitor plasmid, may be the BPV-1 E2 gene product. HPV-6vc enhancer elements, however, are functional in the HeLa cell line which contains a truncated, integrated HPV-18 genome which does not express the E2 ORF (Schwarz et aC, 1986). Similarly, the Siha cell line contains integrated HPV16 DNA sequences also deleted in the E2 ORF (Pater and Pater, 1985) in which the HPV-6vc enhancers are active. Difficult to reconcile is the fact that the enhancers are not active in the Caski cell line which contains a high copy number of intact integrated HPV-16 genomes and a lower copy number of a second related HPV genome (Pater and Pater, 1985). Since all three HPV-associated cell lines contain HPV specific mRNAs (Yee et CZL,1985) the differential expression of the HPVS NCR enhancer in HeLa and Siha cells but not in the Caski cells may be indicative of a preference for a particular undifferentiated cell type and not the presence of HPV or BPV gene products expressed in these tumor cells. Alternatively, Caski cells may produce a suppressor function not expressed in Hela or Siha cells and which acts on the HPV-6vc NCR. The undifferentiated adenovirus Ela transformed 293 and SV40 transformed 324K cell lines were also able to activate the HPV-6vc NCR enhancer element. These transformed human embryonic kidney cell lines may either supply truns-acting factors that activate the NCR
ENHANCER
ELEMENTS
555
enhancer element or may lack a truns-acting suppressor function. The HPV-6vc NCR is being investigated further to determine the exact DNA sequences involved in the transcriptional enhancing function, The presence of transcriptional enhancing elements in other regions of the HPV-6 genome was also indicated by the levels of CAT expression in HeLa cells and C127-53 cells grown in the presence of butyrate when transfected with pV21-1297 and pV21-4421 (Table 2). These regions of the viral genome are being further analyzed for the presence of transcriptional enhancing activity. ACKNOWLEDGMENTS We thank D. Rawlins for providing pSV2cat DNA and for critically reviewing this manuscript, J. Kriedberg for providing pV2lcat, and L. Gissmann for providing cloned HPV-6b DNA. We also thank A. Srinivasan for helpful discussions and J. Patton for technical assistance. R. F. Rando is a National Research Council postdoctoral fellow. This work was supported in part by PHS Grants CA32603 and CA32638 REFERENCES AIELLO, L., GUILFO~LE, R., HUEBNER, K., and WEINMAN, R. (1979). Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryonic kidney cells (HEK-AD-5 or 293). Virology 94,460-469. ANDROPHY, E. J., SCHILLER, J. T., and LOWY, D. R. (1985). Identification of the protein encoded by the E6 transforming gene of bovine papillomavirus.
Science230,442-445. BENOIST, C., and CHAMBON, P. (1981). In vivo sequence requirements of the SV40 early promotor region. Nature (London) 290,304-315. BOSHART, M., and ZUR HAUSEN, H. (1986). Human papillomaviruses in Buschke-Lowenstein tumors: Physical state of the DNA and identification of a tandem duplication in the noncoding region of a human papillomavirus 6 subtype. J. viral 58,963966. COLE, S. T., and STREECK, R. E. (1986). Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer. J. V+oL 58,991-995. DE VILLIERS, J., and SCHAFFNER, W. (1981). A small segment of polyoma virus DNA enhances the expression of a cloned fl-globin gene over a distance of 1400 base pairs. Nucleic Acids Res. 9,6251-6264. DIMAIO, D. (1986). Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA. J. viral.
57,475-480.
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DIMAIO, D., GURALSKI,D., and SCHILLER,J. T. (1986). Translation of open reading frame E5 of bovine papillomavirus is required for its transforming activity. Proc Natl. Acad Sci USA 83,1797-1801. DVORETZKY,I., SHOBER,R., CHATTOPADHYAY,S. K., and Lowy, D. R. (1980).A quantitative in vitro focus assay for bovine papilloma virus. Virobgy 103,369375. FROST,E., and WILLIAMS,J. (1978). Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology 91, 3950. GIDONI,D., KADONAGA,J. T., BARRERA-SALDANA,H., TAKAHASHI, K., CHAMBON,P., and TJIAN, R. (1985). Bidirectional SV40 transcription mediated by tandem Spl binding interactions. Science 230,511-51’7. GIRI, I., DANOS, O., and YANIV, M. (1985). Genomic structure of the cottontail rabbit (Shope) papillomavirus. Proc. Natl Acad Sci USA 82,1580-1584. GORMAN,C. M., and HOWARD,B. H. (1983).Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nu&ic Acids Res. 11, 7631-7648. GORMAN,C. M., MOFFAT,L. F., and HOWARD,B. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mel CeU.Biol. 2,1044-1051. GRAHAM,F. L., and VAN DEREB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456-461. GROFF,D. E., and LANCASTER,W. D. (1986). Genetic analysis of the 3’ early region transformation and replication functions of bovine papillomavirus type 1. Virologg 150,221-230. HEARING, P., and SHENK, T. (1986). The adenovirus type 5 ElA enhancer contains two functionally distinct domains: one is specific for ElA and the other modulates all early units in cis. Cell 45,229-236. HOWLEY,P. M., SCHENBORN, E. T., LUND, E., BYRNE, J. C., and DAHLBERG,J. E. (1985). The bovine papillomavirus distal “enhancer” is not ci.s essential for transformation or for plasmid maintenance. Md Cell Biol. 5,3310-3315. LAW, M. F., LOWY,D. R., DVORETZKY,I., and HOWLEY, P. M. (1981). Mouse cells transformed by bovine papillomavirus contain only extra chromosomal viral DNA sequences. Proc Natl. Acad Sci USA 78, 2727-2731. LUSKY, M., BERG,L., WEIHER, H., and BOTCHAN,M. (1983).Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit. Mol. Cell Biol 3,1108-1122. LUSKY,M., and BOTCHAN,M. (1984). Characterization of the bovine papilloma virus plasmid maintenance sequence. CeU36,391-401. MANIATIS,T., FRITSCH,E. F., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. PATER,M. M., and PATER,A. (1985). Human papillo-
mavirus types 16 and 18 sequences in carcinoma cell lines of the cervix. Virology 145,313-318. RANDO, R. F., GROFF,D. E., CHIRIKJIAN, J. G., and LANCASTER,W. D. (1986a). Isolation and characterization of a novel human papillomavirus type 6 DNA from an invasive vulvar carcinoma. J. V&l 57,353-356. RANDO, R. F., SEDLACEK,T. V., HUNT, J., JENSON, A. B., KURMAN,R. J., and LANCASTER,W. D. (198613). Verrucous carcinoma of the vulva associated with an unusual type 6 human papillomavirus. Obstet. Ggnecol 67,7OS-75s. RHIM, J. S., CHO, H. Y., and HUEBNER,R. J. (1975). Non-producer human cells induced by murine sarcoma virus. Int J. Cancer 15,23-29. ROSL,F., WALDECK,W., and SALJER,G. (1983).Isolation of episomal bovine papillomavirus cbromatin and identification of a DNAse I hypersensitive region. J. Viral 46,567-574. SARVER,N., BYRNE,J. C., and HOWLEY,P. M. (1982). Transformation and replication in mouse cells of a bovine papillomavirus-pML2 plasmid vector that can be rescued in bacteria. Proc. Nat1 Ad Sci USA 79,7147-7151. SCHILLER,J. T., VASS, W. C., VOUSDEN,K. H., and LOWY,D. R. (1986).E5 Open reading frame of bovine papillomavirus type 1 encodes a transforming gene. J. ViroL 57, l-6. SCHWARZ,E., DURST,M., DEMANKOWSKI,C., LA’ITERMAN,O., ZECH,R., WOLFSPERGER, E., SUHAI, S., and ZUR HAUSEN, H. (1983). DNA sequence and genome organization of human genital papillomavirus type 6b. EMBO J. 2.2341-2348. SCHWARZ, E., FREESE, U. K., GISSMANN, L., MAYER, W., ROGGENBUCK, B., STREMLAU,A., and ZURHAUSEN, H. (1985). Structure and transcription of human papillomavirus sequencesin cervical carcinoma cells. Nature (London) 314,111-114. SOUTHERN,E. M. (1975).Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Bid 98, 503-517. SPALHOLZ,B. A., YANG, Y. C., and HOWLEY, P. M. (1985). Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product. CeU42,183-191. TATTERSALL,P., and BRA~ON, J. (1983). Reciprocal productive and restrictive virus-cell interactions of immunosuppressive and prototype strains of minute virus of mice. J. viral. 46,944-955. WALDECK,W., ROSL,R., and ZENTGRAF,H. (1984). Origin of replication in episomal bovine papilloma virus type 1 DNA. EMBO J. 3,2173-2178. YANG, Y. C., SPALHOLZ,B. A., RABSON,M. S., and HOWLEY,P. M. (1985) Dissociation of transforming and trun-s-activation functions for bovine papillomavirus type 1. Nature (Lmdun) 318.575-577. YEE, C., KRISHNAN-HEWLETT,I., BAKER, C. C., SCHLEGEL, R., and HOWLEY,P. M. (1985). Presence and expression of HPV sequences in human cervical carcinoma cell lines. Amer. J. Path01 119.361-366.
155,545-556 (1986)
The Noncoding Region of HPV-6vc Contains Two Distinct Transcriptional Enhancing Elements ROBERT F. RANDO,*,’ WAYNE D. LANCASTER,t AND CARLOS LOPEZ*
PAULA HAN,*
*Viral Exanthems and Herpesvirus Branch 7-206,Centers for Disease Control, 1600CliT Atlanta, Georgia 30&U, and TDepartment of Obstetrics and @newlogy, Gewgetown University Medical Center, Washington D.C. 20007
Road,
Received May 12, 1986;accepted September 3, 1986 HPV-6vc eubgenomic fragments were inserted into an enhancer-dependent expression vector for chloramphenicol acetyltransferase (CAT) and assayed for the presence of transcriptional enhancing elements. A transcriptional enhancing element was detected in the noncoding region (NCR) of the HPV-6vc viral genome when the CAT assays were perfomed in viral transformed human kidney cell lines (293 and 324K), in human cervical carcinoma cell lines (HeLa and Siha), and in bovine papillomavirus type 1 (BPV-1) transformed mouse cells (C127-53). The NCR region of the HPV-6b genome was only capable of enhancing transcription of the CAT gene in the HeLa cell line at a level one-third that of the HPV6vc NCR. The HPV-6vc NCR enhancing activity in Cl2753 cells was further stimulated by the addition of sodium butyrate to the growth medium. Localization of the DNA sequences in the HPV-6vc NCR responsible for enhancing transcription revealed two distinct enhancer elements. One element (HPV-6vc position ‘7218-7544) was active in the 293, HeLa, Siha, and C127-53 cells. The second enhancer element (HPV-6vc position ‘7.544-7971) was only capable of stimulating transcription in HeLa, C127-53, and Siha cells. When the HPV NCR-CAT expression vectors were cotransfected with a competitor plasmid (pNCR75) into Cl27-53 or HeLa cells then transcriptional enhancement decreased, indicating competition of cellular factors which affect both segments of the HPV-6vc NCR.
onstrate productive viral infection in tissue culture. To date, much of the analysis of The regulation of viral replication and these processes with papillomaviruses has centered on BPV-1 transformed rodent gene expression for the papovavirus family has been well characterized for the SV4O cells in vitro (Dvoretzky et al, 1980). Stud(Benoist and Chambon, 1981; Gidoni et aL, ies on BPV-l-transformed mouse cells have 1985), polyoma (de Villiers and Schaffner, contributed to the understanding of the 1981) and the bovine papillomavirus type physical state of the viral genome in la1 (BPV-1) (Lusky et aL, 1983; Spalholz et tently infected cells (Law et al, 1981), the al, 1985) viruses. In these viruses, a com- viral DNA sequences required for mainteplex pattern of regulation is being eluci- nance of the viral genome in infected cells (Lusky and Botchan, 1984), and the mechdated which includes c+ and trans-acting viral functions as well as truns-acting cel- anism by which the genome is replicated (Waldeck et a& 1984). Genetic analysis of lular components. However, investigations of viral replication and regulation of gene the BPV-1 genome has defined specific recontrol in human papillomaviruses have gions of the viral genome which are needed been hampered by the inability to dem- expression of the transformed phenotype (Androphy et al, 1985; Groff and Lancaster, 1986; Schiller et &, 1986; DeMaio et al, i To whom requests for reprints should be addressed 1986; DiMaio, 1986) and regions which act at Pennsylvania Hospital, Department for the Sick in cis (Lusky et al, 1983) or in tram (Spaland Injured, 8th and Spruce Streets, Philadelphia, PA holz et cd, 1985; Yang et cd, 1985; Howley 19147. INTRODUCTION
545
0042-6822/86 $3.00
546
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ET AL.
1983), the adenovirus transformed 293 (Aiello et al, 1979) and the murine leukemia virus transformed 143b (Rhim et aL, 1975) cells. Other cell lines used included the mouse fibroblast Cl27 cell line, the BPV-1 transformed mouse fibroblast cell line C127-53and the cervical carcinoma cell lines HeLa, which contains integrated HPV-18 DNA sequences, Siha, which contains integrated HPV-16 DNA sequences and Caski, which contains a high copy number of integrated HPV-16 genomes and a lower copy number of a related HPV genome (Yee et ah, 1985). HeLa, C127,324K, C127-53, and 143b cells were maintained as monolayer cultures in Dulbecco’s modified minimum essential medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), and L-glutamine (540 pg/ml). Caski, Siha, and 293 cells were maintained as monolayer cultures in Eagle’s minimum essential medium supplemented with nonessential amino acids, 7% fetal calf serum, penicillin (100 U/ml), and L-glutamine (500 dml). Plasmid constructs. The recombinant plasmid pML-BPVl containing bovine papillomavirus type one (BPV-1) and the pBR322 derivative pML2d has been described in detail elsewhere (Sarver et al, 1982). The intact recombinant HPV-6 viral genomes (pHPV-6b and pHPV-6vc) were constructed as previously described (Rando et ak, 1986a). The enhancer-dependent expression vector for CAT, pV2lcat (obtained from J. Kriedberg), contains a 2488bp derivative of pBR322 that is minus the poison sequences, and the SV40 origin of replication, early gene promotor, T antigen splice and polyadenylation sequences. The CAT gene is located 3’ to the SV40 early promoter, and there is a unique SacI recognition site 5’ to the early promoter (Fig. 1). The CAT expression plasmid pSV2cat containing the SV40 72-bp repeat enhancer element has been described previously (Gorman et al, 1982). In order to isolate the HPV-6b NCR the recombinant HPV-6b genome, pHPV-Gb, MATERIALS AND METHODS was cleaved with AvaI (position 6660) and Cell lines. Cell lines used in this study HpaII (position 7860), the overhanging consisted of the simian virus 40 (SV40) ends were repaired, and ligated to Hind111 transformed 324K (Tattersall and Bratton, linkers. The recombinant molecules were
et al, 1985) as transcriptional enhancer elements. Human papillomavirus (HPV) type 6vc was isolated from a highly aggressive vulvar verrucous carcinoma (Rando et al, 1986b) which arose within a condyloma. The HPV-6vc genome was characterized (Rando et al, 1986a) and compared to the well-characterized HPV-6b genome (Schwarz et a.!., 1983) which was cloned from a benign anogenital condyloma. The two HPV-6 DNAs showed a high degree of homology except for sequences in the noncoding region (NCR) of the viral genomes. The major difference between the two genomes was a 74-bp insert in the HPV-6vc genome which caused the formation of a triple 23-bp repeat instead of a 24-bp double repeat in the HPV-6b genome. Recently, Boshart and zur Hausen (1986) reported that the HPV-6b DNA sequence may be lacking sequences which map within the viral NCR due to a cloning artifact and that other HPV-6 subtypes contain DNA sequence alterations in their NCRs. Other investigators have also observed duplications in the NCR of papillomavirus genomes (Giri et al, 1985, and Cole and Streeck, 1986). The following experiments were undertaken to characterize HPV-6 enhancer elements, located in the NCR of the viral genome, using an enhancer-dependent expression vector for chloramphenicol acetyltransferase (CAT). The NCRs of HPV-6vc and HPV-6b were used in these experiments to determine if the DNA sequence differences between these two genomes affected enhancer activity. HPV-6 NCR enhancer activity was detected in specific tissue culture cell lines and evidence for cellular factors that might regulate this enhancer activity was obtained. HPV-6vc enhancer activity was always stronger compared to HPV-6b and multiple viral enhancer sequences were identified in the HPV-6vc NCR.
HPV-6
TRANSCRIPTIONAL
digested with Hind111 and the DNA fragments were fractionated by agarose gel electrophoresis according to standard laboratory procedures (Maniatis et cd, 1982). The DNA fragment corresponding to 1.2 kb was excised from the agarose gel and ligated into the unique Hind111 site on pBR322. The 1.2-kb HPV-6b fragment was amplified in the pBR322 recombinant pNCR12 which was then restricted with Hind111 and HgaI (HPV-6b nucleotide position 7167). The resultant overhanging ends were repaired and attached to Sac1 linkers, The 0.68kb band was excised from an agarose gel and ligated into the unique Sac1 site of pV2lcat (Fig. 1). Sac1 linkers were added to the HPV-6vc 0.75 and 1.3-kb DNA fragments generated by digestion of pHPV-6vc with HpaII and to the 2.1-kb DNA fragment generated by digestion of pHPV-6vc with Hind111 (position 2310) and iVoo1(position 4421). These fragments were then ligated into the Sac1 site of pV2lcat (Fig. 1). The 0.75kb DNA fragment corresponding to the HPV-6vc NCR (position 7218-7971) was also modified by the addition of Hind111 linkers and inserted into the Hind111 site of pBR322 (designated pNCR75). DNA transfections were performed by calcium phosphate coprecipitation (Graham and van der Eb, 1973) of various amounts of plasmid DNA (1 to 20 pg) with sonicated calf thymus DNA (adjusted to bring the final amount of transfected DNA to 30 pg) to 5 X lo5 cells in a 60-mm dish. Six hours post-transfection, the cells were treated with 15% glycerol (Frost and Williams, 1978) for 1 min. In the CAT expression assays, cells were given fresh media 24 hr post-transfection. In the BPV-1 transformation assays, Cl27 cells were split (1:12) 24 hr after transfection with pML-BPVl. In indicated experiments, sodium butyrate was added to the growth media to a final concentration of 5 mM after the glycerol shock treatment. Analysis of cellular DNA. Total cellular DNA from pBR322 or pNCR75 transfected HeLa cells was extracted 24,48, and 72 hr after transfection according to published procedures (Law et aZ., 1981). Fifteen micrograms of each cell extract was digested
ENHANCER
ELEMENTS
547
with HindIII, electrophoresed in 0.8% agarose, transferred to a nitrocellulose filter, and analyzed for the presence of transfected plasmid DNA using 32P-labeled pBR322 as a molecular probe (Southern, 1975). CAT assays. CAT assays were performed essentially as described by Gorman et al (1982). Cell extracts were normalized for degree of cell lysis by monitoring the A260,280 ratio before addition of cell extract to the enzyme assay mixture. Enzyme assays were continued for 1 hr before addition of ethyl acetate. The products of the reaction were separated by ascending thin layer chromatography and quantitated by liquid scintillation. RESULTS
Iaknti&ation of transcriptimal enhancer sequences in the genome of HPV-6vc. Two distinct HPV-6 NCRs (Fig. 1) were ligated into an enhancer-dependent CAT gene expression vector, pV2lcat. In the resulting plasmids, inserted DNA fragments were located 5’ to the CAT gene at the unique Sac1site. Thus, an inserted DNA fragment containing an enhancer element should influence the SV40 early promoter-directed transcription of the CAT gene (Fig. 1). Mouse and human cell lines were transfected with the pVZlcat-HPV recombinants. Forty-eight hours after transfection, cells were harvested and assayed for CAT activity. CAT expression was detected in viral transformed 324K or 293 human kidney cell lines and in the human cervical carcinoma cell lines HeLa and Siha when the CAT gene was located downstream from the inserted HPV-6vc NCR (pV21-75). However, when the CAT gene was located downstream from the HPV-6b NCR (pV2168), transcriptional enhancement was only observed in the HeLa cell line (Table 1). The Cl27 cell line was also transfected with pV21-75a and pV21-68a in the presence of 5 mM sodium butyrate (Gorman and Howard, 1983). Sodium butyrate, which stimulates cellular differentiation, was observed to significantly increase CAT gene expression in the HPV-6 NCR recombinant plasmids. In all cell lines tested, the HPV-
_- -...
-
-
bp
bp
15 19
74bp INSERT
A
--
ATGrTrArrGCCACrGCAr 1
1
ACTACATATTGTATA
13
- -_._ ----
15
-.
11 ,zz
-
---
_--
B I
INSERT
1
A
f3
I f E
TArGrACrGrrA~ArGrGrGrArArArGrGrArGrGrGrArArG~ArArGrArGrGrrG~G~ArA~A~ArGrG~ 1
- - _ _._
bp
-----Inserts----
74
‘V-6vc _----
co E = L I
INSERT
.-
- - - .- -
74
.-
.
--
pv21 - 75a
--m-w-
pv21 - 4421
pv21-
m--w--
s-v---
1297
pV21 - 75b
- -
VECTOR
p
I
s1
L
\ (2a
ltsQFs
$* \** I
Mlu-I
HPV INSERT
J
* d
sv40 1 PROMOTOR I
*
1 CAT I
sensa
Sense
Antisense
sema
Antisense
sense
ORIENTATION
FIG. 1. Comparison of the HPV-6b and HPV-6vc NCRs and flanking DNA sequences (HPV-6b nucleotide position 6660-510) and the nucleotide sequences of the HPV-6vc 74-, 15-, and 19-bp inserts (top). The single base changes which occur between the NCRs of HPV-6vc and HPV-6b (A) and the three HPV-6vc inserts are diagrammed (B). Several restriction endonuclease cleavage sites discussed in the text are included with their positions relative to the two genomes (A). Construction of recombinant pV2lcat plasmids containing HPV-6 DNA sequences inserted 5’ to the CAT gene (C). The HPV-6 subgenomic DNA fragments were generated as described under Materials and Methods and ligated into the unique Sac1 site of pV2lcat after addition of Sac1 linker molecules. The shaded areas in pV21-68 and pV21-‘75 indicate the double and triple 23-bp repeat elements found in the NCRs of HPV-6b and HPV-6vc (Rando et al, 1986a).
c
pV21 - 66b
pv21 - 66a
PLASMID
E z
E
XJ
3
LX
!i G =ii 0
2 b
X
RAND0
550
ET AL.
TABLE
1
ASSAY OF CAT EXPRESSION USING HPV-WpVZleat % Chloramphenicol
Plasmid constructs
RECOMBINANT PLASMIDS’
acetylated
from transfected
cell extracts Caski
Cl27
pV2lcat pSV2cat pV21-68a pV21-68b pV21-75a pV21-75b pv21-1297 pV21-4421
-SB
+SB
143b
293
324K
Hela
Siha
-SB
+SB
0.1 10.5 0.1 0.1
0.3 48.5 7.5 -
0.1 0.8 0.1 0.06
0.9 80.3 0.7 1.1
0.17 74.4 0.2 0.08
0.5 86.3 8.3 11.1
0.3 55.7 1.12
0.16 4.2 0.15 -
0.1 9.8 0.13
0.1 0.1 0.1 0.1
40.2 -
0.4 0.08 -
3.2 3.0 1.5 1.3
3.7 1.6 -
34.2 22.1 2.3 1.5
9.2 -
0.15 -
2.3 -
-
a HPV-6 NCR recombinant plasmids (10 pg) were transfected into the indicated cell lines. The cells were harvested and assayed for CAT activity 48 hr after transfection. Each value indicated is the average of three or more experiments. The Cl27 and Caski cell lines were transfected with the indicated plasmids and then grown with (+SB) or without (-SB) 5 mM sodium butyrate in the growth media.
6vc NCR was a stronger enhancer than the analogous region from the HPV-6b genome (Table 1). HPV-6 enhancer activity in BPV-ltransfmed Cl27 cells. Two different transcriptional enhancer elements have been characterized on the BPV-1 genome (Lusky et ah, 1983; Spalholz et ab, 1985). The enhancer element located in the NCR of the BPV-1 genome is activated by a viral tramacting factor (Spalholz et aL, 1985) encoded by the E2 open reading frame (ORF). Since the amino terminal portion of all the sequenced papillomavirus E2 ORFs have a degree of sequence conservation, it was possible that a BPV-1 encoded E2 protein might enhance HPVS transcription. In addition, the Cl27 cell line could be compared directly to BPV-1 transformed Cl27 cells for differences in the activation of these HPV NCR enhancer elements. The genome of BPV-1 was linearized by digestion with BamHI and ligated into the unique BamHI site of pML2d. The recombinant genome was transfected into Cl27 cells and 3 weeks post-transfection, foci of morphologically transformed cells were picked to establish single cell clones. The cell line C127-53 was able to form colonies in soft agar and harbored episomal pMLBPVl molecules (data not shown).
The C12’7-53cell line was used to test the pVBlcat-HPVB recombinant molecules for expression of the CAT gene. The HPV-6vc NCR (pV21-75a) was able to stimulate CAT activity in this cell line, whereas the HPV6b NCR (pV21-68a) was not (Fig. 2). The C127-53 cells were also transfected with these plasmids in the presence of 5 mMsodium butyrate (Gorman and Howard, 1983). Sodium butyrate was able to stimulate pV21-75a enhancer activity four- to fivefold over that of untreated cells (Fig. 2). Although inactive in untreated C127-53 cells, the HPV-6b NCR enhancer was active in C127-53 cells in the presence of sodium butyrate but at one-third the level of pV2175a (Fig. 2). Localization of the HPV-6vc NCR enhancer element. To further localize the HPV6vc DNA sequences capable of enhancing transcription in the pV2lcat system, the recombinant molecules pV21-75 and pV2168 were partially restricted with Sac1 and fully restricted with MluI (Fig. 3). The overhanging ends were repaired and the molecules were religated. The resultant recombinants, containing either the 5’- or 3’-half of the HPV-6 NCR, were tested for their ability to enhance CAT gene expression. The pV21-7544 recombinant contains a triple 23-bp repeat unit (Rando et aL,
HPV-6
TRANSCRIPTIONAL
3 = pV21
ENHANCER
ELEMENTS
551
-68a
4 = pv21.75a
0 12
34 Cl27
1
2 3 Cl27 Butyrate
4
1234 C127-53
1
2 3 C127-53 Butyrate
4
FIG. 2. Transfection of HPV-6 NCR enhancer plasmids into Cl27 and C127-53 cell lines in the presence or absence of 5 m&f sodium butyrate in the growth media. Each cell line was transfected with 10 pg of the indicated plasmid and 25 ng of sonicated calf thymus DNA and assayed for the presence of CAT activity 48 hr after transfection. The CAT assays were performed as described under Materials and Methods and the CAT activity was measured as the percentage of input chloramphenicol that became acetylated. The data values listed are the average of at least three independent experiments.
1986a) and was capable of enhancing CAT activity, in an orientation independent manner, in HeLa, Siha, 293, 324K, and C12’7-53cells and Cl27 cells transfected in the presence of sodium butyrate (Table 2). The pV21-‘7971 recombinants contain an additional 19-bp which are not found on pV21-7860, the corresponding region on the HPV-6b genome (Rando et aL, 1986a). pV21-7971 was able to enhance CAT activity, in an orientation dependent fashion, in HeLa, C127-53, and Siha cells and Cl27 cells transfected in the presence of sodium butyrate (Table 2). The HPV-6b recombinant pV21-7860a produced minimal enhancing activity in C127-53 and HeLa cells. The pV21-7454 (Fig. 3) construct was never isolated without the recombinant having undergone rearrangements or deletions. This observation is consistent with the fact that this region of the HPV-6b ncr is where deletions may have occurred during cloning of the viral genome (Bosart and zur Hausen, 1986). Cmpetitim of transacting factors. In an attempt to identify the presence of cellular factors that effect the HPV-6vc NCR enhancer element in trams, we cotransfected
the pV21-7971a and pV21-7544a plasmids into HeLa and (X27-53 cell lines with various amounts of a competitor plasmid. The competitor plasmid, pNCR75, contains pBR322 with the HPV-6vc NCR inserted into the Hind111 site (see Materials and Methods). The ability of pNCR75 to maintain itself in the tissue culture system as an episome for the duration of the CAT assay (48 hr) was determined by quantitation of the copy number of recombinant plasmid remaining 24,48, and 72 hr after transfection into C127-53 cells (Fig. 4). C127-53 cells were transfected with 5 pg of pBR322 or pNCR75 and 10 pg of carrier DNA and harvested 24, 48, or 72 hr posttransfection. Total DNA was isolated and analyzed for the presence of transfected plasmid by Southern blot transfer hybridization. In the C127-53 cells, episomal pNCR75 was present at approximately 150 copies per cell 24 hr post-transfection, doubling at 48 hr and decreasing to 100 copies per cell by 72 hr after transfection. Competition of pV21-7544a or pV217971a with pNCR75 in C127-53 cells grown in the presence of sodium butyrate demonstrated a decrease in the enhancing
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ET AL.
sense antisense
pv21-7544a pV21-7544b
pv21-7971 pV21-7971
sense antisense
a b
sense
pv21-74540 ‘% pV21-7860a
48 sense
FIG. 3. Subcloning of the HPV-6vc and HPV-6h NCR CAT expression vector plasmids. The recombinant plasmids pV21-‘75a,b and pV21-68a,b were partially digested with Sac1 and fully digested with M!uI (nucleotide position 7544 in HPV-6vc and position 7454 in HPV-6b). The overhanging ends were repaired, religated, and used to transform E coZi HBlOl to ampicillin resistance. The restriction endonuclease sites for Sac1 (S), BumHI (B), EcoRI (R), and Hind111 (H) are indicated.
ability of these two plasmids which was not observed if pUC9 was used as the competitor plasmid (Fig. 4). The level of pV217544a expression was elevated slightly when competed with 1 pmol of pNCR75, but began to fall as the level of competitor increased whereas the level of expression obtained from pV21-7971a began to decrease as the levels of specific competitor increased above 1 pmol (Fig. 4). Competition of these plasmids in HeLa cells resulted in a decrease in the pV21-7971a and pV217544a enhancer activity when they were competed with pNCR75 but showed no change when pUC9 was used as the competitor plasmid (data not shown). DISCUSSION
The region of the HPV-6vc genome between the end of the late open reading
frames (ORFs, L2 and Ll) and the beginning of the early RNA transcripts (E6) contains transcriptional enhancer elements that are similar in genetic organization to the BPV-1 (Spalholz et al, 1985), SV40 (Benoist and Chambon, 1981) and polyoma (de Villiers and Schaffner, 1981) viruses. The identification of a second enhancer element in this region of the viral genome is similar to the observation of two distinct enhancer elements for the Ela gene of adenovirus type 5 (Hearing and Shenk, 1986). Multiple transcriptional enhancer elements may be used by the viral genome in the temporal regulation of the lytic or latent viral life cycle. The use of two well-characterized HPV6 genomes has helped to localize the DNA sequences responsible for transcriptional enhancement in the viral NCR. The pV2175 and pV21-7544 recombinants contain a
HPV-6 TRANSCRIPTIONAL
ENHANCER
ELEMENTS
553
TABLE 2 ASSAYOFCAT EXPRESSION USINGHPV-6 NCR SunFRAGMENT/pV2lcat RECOMBINANT PLASMIDS’ Piasmid constructs
% Chloramphenicol actylated from transfected cell extracts C127-53
Cl27
pV2lcat psv2 pV21-7971a pV21-7971b pV21-7544a pV21-7544b pV21-7860a pv21-1297 pV21-4421
Caski
-SB
+SB
-SB
+SB
293
Heia
Siha
-SB
+SB
0.1 10.5 0.1 0.1 -
0.1 65.1 25.0 22.0 -
0.15 98.0 6.0 0.1 7.7 13.2 0.1 0.1 0.1
0.2 99.0 35.0 3.1 21.0 42.1 3.7 3.9 5.0
0.9 96.0 0.69 10.5 -
0.5 86.3 22.1 3.2 23.5 18.5 2.0 2.3 1.5
0.3 55.7 8.1 8.9 -
0.1 4.2 0.1 0.1 -
0.1 9.8 0.1 0.1 -
‘HPVS NCR subfragment recombinant plasmids (10 pg) were transfected into the indicated cell lines which were harvested and assayed for CAT activity 48 hr after transfection. Each data point is the average of three or more experiments. The C127, C127-53, and Caski cell lines were grown with (+SB) or without (-SB) 5 mMsodium butyrate in the growth media.
triple 23-bp repeat unit followed by a long stretch of alternating purine and pyrimidine residues. The HPV-6b recombinant, pV21-68, contains only two copies of this repeat unit and a shorter stretch of alternating purines and pyrimidines (Schwarz et ak, 1983). The HPV-6vc genome contains a 19-bp segment at nucleotide position ‘7721, located in pV21-75 and pV21-7971, which is not present in the HPV-6b genome. This 19-bp segment (5’-ATGTTTATTGCCACTGCAT-3’) is part of a structure that contains a 7-bp repeat element which starts at position 7705, TATTGCC-13bp-TATTGCC. This repeat element is similar to transcriptional activator elements found in the BPV-1, SV40, and polyoma virus genomes (Lusky et al, 1983). The effect of sodium butyrate on the HPV-6 NCR enhancer elements is consistent with findings for other papovavirus enhancer elements (Gorman and Howard, 1983; Spalholz et ak, 1985). It is interesting that even in the presence of sodium butyrate the HPV-6vc NCR enhancer always produced a higher level of CAT activity than the HPV-6b NCR enhancer. This result suggests that the additional DNA sequences found in the NCR of HPV-6vc maybe part of a larger viral transcriptional control region.
The pV21-7971 construct was able to enhance CAT activity in C127, C127-53, HeLa and Siha cells in the sense orientation (pV21-7971a) but was only able to slightly elevate CAT activity in C127-53 and HeLa cells when tested in the antisense orientation (pV21-7971b) (Table 2). Since CAT activity was still greater than the pV2lcat control plasmid this suggests that pV217971 is capable of enhancing CAT activity but appears to be orientation dependent. The HPV-6b construct pV21-7860a, in the sense orientation, was only able to slightly elevate CAT activity in HeLa cells and C127-53 cells in the presence of sodium butyrate suggesting that the additional 19 bp found in pV21-7971 may play a role in transcriptional enhancement. The presence of a cellular factor(s) which specifically interact with the HPV-6vc NCR enhancer elements was identified in cotransfection experiments using pNCR75 as a competitor plasmid in HeLa and C127-53 cells. In HeLa cells both pV21-7971a and pV21-7544a showed lowered CAT activity with increasing amounts of cotransfected specific competitor plasmid (data not shown). However, pV21-7544a showed increased CAT activity when cotransfected with low levels of the specific competitor plasmid in C127-53 cells but the activity
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1
2
ET AL.
3
4
5
6
9.8 6.6 4.3 -
pv21-7971a
pv21
- 7544a
100
0.0
1.0
2.5 pMole
5.0
Competitor
0.0
1.0
X
= pNCR
0
= puc9
2.5 pMole
75
5.0
Competitor
FIG. 4. Southern blot analysis of DNA extracted from C127-53 cells 24 hr (lanes l-2), 48 hr (lane 3), and 72 hr (lane 4) after transfection with pBR322 or pNCR’75 (top). C127-53 cells were transfected with 5 ~g of pBR322 (lane 1) or pNCR75 (lanes 2-4) and 20 pg of carrier DNA and were harvested 24, 48, and 72 hr post-transfection. Total cellular DNA was isolated and analyzed by Southern hybridization (Southern, 1975) for the presence of transfected plasmid DNA using q-labeled pBR.322 as a molecular probe. Each lane contains 15 pg of sample DNA digested with HindIII. Lanes 5 and 6 contain 3 and 30 ng, respectively, of pBR322, digested with Hind111 and mixed with 10 pg of sonicated calf thymus DNA. Competition of trots-acting factors using pNCR75 (bottom). C127-53 cells were cotransfected with 2.5 pmol (10 fig) of pV21-7971a or pV21-7544a and with 1, 2.5 or 5.0 pmol of pNCR75. Sonicated calf thymus DNA was added to the transfection to bring the total amount of DNA in each transfection up to 30 pg. Cell extracts were assayed for CAT activity 48 hr post-transfection as described under Materials and Methods. The CAT activity was measured as the percentage of input chloramphenicol that became acetylated. When pUC 9 was used in the cotransfection experiments instead of pNCR75 then no changes were observed in the enhancing capabilities of pV21-7971a or pV21-7544 in the C127-53 cell line. Each data point is the average of at least three experiments.
decreased upon addition of higher concentrations of competitor plasmid. One possible explanation for this result is that a suppressing factor has been competed from pV21-7544a followed by a gradual titration of an activating factor away from the CAT
plasmid. Alternatively, cellular factors associated with DNA replication may be responsible for this phenomenon if the positioning of the HPVB origin of viral replication is similar to that found with the BPV-1 genome (Waldeck et d, 1984). If this
HPV-6
TRANSCRIPTIONAL
were true then the pV21-7544a recombinant may contain the origin of viral replication. This region of pV21-7544a does contain nucleotides exhibiting the ability to assume the Z-DNA conformation (Rando et ok, 1986a) which would make this region nuclease sensitive (Rosl et al, 1983). This section of the HPV-6vc genome would be more conformationally relaxed and accessible to factors affecting viral replication which might interact with factors affecting transcription. The HPV-6vc enhancer elements are active in the C12’7-53 cells but not in Cl27 cells when CAT expression assays were performed in the absence of sodium butyrate. One of the activating factors in the C127-53 cells, which becomes competed away from the HPV-6vc NCR enhancer elements upon cotransfection with competitor plasmid, may be the BPV-1 E2 gene product. HPV-6vc enhancer elements, however, are functional in the HeLa cell line which contains a truncated, integrated HPV-18 genome which does not express the E2 ORF (Schwarz et aC, 1986). Similarly, the Siha cell line contains integrated HPV16 DNA sequences also deleted in the E2 ORF (Pater and Pater, 1985) in which the HPV-6vc enhancers are active. Difficult to reconcile is the fact that the enhancers are not active in the Caski cell line which contains a high copy number of intact integrated HPV-16 genomes and a lower copy number of a second related HPV genome (Pater and Pater, 1985). Since all three HPV-associated cell lines contain HPV specific mRNAs (Yee et CZL,1985) the differential expression of the HPVS NCR enhancer in HeLa and Siha cells but not in the Caski cells may be indicative of a preference for a particular undifferentiated cell type and not the presence of HPV or BPV gene products expressed in these tumor cells. Alternatively, Caski cells may produce a suppressor function not expressed in Hela or Siha cells and which acts on the HPV-6vc NCR. The undifferentiated adenovirus Ela transformed 293 and SV40 transformed 324K cell lines were also able to activate the HPV-6vc NCR enhancer element. These transformed human embryonic kidney cell lines may either supply truns-acting factors that activate the NCR
ENHANCER
ELEMENTS
555
enhancer element or may lack a truns-acting suppressor function. The HPV-6vc NCR is being investigated further to determine the exact DNA sequences involved in the transcriptional enhancing function, The presence of transcriptional enhancing elements in other regions of the HPV-6 genome was also indicated by the levels of CAT expression in HeLa cells and C127-53 cells grown in the presence of butyrate when transfected with pV21-1297 and pV21-4421 (Table 2). These regions of the viral genome are being further analyzed for the presence of transcriptional enhancing activity. ACKNOWLEDGMENTS We thank D. Rawlins for providing pSV2cat DNA and for critically reviewing this manuscript, J. Kriedberg for providing pV2lcat, and L. Gissmann for providing cloned HPV-6b DNA. We also thank A. Srinivasan for helpful discussions and J. Patton for technical assistance. R. F. Rando is a National Research Council postdoctoral fellow. This work was supported in part by PHS Grants CA32603 and CA32638 REFERENCES AIELLO, L., GUILFO~LE, R., HUEBNER, K., and WEINMAN, R. (1979). Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryonic kidney cells (HEK-AD-5 or 293). Virology 94,460-469. ANDROPHY, E. J., SCHILLER, J. T., and LOWY, D. R. (1985). Identification of the protein encoded by the E6 transforming gene of bovine papillomavirus.
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