VIROLOGY
185,
536-543
(1991)
Human
Papillomavirus Type 16 E6 Proteins with Glycine Substitution for Cysteine in the Metal-Binding Motif
TADAHITO
‘Department
KANDA,*a’ SUMIE WATANABE,* SOICHI ZANMA,t AKEMI FURUNO,* AND KUNITO YOSHIIKE*
of Enteroviruses, National Institute of Health, Technology Laboratories, Chugai Pharmaceutical Received
April
Kamiosaki, Company,
8, 799 1; accepted
HIRONORI
SATO,*,’
Shinagawa-ku, Tokyo 14 1; and tDiagnostics Ltd., Toshima-ku, Tokyo, 171, Japan August
9, 799 1
The human papillomavirus type 16 (HPV 16) E6 is a 151 amino acid protein containing four metal-binding motifs, Cys-X-X-Cys. We constructed and characterized three mutants with Gly substitutions for Cys within the motif; for Cys-66, for Cys-136. and for both, respectively. Zinc binding to bacterially expressed E6 was markedly reduced by the substitution for Cys-66, but DNA binding was unaffected by any of these mutations. immunofluorescence staining showed that, whereas the E6 expressed in monkey COS-1 cells appeared mostly nuclear, the Cys-88 mutant appeared cytoplasmic. Subcellular fractionation followed by immunoprecipitation showed that the E6 in COS-1 ceils was located in the membrane, nuclear, and nuclear-wash fractions, but not in the soluble cytoplasmic fraction, and that the nuclear Cys-86 protein was markedly reduced. The mutant proteins in COS-1 cells appeared to be less stable than the wild type, because the immunofluorescent cells were fewer and because the E6 bands in autoradiograms were less dense. The substitution mutants lost their capacity to enhance HPV 18 E7 transformation of rat 3Yl cells. The data indicate that Cys-66 plays a crucial role for zinc binding and nuclear localization of E6 and that both Cys-66 and Cys-136 are required for a stable or functional structure of E6. o 1991 Academic PWSS, IIIC.
INTRODUCTION
The E6 and E7 proteins have been shown to be expressed in CaSki and SiHa cells, human cell lines derived from cervical carcinomas (Androphy et al., 1987; Seedorf et a/., 1987). Analyses of transcripts have shown that the cap site for viral mRNAs is at nucleotide 97 (Smotkin and Wettstein, 1986; Smotkin eta/., 1989), indicating that the viral P97 transcripts encode 151 and 98 amino acid (AA) proteins for E6 (translated from the second ATG of the open reading frame) and E7 genes, respectively. Presence of metal-binding motifs, Cys-X-X-Cys, is a feature common to these two oncoproteins, the AA sequences of which can be deduced from the entire nucleotide sequence of HPV 16 (Seedorf et a/., 1985). Presence of the four metal-binding motifs is characteristic of and common to the E6 proteins from human and animal papillomaviruses (Cole and Danos, 1987). The motifs of the HPV 16 E6 are at Cys at AAs 30 (Cys-30) to Cys-33, at Cys-63 to Cys-66, at Cys-103 to Cys-106, and at Cys-136 to Cys-139 (Fig. 1). The intervals between the N-terminal pair and between the Cterminal pair are 29 AAs long and are conserved among papillomaviruses (Cole and Danos. 1987). The conserved pattern of the distribution of the motifs suggests an importance of these cysteines in the functional E6 protein. In this study, we examined biochemical and biological properties of the HPV 16 E6 proteins with Gly substitutions for Cys (Cys-66 and Cys-136) in the metal-binding motif.
Human papillomavirus type 16 (HPV 16) (Dllrst eta/., 1983), closely associated with cervical carcinoma (zur Hausen, 1989), encodes two transforming genes E6 and E7. Expression of the transactivating (Phelps eta/., 1988), mitogenic (Sato et al., 1989a), nuclear (Sato et a/., 1989b) E7 protein alone is sufficient for transformation of rodent cell lines (Kanda eta/., 1988a; Yutsudo et a/., 1988; Vousden et al., 1988; Tanaka et al., 1989), primary rat cells (Kanda et al., 1988b), and primary rat cells in conjunction with the expression of activated ras (Phelps et al., 1988; Storey et al., 1988; Crook et a/., 1989). However, expression of both E7 and E6 genes is required for extension of the life span of human fibroblasts (Watanabe et a/., 1989) and for immortalization of human foreskin keratinocytes (MUnger eta/., 1989a; Hawley-Nelson et al., 1989), or E6 can significantly enhance the E7 immortalizing function for human foreskin keratinocytes (Halbet-t et al., 1991). The E7 and E6 proteins are capable of binding to antioncogene products Rb (Dyson et al., 1989; M(lnger et a/., 198913; Gage et al., 1990; Barbosa et al., 1990) and p53 (Werness et a/., 1990), respectively.
’ To whom requests for reprints should be addressed. * Present address: Laboratory of Molecular Microbiology, tional Institute of Allergy and Infectious Diseases, Bethesda, 20892. 0042-6822/91
$3.00
Copynght 0 1991 by Academic Press, Inc. All rights of reproductaon in any form reserved
NaMD
536
HPV
1-MFQDPQERPR
KLPQLCTELQ
16 E6 MUTANT
TTIHDIILE~~QQLLRR
RVYDfAFRDL
G CIVYRDGNPY AVWLKFY
SKISEYRRYC
YSLYGTTLEQ
QYNKPLCDLL
CPEEKQRRLD
PROTEINS
and purified 1982). Preparation
G KKQRFRNIRG RWTGW
IR-QKPL
RSSRTRRETQ L-151
FIG. 1. AA sequence of HPV 16 E6 protein. motifs (Cys-X-X-Cys) are underlined. Cys-66 been replaced with Gly in mutants.
MATERIALS Construction
The metal-binding and Cys-136 have
AND METHODS
of expression
plasmids
The plasmids used to express mutant E6 proteins were constructed by site-directed mutagenesis (Kramer and Fritz, 1987) to replace appropriate nucleotides in the E6 gene, with Mutan-G (Takara Shuzo Co., Ltd., Kyoto, Japan). The E6 mutants constructed in this study had Gly substitutions for Cys-66 (E6C66G), Cys136 (E6Cl36G), and both (E6C66G/C136G). A bacterial expression plasmid for wild-type E6 protein, pKK-E6, was constructed by insertion of the E6 coding region of HPV 16 DNA into pKK233-2 (Pharmacia LKB Biotechnology, Sweden). A new Ncol site was introduced into the HindIll fragment from pSV2-E6 by replacement of A at nucleotides 103 (A-103) and T-107 with C and G, respectively. The Ncol to Hindlll fragment (from nt 103 to nt 657) was cloned into pKK233-2 between its Ncol and /-/indIll sites. G-107 was then restored to T by mutagenesis to obtain pKKE6. Mutants pKK-E6C66G, pKK-E6Cl36G, and pKKE6C66G/C136G were constructed from pKK-E6 by replacements of T-299 and T-509 with Gs, respectively. Eucaryotic expression plasmid pSRa-E6 was constructed by insertion of the HindIll fragment from pSV2-E6 (Kanda et a/., 1988a) (HPV 16 DNA nt 25 to 657) into the HindIll site of pSRa-0 (Kanda et a/., 1991) which contains SV40 transcriptional signals and R-U5 sequence from the LTR of human T-cell leukemia virus type 1 (Takebe et a/., 1988). The E6 protein expressed from pSRa-E6 in monkey COS-1 cells (Gluzman, 1981) migrated together with the bacterially expressed 151 AA E6 protein in gel electrophoresis (data not shown). In mutants pSRa-EGC66G, pSRa-EGCl36G, and pSRa-E6C66G/Cl36G, T-299, T-509, and both Ts were replaced with Gs, respectively, as described above. pSRa-E7 for the expression of the E7 gene was described previously (Kanda et al., 1991). The structures of plasmids newly constructed in this study were verified by DNA sequencing (Sanger et al., 1977; Messing, 1983). Plasmids were grown in E. co/i RR1
637
by standard
methods
(Maniatis
et a/.,
of the E6 proteins
E. co/i strain YA21 (Mizushima and Yamada, 1975) transformed with pKK-E6, pKK-E6C66G, pKK-EGCl36G, or pKK-E6C66G/Cl36G was grown overnight in LBroth (Maniatis et al., 1982) containing 1 mll/l isopropyl-fl-b(-)-thiogalactopyranoside, washed once with 10 rnM Tris-HCI (pH 7.5) 50 mll/l NaCI, 1 mll/l phenylmethylsulfonyl fluoride (PMSF), and sonicated in the same buffer. The lysate was centrifuged at 10,000 gfor 30 min. The pellet (Pl 0) was suspended in 6 M guanidine-HCI, 50 mM Tris (pH 7.5), 2 mM 2-mercaptoethanol(2ME) and centrifuged at 10,000 gfor 10 min. The supernatant was loaded onto a Sephadex Gl50 (Pharmacia LKB Biotechnology) column equilibrated in the same buffer. Collected protein was dialyzed against 10 mM Tris-HCI (pH 7.4) 0.2 m/M 2ME. This material formed a single band in SDS-polyacrylamide gel electrophoresis. The amino acid sequence from the N-terminus of purified E6 protein was determined by the Edoman degradation method using an automatic protein sequencer (PSQ-1, Shimadzu, Tokyo, Japan). The first 12 residues analyzed agreed with those expected from the DNA sequence (Fig. 1). Preparation of monoclonal the E6 protein
antibody
(MAb) against
A cell clone producing anti-E6 immunogloblin was established from a hybridoma between spleen cells of an immunized BALB/C-AnN mouse and the X63Ag8.653 myeloma line by standard methods (Prabhakar et al., 1984). The PlO fraction of bacterially expressed E6 was suspended in 7 n/l urea, 1.25% sarcosine, dialyzed against 10 mlLITris-HCI (pH 7.5) 50 rnM NaCI, and centrifuged at 10,000 g for 30 min. The supernatant (100 to 200 pg) was mixed with Freund’s complete adjuvant and used for subcutaneous injections into a mouse five times at 2-week intervals. Then, 1 mg of E6 protein which was electrophoretically purified from the PlO fraction, precipitated with cold acetone, and suspended in PBS was injected into the mouse intraperitoneally twice at 2-week intervals. The spleen was removed from the immunized mouse 4 days after the final immunization and fused with X68Ag8.653 myeloma cells with polyethylene glycol. The cell clone producing MAb against the E6 protein (MAb 618) was selected by ELISA (purified E6 protein was used for antigen) and injected into BALB/C mice for preparation of ascites containing MAb, which was used in this study. The isotype of MAb 618 was lgG2b which was determined with the Mouse Monoclonal
538
KANDA
Sub-lsotyping kit (American Qualex International, Inc., La Mirada, CA). The epitope for MAb 618 was in the region from AA 1 to AA 13 of the E6 protein, which was determined by competitive binding assay using synthesized oligopeptides (Kanda et al., 1991). Assay for zinc binding
to the E6 protein
The zinc-binding capacities of bacterially expressed E6 proteins were examined by the zinc-blotting method described by Barbosa et al. (1989). Five micrograms of purified wild-type protein or mutant E6 protein was electrophoresed on a 15% SDS-polyacrylamide gel and transferred to a nitrocellulose filter. The transfer and the retention of E6 proteins were confirmed by staining of the membrane with Ponceau red (Sigma, St. Louis, MO). The filter was incubated in renaturing buffer [lo0 mNI Tris-HCI (pH 6.8) 50 mM NaCI, 10 mM dithiothreitol (DlT)] for 1 hr with three changes of buffer, rinsed with labeling buffer [lo0 mM Tris-HCI (pH 6.8) 50 mM NaCI] twice, and incubated in labeling buffer containing 10 PLM of 65ZnCI, (150 MBq/mg) for 15 min. The filter was then rinsed twice with wash buffer [ 100 mMTris-HCI (pH 7.5) 50 mM NaCI, 1 mM DTT] and washed for 1 hr with three changes of buffer. Binding of 65Zn was detected by autoradiography. Assay for DNA binding
to the E6 protein
DNA-binding capacities of bacterially expressed wild-type or mutated E6 proteins were analyzed by protein (Southwestern) blotting with 32P-labeled DNA probes (Grossman et a/., 1989). Five micrograms of purified wild-type protein or mutated E6 protein was electrophoresed on a 15O$1SDS-polyacrylamide gel and transferred to a nylon membrane (Clear Blot Membrane-P, ATTO, Tokyo, Japan). A nitrocellulose membrane was not used because it did not retain the protein during the following incubation. The membrane with proteins was incubated in 10 mM Tris-HCI (pH 7.5) 10% glycerol, 2% bovine serum albumin (BSA), 2.5% Nonidet P-40 (NP-40) 1 mNI DTT, 100 PIMZnCI, for 6 hr at room temperature. After a brief rinse in binding buffer [lo mll/l Tris-HCI (pH 7.2) 50 mM NaCI, 0.25% BSA, 1 mA# DlT, 100 @I ZnCI,, 2% polylC (Pharmacia LKB Biotechnology), the membrane was incubated in binding buffer for 1 hr at room temperature, incubated in binding buffer containing 1 O6 cpm of 32P-end-labeled restriction fragments of pBR322 DNA (digested with Hinfl and Haelll) or HPV 16 DNA (the 959-bp segment from nt 7003 to nt 57, digested with /-/haI and PmaCI) for 2 hr at room temperature, and then washed for 30 min with three changes of 10 m/l/l Tris-HCI (pH 7.5) 150 mM NaCI, 1 ml\/l DlT. DNA binding was detected byautoradiography. The radioac-
ET AL.
tivity of the membrane which produced a band on the autoradiogram was determined by Serenkov count. lmmunofluorescence
staining
Monkey COS-1 cells (on coverslips), which were grown in Dulbecco’s modified Eagle medium with 10% calf serum, were transfected with pSRa plasmid (1 pg of DNA per coverslip) by the DEAE-dextran method (McCutchan and Pagano, 1968). Forty-eight hours later, cells were fixed with 5% Formalin in PBS at room temperature for 20 min, rinsed with PBS, incubated in 1% NP-40 in PBS for 20 min at room temperature, reacted with ascites containing MAb 618 for 40 min at 37”, and then reacted with fluorescein-conjugated anti-mouse IgG (Cappel-Organin Teknika Corp., West Chester, PA) for 30 min at 37”. Stained cultures were examined and photographed under a Nikon uv microscope (EFD2). lmmunoprecipitation COS-1 cells (5 X 107) were transfected with 50 pg of pSRa plasmid by electroporation [0.2 mMTris-HCI (pH 7.4) 0.25 M-mannitol, 0.1 mM CaCI,, 0.1 mM MgCI,, pulse height 200 V, pulse width 800 psec three pulses at I-set interval] using GTE-l (Shimadzu, Tokyo, Japan). Forty hours later, mock-transfected or transfected cells were labeled with a [35S]methioninecysteine mixture (3.7 MBq/ml) (Trans 35S-label; ICN Radiochemicals Inc., Irvine, CA) for 3 hr. Cells were scraped off culture dishes, suspended in 1 ml of RIPA buffer [20 mM Tris-HCI (pH 7.5) 150 mll/l NaCI, 1% NP-40, 19/o sodium deoxycholate, 0.1% SDS, 0.2 mNI PMSF, 0.005% aprotinin], and sonicated for 2 min at 150 W (whole cell extract) or fractionated into the soluble cy-toplasmic, membrane, nuclear, and nuclearwash fractions (Barbosa and Wettstein, 1988). The whole cell extract was mixed with 20 ~1 of mouse ascites containing anti-E7 MAb (Mab 730) (Kanda et a/., 1991) kept on ice for 4 hr, mixed with 50 ~1 (bed volume) of protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology) by gentle rocking for 1 hr at 4”, and then centrifuged at 10,000 g for 10 min. The supernatant or the subcellular fractions were mixed with 5 ~1 of ascites containing MAb 6 18 and kept on ice for 4 hr. lmmunocomplexes were collected with protein A-Sepharose CL-4B, electrophoresed on 15% SDS-polyacrylamide gels, and autoradiographed as described previously (Sato et al., 1989b). Assay for E6 transforming
functions
The transforming function of the E6 gene was examined by focal transformation of rat 3Yl cells (Kanda et al., 1987) in cooperation with the E7 gene. Ten micro-
HPV 16 E6 MUTANT
A
B
c FIG. 2. Zinc and DNA binding to bacterially expressed E6 proteins: Autoradiograms of zinc blotting (A), DNA blotting with pBR322 DNA (B), and DNA blotting with HPV 16 DNA (C). Bindings of ‘j6Zn or 3*P-labeled DNA to wild-type E6 (lane l), E6C66G (lane 2) E6C136G (lane 3) and E6C66G/C136G (lane 4) proteins were visualized.
grams of pSRcu-E6 plasmid (with orwithout mutation) or pSRa-0 was mixed with 2 pg of pSRa-E7 and transfected to subconfluent 3Yl cells grown in minimum essential medium with 109’0 fetal bovine serum, by the calcium phosphate method (Graham and van der Eb, 1973). Foci were scored in cultures stained with 5% Giemsa, 25 days after transfection. RESULTS Detection
of zinc-binding
ability of the E6 proteins
To determine their capacity to bind zinc, we compared the bacterially expressed, wild-type and mutant E6 proteins with Gly substitutions for Cys-66 (E6C66G), for Cys-136 (E6Cl36G), and for both (E6C66GKl36G). Binding of 65Zn to immobilized E6 proteins on a nitrocellulose membrane was detected by autoradiography. The wild-type and mutated proteins were transferred to and retained on the membrane at a similar efficiency. The wild-type E6 protein was capable of binding zinc (Fig. 2A, lane 1). Zinc binding to the E6C66G (lane 2) and E6C66G/Cl36G (lane 4) proteins was markedly reduced, but the level of binding to the E6C136G protein was similar to that of the wildtype E6 protein (lanes 1 and 3). These results were consistent with those in the repeated experiments. Thus, it was concluded that Cys-66 is essential for the binding of zinc to the E6 protein. Detection
of DNA-binding
ability of the E6 proteins
DNA binding to bacterially expressed E6 protein was analyzed by the Southwestern blotting method (Grossman et a/., 1989). Figures 2B and 2C show the results of a representative set of four experiments, in which purified E6 protein (5 pg per lane) was electrophor-
PROTEINS
539
esed, transferred to nylon membranes, and allowed to bind 32P-labeled DNA fragments from the entire pBR322 (Fig. 2B) and from an HPV 16 segment containing the long control region (Fig. 2C). The radioactivity for each band varied at most by 20% from experiment to experiment, but the averages for wild-type and mutant proteins were approximately the same (data not shown). The radioactive DNA bound to E6 proteins was eluted from the filters by incubation in IO rnM Tris-HCI (pH 7.5) 1 mM EDTA, 1% SDS and separated on agarose gels. No specific restriction fragments were found to bind preferentially to the E6 protein (data not shown). The data indicate that the DNA-binding capacity of the E6 protein is not affected by the mutation in the metal-binding motif and that the HPV 16 E6 protein does not show a specific affinity for HPV 16 DNA in this assay. lmmunofluorescence staining expressed in COS-1 cells
of E6 protein
We examined the E6 protein transiently expressed in COS-1 cells by indirect immunofluorescence staining. Monkey COS-1 cells transfected with pSRa-E6 plasmids were fixed with Formalin in PBS at 48 hr after transfection and immunostained with anti-E6 mouse monoclonal antibody (MAb 618) (Fig. 3). In the culture transfected with pSRa-E6, EG-producing cells showed strong nuclear immunofluorescence, and approximately 70°b of the positive cells were accompanied by cytoplasmic immunofluorescence (Figs. 3A and 3B). In the cultures transfected with pSRa-EGC66G, pSRa-EGC136G, and pSRa-EGC66GICl36G, the numbers of immunofluorescent cells were approximately;, i, and i, respectively, of that in the culture transfected with pSRa-E6. lmmunofluorescence features of the cells expressing E6C66G protein were markedly different from those of the cells producing wild-type E6 protein (Figs, 3C and 3D) in that immunofluorescent dots with variable density were distributed in the cytoplasm without apparent nuclear staining. The cells producing E6C136G protein showed weaker nuclear immunofluorescence alone when compared with the cells transfected with pSRa-E6 (Figs. 3E and 3F). lmmunoprecipitation COS-1 cells
of E6 protein
expressed
in
With MAb 618, we attempted to immunoprecipitate E6 protein expressed in COS-1 cells. Mock- or pSRaEG-transfected COS-1 cells, which were metabolically labeled with the [35S]methionine-cysteine mixture for 3 hr at 40 hr after transfection, were scraped off a culture dish and lysed in RIPA buffer with brief sonication. The lysate, after being preadsorbed with ascites containing
540
KANDA
ET AL
located in the membrane, nuclear, and nuclear-wash fractions (lanes 8 to lo), but the band from the nuclear fraction was faint (lane 9). The localization of the E6C 136G protein was similar to that of the wild-type E6 protein (lanes 11 to 14). The level of precipitated E6C66G, E6C136G, or E6C66GIC136G protein was lower than that of precipitated wild-type E6 protein (lanes 3 to 14). Although the bands from E6C66G/ Cl 36G were less dense than those from E6C66G, the immunoprecipitation features were common to the two proteins (data not shown). The data indicate that replacement of Cys-66 with Gly alters the subcellular localization of the E6 protein and suggest that substitution of Gly for either Cys-66 or Cys-136 makes the E6 protein unstable.
Focal transformation of rat 3Yl cells by the cooperation of E6 and E7 We examined the E6 and the mutant genes for their capacity to enhance focal transformation of rat 3Yl cells by HPV 16 E7. Focus formation mediated by cotransfection with pSRa-E7 and ~SRLY-E6 was three to four times as efficient as that with ~SRCX-E7 and pSRar0 (Table 1). The cells cotransformed with E6 and E7 were smaller and more densely packed than those transformed with E7 alone. The transforming activities of pSRa-E6C66G and pSRa-EGCl36G, in conjunction FIG. 3. lmmunofluorescence staining of monkey COS-1 cells expressing HPV 16 E6 proteins. Cells transfected with pSRa-E6 (A and B), pSRa-E6C66G (C and D), and pSRa-EGC136G (E and F) were fixed with 5% Formalin and stained indirectly with anti-E6 MAb 6 18.
anti-HPV 16 E7 monoclonal antibody (MAb 730) to reduce nonspecific reactions, was allowed to react with MAb 618. A single protein band with an apparent molecular weight of 18 kDa was immunoprecipitated from the lysate of the COS-1 cells transfected with pSRa-E6 (Fig. 4, lane l), but no band was detected from the lysate of mock-transfected cells (lane 2). Therefore, we concluded that the 18-kDa protein was the E6 protein. We examined subcellular localization of wild-type or mutated E6 protein by immunoprecipitation. The COS1 cells transfected with pSRa plasmids and metabolically labeled with the [35S]methionine-cysteine mixture were fractionated into the soluble cytoplasmic, membrane, nuclear, and nuclear-wash fractions. The E6 protein in each subcellular fraction was immunoprecipitated with MAb 618. We omitted the preadsorption step in order to minimize possible degradation of the E6 proteins. The wild-type E6 protein was located in the membrane, nuclear, and nuclear-wash fractions (lanes 4 to 6) and was not detected in the soluble cytoplasmic fraction (lane 3). The E6C66G protein was also
FIG. 4. lmmunoprecipitation of E6 proteins produced in COS-1 cells. 35S-Labeled whole cell lysate of pSRa-E6 (lane 1) or mock (lane 2).transfected COS-1 cells were preadsorbed with mouse ascites containing anti-HPV 16 E7 protein MAb 730 and immunoprecipitated with anti-E6 MAb 618. COS-1 cells transfected with ~SRL~ plasmids and labeled with 35S were subjected to subcellular fractionation, and the E6 protein in each fraction was immunoprecipitated. Cytoplasmic (lane 3), membrane (lane 4) nuclear (lane 5) and nuclear-wash (lane 6) fractions of cells transfected with pSRa-E6. Cytoplasmic (lane 7) membrane (lane 8). nuclear (lane 9) and nuclearwash (lane 10) fractions of cells transfected with pSRa-EGC66G. Cytoplasmic (lane 1 l), membrane (lane 12) nuclear (lane 13) and nuclear-wash (lane 14) fractions of cells transfected with pSRaE6C136G. Bars on the left Indicate the positions for molecular size markers of 12.3K, 18.4K, and 30K (from the bottom). The E6 protern is denoted by the arrow.
HPV TABLE FOCAL TRANSFORMATION
16 E6 MUTANT
1
OF RAT 3Y 1 CELLS WITH E6 AND E7 OF HPV 16 Number of foci/l fig of pSRa-E7 DNAB
Plasmids
with pSR&-E7 Expt
pSRa-0 pSRa-E6 pSRa-E6C66G pSRa-EGCl36G pSRa-EGC66GK136G
66 284 43 114 103
1
Expt
2
75 249 25 28 92
’ Rat 3Yl cells (2 X 1 Or’) cotransfected with 2 pg of pSRa-E7 and 10 Pg of indicated plasmid were subcultured in six plates. Foci were stained and counted on Day 25. The sum of the number of foci in all plates was divided by 2.
with pSRa-E7, were significantly lower than that of the plasmid expressing wild-type E6, and the cells cotransformed by the E6 mutants and E7 were morphologically similar to those transformed by E7 alone. Thus, both Cys-66 and Cys-136 are important for the E6 transforming function for 3Y 1 cells. DISCUSSION The HPV 16 E6 protein composed of 151 AAs contains four metal-binding motifs Cys-X-X-Cys (Fig. l), one pair of which can form, by binding to zinc, a finger structure (Berg, 1986; Evans and Hollenberg, 1988) possibly mediating sequence-specific DNA binding (Berg, 1986; Evans and Hollenberg, 1988) or proteinprotein interactions (Frankel et a/., 1988). From the conserved distance between the two motifs (29 AAs) among papillomaviruses, it is postulated that the E6 protein potentially forms two fingers, each consisting of 29 AAs. Interestingly, a probable zinc finger of the E7 protein is identical in size to an E6 finger (Cole and Danos, 1987). In this study we constructed three E6 mutants with Gly substitutions for Cys in the motif (for Cys-66, for Cys-136, and for both). The substitutions for cysteines are expected to interrupt the formation of one or two putative zinc fingers of the E6 protein. Biochemical and biological characterization of these mutant proteins has revealed the importance of the motif, especially for their stability and biological functions. Zinc-blotting experiments (Fig. 2A) have shown that Cys-66 is one of the probable essential amino acids for the zinc-binding capacity of the E6 protein. Like Barbosa et al. (1989) and Grossman and Laimins (1989) who used a fusion or nonfusion E6 protein of HPV 18, we found that the nonfusion E6 protein (bacterially expressed) of HPV 16 can bind zinc. Since zinc binding was markedly reduced by Gly substitution for Cys-66,
PROTEINS
541
but not by Gly substitution for Cys-136, the motif from Cys-63 to Cys-66 is more important for zinc binding than the motif from Cys-133 to Cys-136. The results suggest that the N-terminal pair of the motifs may form a finger, but that the C-terminal pair may not. Despite the probable conformational change, DNA binding of the Cys-66 mutant protein was not much different from that of the wild-type E6 or the other mutants (Fig. 2B). Like Mallon et al. (1987), we found that DNA binding was not sequence-specific. DNA fragments from both pBR322 and HPV 16 were found to bind similarly to the bacterially expressed wild-type and mutant E6 proteins. Since the HPV 18 E6 gene is transactivating (Gius eT al., 1988) it possibly interacts with DNA by an unidentified mechanism. The data obtained in this study do not rule out the possibility that a complex of E6 and a cellular protein may bind to a specific segment of HPV chromatin. The replacement of Cys-66 with Gly was found to alter the subcellular localization of the E6 protein, from immunofluorescence staining of COS-1 cells expressing E6 proteins (Fig. 3) and from subcellular fractionation followed by immunoprecipitation of E6 proteins in COS-1 cells (Fig. 4). The observation that the E6 protein is nuclear and membrane-associated is consistent with the subcellular localization of HPV 18 E6 protein expressed in insect cells with a baculovirus vector (Grossman et al., 1989). Whereas the subcellular localization of the E6C136G protein was the same as that of wild-type E6 protein, the E6C66G protein was located predominantly in the membrane and nuclear-wash fractions, but not apparently in the nuclear fraction. These findings indicate that Cys-66 is essential for the E6 protein to be present in nuclei and suggest that the putative N-terminal finger is possibly required for interaction of the E6 protein with a yet unidentified nuclear protein(s). Substitution of Gly for either Cys-66 or Cys-136 appeared to lower the stability of the E6 protein transiently expressed in COS-1 cells. The numbers of immunofluorescent cells in COS-1 cultures transfected with pSRa-EGC66G, pSRa-EGCl36G, or pSRaE6C66G/Ci 36G were less than half of that in the culture transfected with pSRcu-E6. The level of 35S-labeled, immunoprecipitated C66G orC136G protein was markedly lower (as estimated from darkening of autoradiograms) than that of the wild-type E6 protein (Fig. 4). The mutated E6 proteins may be more sensitive than the wild-type E6 to degradation such as proteolysis in eucaryotic cells, because they were as stable as the wildtype in bacteria and in vitro (data not shown). The E6 transforming function for rat cells was impaired by the substitutions for any of the Cys tested (Table 1). Previously, we reported that the E7 gene
KANDA
542
alone is sufficient for focal transformation of rat 3Yl cells, but could not find any function of the E6 for 3Yl cells when the HPV genes were expressed under the control of the SV40 promoter. Replacement of the SV40 promoter with the SRa promoter (Takebe et al., 1988) has greatly enhanced transcription and expression of the HPV genes in 3Y 1 cells and made cooperative transformation by E6 and E7 apparent (details will be described elsewhere). Use of this system enabled us to examine the effect of the substitutions on the E6 transforming function. The lowered E6 transforming activities could be ascribed to instability, change of subcellular localization, and conformational changes of the protein. Whether the E6 transforming function for rat cells is related to its binding to ~53 also remains to be investigated. In this study we characterized the E6 protein with Gly substitution for Cys-66 or Cys-136. The Cys-66 mutation reduced zinc binding, nuclear localization, stability, and transforming capacity, and the Cys-136 mutation reduced stability and transforming capacity. These changes caused by the substitutions may have resulted primarily from the conformational changes of the E6 protein without complete sets of the metal-binding motif. Previously, we reported that the changes of Cys within the two metal-binding motifs in the HPV 16 E7 protein cause the loss of stability and transforming functions and that the functional domain of the E7 protein appears to be in the N-terminal half not containing the metal-binding motifs (Edmonds and Vousden, 1989; Watanabe eta/., 1990). Further genetic analyses of the E6 protein are needed to clarify the functional domains that possibly interact with cellular proteins. ACKNOWLEDGMENTS This work was supported by a grant-in-aid from the Ministry of Health and Welfare for the Comprehensive lo-Year Strategy for Cancer Control, by a grant from the Japan Health Sciences Foundation, by a research grant for aging and health, the Ministry of Health and Welfare, and by cancer research grants from the Ministry of Education, Science, and Culture.
REFERENCES ANDROPHY, E. J., HUBBERT, N. L., SCHILLER, J. T., and Low-y, D. R. (1987). Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines. EMBO /. 6, 989-992. BARBOSA, M. S., EDMONDS, C., FISHER, C., SCHILLER, J. T., Lowv, D. R.. and VOUSDEN, K. H. (1990). The region of the HPV E7 oncoprotein homologous to adenovirus El a and SV40 large T antigen contains separate domains for Rb binding and casein kinase II phosphorylation. EMBO /. 9, 153-l 60. BARBOSA, M. S., Lowv, D. R., and SCHILLER, J. T. (1989). Papillomavirus polypeptides E6 and E7 are zinc-binding proteins. 1. Viral. 63, 1404-1407.
ET AL BARBOSA, M. S., and WETSTEIN, F. 0. (1988). E2 of cottontail rabbit papillomavirus is a nuclear phosphoprotein translated from an mRNA encoding multiple open reading frames. 1. viral. 62, 32423249. BERG, J. M. (1986). Potential metal-binding domains in nucleic acid binding proteins. Science 232, 485-487. COLE, S. T., and DANOS, 0. (1987). Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome. /. Mol. Biol. 193, 599-608. CROOK, T.. MORGENSTERN, J. P., CRAWFORD, L., and BANKS, L. (1989). Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras. EMBO J. 8, 513-519. DORST. M., GISSMANN, L., IKENBERG, H., and ZUR HAUSEN, H. (1983). A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc. Nat/ Acad. Sci. USA 80, 3812-3815. DYSON, N., HOWLEY, P. M., MONGER, K., and HARLOW, E. (1989). The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243, 934-937. EDMONDS, C., and VOUSDEN, K. H. (1989). A point mutational analysis of human papillomavirus type 16 E7 protein. 1. Viral. 63, 26502656. EVANS, R. M., and HOLLENBERG, S. M. (1988). Zinc fingers: Gilt by association. Cell 52, l-3. FRANKEL, A. D.. BREDT, D. S., and PABO, C. 0. (1988). Tat protein from human immunodeficiency virus forms a metal-linked dimer. Science 240, 70-73. GAGE, J. R., MEYERS, C., and WET~STEIN, F. 0. (1990). The E7 proteins of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinoblastoma protein binding and other properties. J. Viral. 64, 723-730. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456467. GIUS, D., GROSSMAN, S., BEDELL, M. A., and LAIMINS, L. A. (1988). Inducible and constitutive enhancer domains in the noncoding region of human papillomavirus type 18. /. Wok 62, 665-672. GLUZMAN, Y. (1981). SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23, 175-l 82. GROSSMAN, S. R., and LAIMINS, L. A. (1989). E6 protein of human papillomavirus type 18 binds zinc. Oncogene 4, 1089-l 093. GROSSMAN, S. R., MORA, R., and LAIMINS, L. A. (1989). Intracellular localization and DNA-binding properties of human papillomavirus type 18 E6 protein expressed with a baculovirus vector. /. Viral. 63, 366-374. HAWLEY-NELSON, P., VOUSDEN, K. H., HUBBERT, N. L., Lowv, D. R., and SCHILLER. J. T. (1989). HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO /. 8, 39053910. HALBERT, C. L., DEMERS, G. W.. and GALLOWAY, D. A. (1991). The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. 1. Viral. 65, 473-478. KANDA, T., FURUNO, A., and YOSHIIKE, K. (1988a). Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Yl cells. 1. Viral. 62, 61 O-61 3. KANDA, T., WATANABE, S., and YOSHIIKE, K. (1987). Human papillomavirus type 16 transformation of rat 3Y 1 cells. Ipn. /. Cancer Res. (Gann) 78, 103-l 08. KANDA, T.. WATANABE, S., and YOSHIIKE, K. (1988b). lmmortalizatlon of primary rat cells by human papillomavirus type 16 subgenomic DNA fragments controlled by the SV40 promoter. Virology 165, 321-325. KANDA, T., ZANMA, S., WATANABE, S., FURUNO. A., and YOSHIIKE, K.
HPV
16 E6 MUTANT
(1991). Two immunodominant regions of the human papillomavirus type 16 E7 protein are masked in the nuclei of monkey COS-1 cells. Virology 182, 723-731. KIMURA, G., ITAGAKI, A., and SUMMERS, J. (1975). Rat cell line 3Yl and its virogenic polyomaand SV40-transformed derivatives. Inf. J. Cancer 15, 694-706. KRAMER, W., and FRITZ, H.-J. (1987). Oligonucleotide-directed construction of mutations via gapped duplex DNA. In “Methods in Enzymology” (R. Wu and L. Grossman, Eds.), Vol. 154, pp. 350367. Academic Press, San Diego. MALLON, R. G., WOJCIECHOWICZ, D., and DEFENDI, V. (1987). DNAbinding activity of papillomavirus proteins. J. l&o/. 61, 1655-l 660. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratoty, Cold Spring Harbor, New York. MCCUTCHAN, J. H., and PAGANO, J. S. (1968). Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. Nat/. Cancer Inst. 41, 351-357. MESSING, J. (1983). New Ml3 vectors for cloning. In “Methods in Enzymology” (R. Wu, L. Grossman, and K. Moldave, Eds.), Vol. 101, pp. 20-78. Academic Press, San Diego. MIZUSHIMA, S., and YAMADA, H. (1975). Isolation and characterization of two outer membrane preparations from Escherichia Co/i. Bio-
chim. Biophys. Acta 375, 44-53. M~JNGER, K., PHELPS, W. C., BUBB, V., HOWLEY, P. M., and SCHLEGEL, R. (1989a). The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Viral. 63, 4417-4421. MONGER, K., WERNESS, B. A., DYSON, N., PHELPS, W. C., HARLOW, E., and HOWLEY, P. M. (198913). Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 8,4099-4105. PHELPS, W. C., YEE, C. L., MONGER, K., and HOWLEY, P. M. (1988). The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E 1 A. Cell 53, 539-547. PRABHAKAR, B. S., HASPEL, M. V.. and NOTKINS. A. L. (1984). Monoclonal antibody techniques applied to viruses. Methods Viral. 7, 1-18. SANGER, F., NICKLEN, S., and COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Nat/. Acad. Sci. USA 74, 5463-5467. SATO, H., FURUNO, A., and YOSHIIKE, K. (1989a). Expression of human papillomavirus type 16 E7 gene induces DNA synthesis of rat 3Y 1 cells. Virology 168, 195-l 99. SATO, H., WATANABE, S., FURUNO, A., and YOSHIIKE, K. (1989b). Human papillomavirus type 16 E7 protein expressed in Escherichia
PROTEINS
543
co/i and monkey COS-1 cells: lmmunofluorescence detection of the nuclear E7 protein. Virology 170, 3 1 l-3 15. SEEDORF, K., K~~MMER, G., DIIRsT, M., SUHAI, S., and R~WEKAMP, W. G. (1985). Human papillomavirus type 16 DNA sequence. Viralogy 145, 181-185. SEEDORF, K., OLTERSDORF, T., K~~MMER, G., and R~WEKAMP, W. (1987). Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells. EMBO J. 6, 139-l 44. SMOTKIN, D., and WEITSTEIN, F. 0. (1986). Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proc. Nat/. Acad. Sci. USA 83, 4680-4684. SMOTKIN, D., PROKOPH, H., and WETSTEIN, F. 0. (1989). Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. J. Viral. 63, 1441-l 447. STOREY, A., PIM, D., MURRAY, A., OSBORN, K., BANKS, L., and CRAWFORD, L. (1988). Comparison of the in vitro transforming activities of human papillomavirus types. EMBO 1. 7, 1815-l 820. TAKEBE, Y., SEIKI, M., FUJISAWA, J., HOY, P., YOKOTA, K., AFUII, K., YoSHIDA, M., and ARAI, N. (1988). SRa promoter: An efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8, 466-472. TANAKA, A., NODA, T., YAJIMA, H., HATANAKA, M., and ITO, Y. (1989). Identification of a transforming gene of human papillomavirus type 16. J. Viral. 63, 1465-1469. VOUSDEN, K. H., DONIGER, J., DIPAOLO, J. A., and Lowv, D. R. (1988). The E7 open reading frame of human papillomavirus type 16 encodes a transforming gene. Oncobene Res. 3, 167-l 75. WATANABE, S., KANDA, T., SATO, H., FURUNO, A., and YOSHIIKE, K. (1990). Mutational analysis of human papillomavirus type 16 E7 functions. J. Viral. 64, 207-2 14. WATANABE, S., KANDA, T., and YOSHIIKE, K. (1989). Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7. J.
Viral. 63, 965-969. WERNESS, B. A., LEVINE, A. J., and HOWLEY, P. M. (1990). Association of human papillomavirus type 16 and 18 E6 proteins with ~53.
Science 248, 76-79. YUTSUDO, M., OKAMOTO, ciation of transforming
Y., and HAKURA, A. (1988). Functional dissogenes of human papillomavirus type 16.
Virology 166, 594-597. ZUR HAUSEN, H. (1989). model to understand
Res. 49,4677-4681.
Papillomaviruses the role of viruses
in anogenital cancer as a in human cancers. Cancer
185,
536-543
(1991)
Human
Papillomavirus Type 16 E6 Proteins with Glycine Substitution for Cysteine in the Metal-Binding Motif
TADAHITO
‘Department
KANDA,*a’ SUMIE WATANABE,* SOICHI ZANMA,t AKEMI FURUNO,* AND KUNITO YOSHIIKE*
of Enteroviruses, National Institute of Health, Technology Laboratories, Chugai Pharmaceutical Received
April
Kamiosaki, Company,
8, 799 1; accepted
HIRONORI
SATO,*,’
Shinagawa-ku, Tokyo 14 1; and tDiagnostics Ltd., Toshima-ku, Tokyo, 171, Japan August
9, 799 1
The human papillomavirus type 16 (HPV 16) E6 is a 151 amino acid protein containing four metal-binding motifs, Cys-X-X-Cys. We constructed and characterized three mutants with Gly substitutions for Cys within the motif; for Cys-66, for Cys-136. and for both, respectively. Zinc binding to bacterially expressed E6 was markedly reduced by the substitution for Cys-66, but DNA binding was unaffected by any of these mutations. immunofluorescence staining showed that, whereas the E6 expressed in monkey COS-1 cells appeared mostly nuclear, the Cys-88 mutant appeared cytoplasmic. Subcellular fractionation followed by immunoprecipitation showed that the E6 in COS-1 ceils was located in the membrane, nuclear, and nuclear-wash fractions, but not in the soluble cytoplasmic fraction, and that the nuclear Cys-86 protein was markedly reduced. The mutant proteins in COS-1 cells appeared to be less stable than the wild type, because the immunofluorescent cells were fewer and because the E6 bands in autoradiograms were less dense. The substitution mutants lost their capacity to enhance HPV 18 E7 transformation of rat 3Yl cells. The data indicate that Cys-66 plays a crucial role for zinc binding and nuclear localization of E6 and that both Cys-66 and Cys-136 are required for a stable or functional structure of E6. o 1991 Academic PWSS, IIIC.
INTRODUCTION
The E6 and E7 proteins have been shown to be expressed in CaSki and SiHa cells, human cell lines derived from cervical carcinomas (Androphy et al., 1987; Seedorf et a/., 1987). Analyses of transcripts have shown that the cap site for viral mRNAs is at nucleotide 97 (Smotkin and Wettstein, 1986; Smotkin eta/., 1989), indicating that the viral P97 transcripts encode 151 and 98 amino acid (AA) proteins for E6 (translated from the second ATG of the open reading frame) and E7 genes, respectively. Presence of metal-binding motifs, Cys-X-X-Cys, is a feature common to these two oncoproteins, the AA sequences of which can be deduced from the entire nucleotide sequence of HPV 16 (Seedorf et a/., 1985). Presence of the four metal-binding motifs is characteristic of and common to the E6 proteins from human and animal papillomaviruses (Cole and Danos, 1987). The motifs of the HPV 16 E6 are at Cys at AAs 30 (Cys-30) to Cys-33, at Cys-63 to Cys-66, at Cys-103 to Cys-106, and at Cys-136 to Cys-139 (Fig. 1). The intervals between the N-terminal pair and between the Cterminal pair are 29 AAs long and are conserved among papillomaviruses (Cole and Danos. 1987). The conserved pattern of the distribution of the motifs suggests an importance of these cysteines in the functional E6 protein. In this study, we examined biochemical and biological properties of the HPV 16 E6 proteins with Gly substitutions for Cys (Cys-66 and Cys-136) in the metal-binding motif.
Human papillomavirus type 16 (HPV 16) (Dllrst eta/., 1983), closely associated with cervical carcinoma (zur Hausen, 1989), encodes two transforming genes E6 and E7. Expression of the transactivating (Phelps eta/., 1988), mitogenic (Sato et al., 1989a), nuclear (Sato et a/., 1989b) E7 protein alone is sufficient for transformation of rodent cell lines (Kanda eta/., 1988a; Yutsudo et a/., 1988; Vousden et al., 1988; Tanaka et al., 1989), primary rat cells (Kanda et al., 1988b), and primary rat cells in conjunction with the expression of activated ras (Phelps et al., 1988; Storey et al., 1988; Crook et a/., 1989). However, expression of both E7 and E6 genes is required for extension of the life span of human fibroblasts (Watanabe et a/., 1989) and for immortalization of human foreskin keratinocytes (MUnger eta/., 1989a; Hawley-Nelson et al., 1989), or E6 can significantly enhance the E7 immortalizing function for human foreskin keratinocytes (Halbet-t et al., 1991). The E7 and E6 proteins are capable of binding to antioncogene products Rb (Dyson et al., 1989; M(lnger et a/., 198913; Gage et al., 1990; Barbosa et al., 1990) and p53 (Werness et a/., 1990), respectively.
’ To whom requests for reprints should be addressed. * Present address: Laboratory of Molecular Microbiology, tional Institute of Allergy and Infectious Diseases, Bethesda, 20892. 0042-6822/91
$3.00
Copynght 0 1991 by Academic Press, Inc. All rights of reproductaon in any form reserved
NaMD
536
HPV
1-MFQDPQERPR
KLPQLCTELQ
16 E6 MUTANT
TTIHDIILE~~QQLLRR
RVYDfAFRDL
G CIVYRDGNPY AVWLKFY
SKISEYRRYC
YSLYGTTLEQ
QYNKPLCDLL
CPEEKQRRLD
PROTEINS
and purified 1982). Preparation
G KKQRFRNIRG RWTGW
IR-QKPL
RSSRTRRETQ L-151
FIG. 1. AA sequence of HPV 16 E6 protein. motifs (Cys-X-X-Cys) are underlined. Cys-66 been replaced with Gly in mutants.
MATERIALS Construction
The metal-binding and Cys-136 have
AND METHODS
of expression
plasmids
The plasmids used to express mutant E6 proteins were constructed by site-directed mutagenesis (Kramer and Fritz, 1987) to replace appropriate nucleotides in the E6 gene, with Mutan-G (Takara Shuzo Co., Ltd., Kyoto, Japan). The E6 mutants constructed in this study had Gly substitutions for Cys-66 (E6C66G), Cys136 (E6Cl36G), and both (E6C66G/C136G). A bacterial expression plasmid for wild-type E6 protein, pKK-E6, was constructed by insertion of the E6 coding region of HPV 16 DNA into pKK233-2 (Pharmacia LKB Biotechnology, Sweden). A new Ncol site was introduced into the HindIll fragment from pSV2-E6 by replacement of A at nucleotides 103 (A-103) and T-107 with C and G, respectively. The Ncol to Hindlll fragment (from nt 103 to nt 657) was cloned into pKK233-2 between its Ncol and /-/indIll sites. G-107 was then restored to T by mutagenesis to obtain pKKE6. Mutants pKK-E6C66G, pKK-E6Cl36G, and pKKE6C66G/C136G were constructed from pKK-E6 by replacements of T-299 and T-509 with Gs, respectively. Eucaryotic expression plasmid pSRa-E6 was constructed by insertion of the HindIll fragment from pSV2-E6 (Kanda et a/., 1988a) (HPV 16 DNA nt 25 to 657) into the HindIll site of pSRa-0 (Kanda et a/., 1991) which contains SV40 transcriptional signals and R-U5 sequence from the LTR of human T-cell leukemia virus type 1 (Takebe et a/., 1988). The E6 protein expressed from pSRa-E6 in monkey COS-1 cells (Gluzman, 1981) migrated together with the bacterially expressed 151 AA E6 protein in gel electrophoresis (data not shown). In mutants pSRa-EGC66G, pSRa-EGCl36G, and pSRa-E6C66G/Cl36G, T-299, T-509, and both Ts were replaced with Gs, respectively, as described above. pSRa-E7 for the expression of the E7 gene was described previously (Kanda et al., 1991). The structures of plasmids newly constructed in this study were verified by DNA sequencing (Sanger et al., 1977; Messing, 1983). Plasmids were grown in E. co/i RR1
637
by standard
methods
(Maniatis
et a/.,
of the E6 proteins
E. co/i strain YA21 (Mizushima and Yamada, 1975) transformed with pKK-E6, pKK-E6C66G, pKK-EGCl36G, or pKK-E6C66G/Cl36G was grown overnight in LBroth (Maniatis et al., 1982) containing 1 mll/l isopropyl-fl-b(-)-thiogalactopyranoside, washed once with 10 rnM Tris-HCI (pH 7.5) 50 mll/l NaCI, 1 mll/l phenylmethylsulfonyl fluoride (PMSF), and sonicated in the same buffer. The lysate was centrifuged at 10,000 gfor 30 min. The pellet (Pl 0) was suspended in 6 M guanidine-HCI, 50 mM Tris (pH 7.5), 2 mM 2-mercaptoethanol(2ME) and centrifuged at 10,000 gfor 10 min. The supernatant was loaded onto a Sephadex Gl50 (Pharmacia LKB Biotechnology) column equilibrated in the same buffer. Collected protein was dialyzed against 10 mM Tris-HCI (pH 7.4) 0.2 m/M 2ME. This material formed a single band in SDS-polyacrylamide gel electrophoresis. The amino acid sequence from the N-terminus of purified E6 protein was determined by the Edoman degradation method using an automatic protein sequencer (PSQ-1, Shimadzu, Tokyo, Japan). The first 12 residues analyzed agreed with those expected from the DNA sequence (Fig. 1). Preparation of monoclonal the E6 protein
antibody
(MAb) against
A cell clone producing anti-E6 immunogloblin was established from a hybridoma between spleen cells of an immunized BALB/C-AnN mouse and the X63Ag8.653 myeloma line by standard methods (Prabhakar et al., 1984). The PlO fraction of bacterially expressed E6 was suspended in 7 n/l urea, 1.25% sarcosine, dialyzed against 10 mlLITris-HCI (pH 7.5) 50 rnM NaCI, and centrifuged at 10,000 g for 30 min. The supernatant (100 to 200 pg) was mixed with Freund’s complete adjuvant and used for subcutaneous injections into a mouse five times at 2-week intervals. Then, 1 mg of E6 protein which was electrophoretically purified from the PlO fraction, precipitated with cold acetone, and suspended in PBS was injected into the mouse intraperitoneally twice at 2-week intervals. The spleen was removed from the immunized mouse 4 days after the final immunization and fused with X68Ag8.653 myeloma cells with polyethylene glycol. The cell clone producing MAb against the E6 protein (MAb 618) was selected by ELISA (purified E6 protein was used for antigen) and injected into BALB/C mice for preparation of ascites containing MAb, which was used in this study. The isotype of MAb 618 was lgG2b which was determined with the Mouse Monoclonal
538
KANDA
Sub-lsotyping kit (American Qualex International, Inc., La Mirada, CA). The epitope for MAb 618 was in the region from AA 1 to AA 13 of the E6 protein, which was determined by competitive binding assay using synthesized oligopeptides (Kanda et al., 1991). Assay for zinc binding
to the E6 protein
The zinc-binding capacities of bacterially expressed E6 proteins were examined by the zinc-blotting method described by Barbosa et al. (1989). Five micrograms of purified wild-type protein or mutant E6 protein was electrophoresed on a 15% SDS-polyacrylamide gel and transferred to a nitrocellulose filter. The transfer and the retention of E6 proteins were confirmed by staining of the membrane with Ponceau red (Sigma, St. Louis, MO). The filter was incubated in renaturing buffer [lo0 mNI Tris-HCI (pH 6.8) 50 mM NaCI, 10 mM dithiothreitol (DlT)] for 1 hr with three changes of buffer, rinsed with labeling buffer [lo0 mM Tris-HCI (pH 6.8) 50 mM NaCI] twice, and incubated in labeling buffer containing 10 PLM of 65ZnCI, (150 MBq/mg) for 15 min. The filter was then rinsed twice with wash buffer [ 100 mMTris-HCI (pH 7.5) 50 mM NaCI, 1 mM DTT] and washed for 1 hr with three changes of buffer. Binding of 65Zn was detected by autoradiography. Assay for DNA binding
to the E6 protein
DNA-binding capacities of bacterially expressed wild-type or mutated E6 proteins were analyzed by protein (Southwestern) blotting with 32P-labeled DNA probes (Grossman et a/., 1989). Five micrograms of purified wild-type protein or mutated E6 protein was electrophoresed on a 15O$1SDS-polyacrylamide gel and transferred to a nylon membrane (Clear Blot Membrane-P, ATTO, Tokyo, Japan). A nitrocellulose membrane was not used because it did not retain the protein during the following incubation. The membrane with proteins was incubated in 10 mM Tris-HCI (pH 7.5) 10% glycerol, 2% bovine serum albumin (BSA), 2.5% Nonidet P-40 (NP-40) 1 mNI DTT, 100 PIMZnCI, for 6 hr at room temperature. After a brief rinse in binding buffer [lo mll/l Tris-HCI (pH 7.2) 50 mM NaCI, 0.25% BSA, 1 mA# DlT, 100 @I ZnCI,, 2% polylC (Pharmacia LKB Biotechnology), the membrane was incubated in binding buffer for 1 hr at room temperature, incubated in binding buffer containing 1 O6 cpm of 32P-end-labeled restriction fragments of pBR322 DNA (digested with Hinfl and Haelll) or HPV 16 DNA (the 959-bp segment from nt 7003 to nt 57, digested with /-/haI and PmaCI) for 2 hr at room temperature, and then washed for 30 min with three changes of 10 m/l/l Tris-HCI (pH 7.5) 150 mM NaCI, 1 ml\/l DlT. DNA binding was detected byautoradiography. The radioac-
ET AL.
tivity of the membrane which produced a band on the autoradiogram was determined by Serenkov count. lmmunofluorescence
staining
Monkey COS-1 cells (on coverslips), which were grown in Dulbecco’s modified Eagle medium with 10% calf serum, were transfected with pSRa plasmid (1 pg of DNA per coverslip) by the DEAE-dextran method (McCutchan and Pagano, 1968). Forty-eight hours later, cells were fixed with 5% Formalin in PBS at room temperature for 20 min, rinsed with PBS, incubated in 1% NP-40 in PBS for 20 min at room temperature, reacted with ascites containing MAb 618 for 40 min at 37”, and then reacted with fluorescein-conjugated anti-mouse IgG (Cappel-Organin Teknika Corp., West Chester, PA) for 30 min at 37”. Stained cultures were examined and photographed under a Nikon uv microscope (EFD2). lmmunoprecipitation COS-1 cells (5 X 107) were transfected with 50 pg of pSRa plasmid by electroporation [0.2 mMTris-HCI (pH 7.4) 0.25 M-mannitol, 0.1 mM CaCI,, 0.1 mM MgCI,, pulse height 200 V, pulse width 800 psec three pulses at I-set interval] using GTE-l (Shimadzu, Tokyo, Japan). Forty hours later, mock-transfected or transfected cells were labeled with a [35S]methioninecysteine mixture (3.7 MBq/ml) (Trans 35S-label; ICN Radiochemicals Inc., Irvine, CA) for 3 hr. Cells were scraped off culture dishes, suspended in 1 ml of RIPA buffer [20 mM Tris-HCI (pH 7.5) 150 mll/l NaCI, 1% NP-40, 19/o sodium deoxycholate, 0.1% SDS, 0.2 mNI PMSF, 0.005% aprotinin], and sonicated for 2 min at 150 W (whole cell extract) or fractionated into the soluble cy-toplasmic, membrane, nuclear, and nuclearwash fractions (Barbosa and Wettstein, 1988). The whole cell extract was mixed with 20 ~1 of mouse ascites containing anti-E7 MAb (Mab 730) (Kanda et a/., 1991) kept on ice for 4 hr, mixed with 50 ~1 (bed volume) of protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology) by gentle rocking for 1 hr at 4”, and then centrifuged at 10,000 g for 10 min. The supernatant or the subcellular fractions were mixed with 5 ~1 of ascites containing MAb 6 18 and kept on ice for 4 hr. lmmunocomplexes were collected with protein A-Sepharose CL-4B, electrophoresed on 15% SDS-polyacrylamide gels, and autoradiographed as described previously (Sato et al., 1989b). Assay for E6 transforming
functions
The transforming function of the E6 gene was examined by focal transformation of rat 3Yl cells (Kanda et al., 1987) in cooperation with the E7 gene. Ten micro-
HPV 16 E6 MUTANT
A
B
c FIG. 2. Zinc and DNA binding to bacterially expressed E6 proteins: Autoradiograms of zinc blotting (A), DNA blotting with pBR322 DNA (B), and DNA blotting with HPV 16 DNA (C). Bindings of ‘j6Zn or 3*P-labeled DNA to wild-type E6 (lane l), E6C66G (lane 2) E6C136G (lane 3) and E6C66G/C136G (lane 4) proteins were visualized.
grams of pSRcu-E6 plasmid (with orwithout mutation) or pSRa-0 was mixed with 2 pg of pSRa-E7 and transfected to subconfluent 3Yl cells grown in minimum essential medium with 109’0 fetal bovine serum, by the calcium phosphate method (Graham and van der Eb, 1973). Foci were scored in cultures stained with 5% Giemsa, 25 days after transfection. RESULTS Detection
of zinc-binding
ability of the E6 proteins
To determine their capacity to bind zinc, we compared the bacterially expressed, wild-type and mutant E6 proteins with Gly substitutions for Cys-66 (E6C66G), for Cys-136 (E6Cl36G), and for both (E6C66GKl36G). Binding of 65Zn to immobilized E6 proteins on a nitrocellulose membrane was detected by autoradiography. The wild-type and mutated proteins were transferred to and retained on the membrane at a similar efficiency. The wild-type E6 protein was capable of binding zinc (Fig. 2A, lane 1). Zinc binding to the E6C66G (lane 2) and E6C66G/Cl36G (lane 4) proteins was markedly reduced, but the level of binding to the E6C136G protein was similar to that of the wildtype E6 protein (lanes 1 and 3). These results were consistent with those in the repeated experiments. Thus, it was concluded that Cys-66 is essential for the binding of zinc to the E6 protein. Detection
of DNA-binding
ability of the E6 proteins
DNA binding to bacterially expressed E6 protein was analyzed by the Southwestern blotting method (Grossman et a/., 1989). Figures 2B and 2C show the results of a representative set of four experiments, in which purified E6 protein (5 pg per lane) was electrophor-
PROTEINS
539
esed, transferred to nylon membranes, and allowed to bind 32P-labeled DNA fragments from the entire pBR322 (Fig. 2B) and from an HPV 16 segment containing the long control region (Fig. 2C). The radioactivity for each band varied at most by 20% from experiment to experiment, but the averages for wild-type and mutant proteins were approximately the same (data not shown). The radioactive DNA bound to E6 proteins was eluted from the filters by incubation in IO rnM Tris-HCI (pH 7.5) 1 mM EDTA, 1% SDS and separated on agarose gels. No specific restriction fragments were found to bind preferentially to the E6 protein (data not shown). The data indicate that the DNA-binding capacity of the E6 protein is not affected by the mutation in the metal-binding motif and that the HPV 16 E6 protein does not show a specific affinity for HPV 16 DNA in this assay. lmmunofluorescence staining expressed in COS-1 cells
of E6 protein
We examined the E6 protein transiently expressed in COS-1 cells by indirect immunofluorescence staining. Monkey COS-1 cells transfected with pSRa-E6 plasmids were fixed with Formalin in PBS at 48 hr after transfection and immunostained with anti-E6 mouse monoclonal antibody (MAb 618) (Fig. 3). In the culture transfected with pSRa-E6, EG-producing cells showed strong nuclear immunofluorescence, and approximately 70°b of the positive cells were accompanied by cytoplasmic immunofluorescence (Figs. 3A and 3B). In the cultures transfected with pSRa-EGC66G, pSRa-EGC136G, and pSRa-EGC66GICl36G, the numbers of immunofluorescent cells were approximately;, i, and i, respectively, of that in the culture transfected with pSRa-E6. lmmunofluorescence features of the cells expressing E6C66G protein were markedly different from those of the cells producing wild-type E6 protein (Figs, 3C and 3D) in that immunofluorescent dots with variable density were distributed in the cytoplasm without apparent nuclear staining. The cells producing E6C136G protein showed weaker nuclear immunofluorescence alone when compared with the cells transfected with pSRa-E6 (Figs. 3E and 3F). lmmunoprecipitation COS-1 cells
of E6 protein
expressed
in
With MAb 618, we attempted to immunoprecipitate E6 protein expressed in COS-1 cells. Mock- or pSRaEG-transfected COS-1 cells, which were metabolically labeled with the [35S]methionine-cysteine mixture for 3 hr at 40 hr after transfection, were scraped off a culture dish and lysed in RIPA buffer with brief sonication. The lysate, after being preadsorbed with ascites containing
540
KANDA
ET AL
located in the membrane, nuclear, and nuclear-wash fractions (lanes 8 to lo), but the band from the nuclear fraction was faint (lane 9). The localization of the E6C 136G protein was similar to that of the wild-type E6 protein (lanes 11 to 14). The level of precipitated E6C66G, E6C136G, or E6C66GIC136G protein was lower than that of precipitated wild-type E6 protein (lanes 3 to 14). Although the bands from E6C66G/ Cl 36G were less dense than those from E6C66G, the immunoprecipitation features were common to the two proteins (data not shown). The data indicate that replacement of Cys-66 with Gly alters the subcellular localization of the E6 protein and suggest that substitution of Gly for either Cys-66 or Cys-136 makes the E6 protein unstable.
Focal transformation of rat 3Yl cells by the cooperation of E6 and E7 We examined the E6 and the mutant genes for their capacity to enhance focal transformation of rat 3Yl cells by HPV 16 E7. Focus formation mediated by cotransfection with pSRa-E7 and ~SRLY-E6 was three to four times as efficient as that with ~SRCX-E7 and pSRar0 (Table 1). The cells cotransformed with E6 and E7 were smaller and more densely packed than those transformed with E7 alone. The transforming activities of pSRa-E6C66G and pSRa-EGCl36G, in conjunction FIG. 3. lmmunofluorescence staining of monkey COS-1 cells expressing HPV 16 E6 proteins. Cells transfected with pSRa-E6 (A and B), pSRa-E6C66G (C and D), and pSRa-EGC136G (E and F) were fixed with 5% Formalin and stained indirectly with anti-E6 MAb 6 18.
anti-HPV 16 E7 monoclonal antibody (MAb 730) to reduce nonspecific reactions, was allowed to react with MAb 618. A single protein band with an apparent molecular weight of 18 kDa was immunoprecipitated from the lysate of the COS-1 cells transfected with pSRa-E6 (Fig. 4, lane l), but no band was detected from the lysate of mock-transfected cells (lane 2). Therefore, we concluded that the 18-kDa protein was the E6 protein. We examined subcellular localization of wild-type or mutated E6 protein by immunoprecipitation. The COS1 cells transfected with pSRa plasmids and metabolically labeled with the [35S]methionine-cysteine mixture were fractionated into the soluble cytoplasmic, membrane, nuclear, and nuclear-wash fractions. The E6 protein in each subcellular fraction was immunoprecipitated with MAb 618. We omitted the preadsorption step in order to minimize possible degradation of the E6 proteins. The wild-type E6 protein was located in the membrane, nuclear, and nuclear-wash fractions (lanes 4 to 6) and was not detected in the soluble cytoplasmic fraction (lane 3). The E6C66G protein was also
FIG. 4. lmmunoprecipitation of E6 proteins produced in COS-1 cells. 35S-Labeled whole cell lysate of pSRa-E6 (lane 1) or mock (lane 2).transfected COS-1 cells were preadsorbed with mouse ascites containing anti-HPV 16 E7 protein MAb 730 and immunoprecipitated with anti-E6 MAb 618. COS-1 cells transfected with ~SRL~ plasmids and labeled with 35S were subjected to subcellular fractionation, and the E6 protein in each fraction was immunoprecipitated. Cytoplasmic (lane 3), membrane (lane 4) nuclear (lane 5) and nuclear-wash (lane 6) fractions of cells transfected with pSRa-E6. Cytoplasmic (lane 7) membrane (lane 8). nuclear (lane 9) and nuclearwash (lane 10) fractions of cells transfected with pSRa-EGC66G. Cytoplasmic (lane 1 l), membrane (lane 12) nuclear (lane 13) and nuclear-wash (lane 14) fractions of cells transfected with pSRaE6C136G. Bars on the left Indicate the positions for molecular size markers of 12.3K, 18.4K, and 30K (from the bottom). The E6 protern is denoted by the arrow.
HPV TABLE FOCAL TRANSFORMATION
16 E6 MUTANT
1
OF RAT 3Y 1 CELLS WITH E6 AND E7 OF HPV 16 Number of foci/l fig of pSRa-E7 DNAB
Plasmids
with pSR&-E7 Expt
pSRa-0 pSRa-E6 pSRa-E6C66G pSRa-EGCl36G pSRa-EGC66GK136G
66 284 43 114 103
1
Expt
2
75 249 25 28 92
’ Rat 3Yl cells (2 X 1 Or’) cotransfected with 2 pg of pSRa-E7 and 10 Pg of indicated plasmid were subcultured in six plates. Foci were stained and counted on Day 25. The sum of the number of foci in all plates was divided by 2.
with pSRa-E7, were significantly lower than that of the plasmid expressing wild-type E6, and the cells cotransformed by the E6 mutants and E7 were morphologically similar to those transformed by E7 alone. Thus, both Cys-66 and Cys-136 are important for the E6 transforming function for 3Y 1 cells. DISCUSSION The HPV 16 E6 protein composed of 151 AAs contains four metal-binding motifs Cys-X-X-Cys (Fig. l), one pair of which can form, by binding to zinc, a finger structure (Berg, 1986; Evans and Hollenberg, 1988) possibly mediating sequence-specific DNA binding (Berg, 1986; Evans and Hollenberg, 1988) or proteinprotein interactions (Frankel et a/., 1988). From the conserved distance between the two motifs (29 AAs) among papillomaviruses, it is postulated that the E6 protein potentially forms two fingers, each consisting of 29 AAs. Interestingly, a probable zinc finger of the E7 protein is identical in size to an E6 finger (Cole and Danos, 1987). In this study we constructed three E6 mutants with Gly substitutions for Cys in the motif (for Cys-66, for Cys-136, and for both). The substitutions for cysteines are expected to interrupt the formation of one or two putative zinc fingers of the E6 protein. Biochemical and biological characterization of these mutant proteins has revealed the importance of the motif, especially for their stability and biological functions. Zinc-blotting experiments (Fig. 2A) have shown that Cys-66 is one of the probable essential amino acids for the zinc-binding capacity of the E6 protein. Like Barbosa et al. (1989) and Grossman and Laimins (1989) who used a fusion or nonfusion E6 protein of HPV 18, we found that the nonfusion E6 protein (bacterially expressed) of HPV 16 can bind zinc. Since zinc binding was markedly reduced by Gly substitution for Cys-66,
PROTEINS
541
but not by Gly substitution for Cys-136, the motif from Cys-63 to Cys-66 is more important for zinc binding than the motif from Cys-133 to Cys-136. The results suggest that the N-terminal pair of the motifs may form a finger, but that the C-terminal pair may not. Despite the probable conformational change, DNA binding of the Cys-66 mutant protein was not much different from that of the wild-type E6 or the other mutants (Fig. 2B). Like Mallon et al. (1987), we found that DNA binding was not sequence-specific. DNA fragments from both pBR322 and HPV 16 were found to bind similarly to the bacterially expressed wild-type and mutant E6 proteins. Since the HPV 18 E6 gene is transactivating (Gius eT al., 1988) it possibly interacts with DNA by an unidentified mechanism. The data obtained in this study do not rule out the possibility that a complex of E6 and a cellular protein may bind to a specific segment of HPV chromatin. The replacement of Cys-66 with Gly was found to alter the subcellular localization of the E6 protein, from immunofluorescence staining of COS-1 cells expressing E6 proteins (Fig. 3) and from subcellular fractionation followed by immunoprecipitation of E6 proteins in COS-1 cells (Fig. 4). The observation that the E6 protein is nuclear and membrane-associated is consistent with the subcellular localization of HPV 18 E6 protein expressed in insect cells with a baculovirus vector (Grossman et al., 1989). Whereas the subcellular localization of the E6C136G protein was the same as that of wild-type E6 protein, the E6C66G protein was located predominantly in the membrane and nuclear-wash fractions, but not apparently in the nuclear fraction. These findings indicate that Cys-66 is essential for the E6 protein to be present in nuclei and suggest that the putative N-terminal finger is possibly required for interaction of the E6 protein with a yet unidentified nuclear protein(s). Substitution of Gly for either Cys-66 or Cys-136 appeared to lower the stability of the E6 protein transiently expressed in COS-1 cells. The numbers of immunofluorescent cells in COS-1 cultures transfected with pSRa-EGC66G, pSRa-EGCl36G, or pSRaE6C66G/Ci 36G were less than half of that in the culture transfected with pSRcu-E6. The level of 35S-labeled, immunoprecipitated C66G orC136G protein was markedly lower (as estimated from darkening of autoradiograms) than that of the wild-type E6 protein (Fig. 4). The mutated E6 proteins may be more sensitive than the wild-type E6 to degradation such as proteolysis in eucaryotic cells, because they were as stable as the wildtype in bacteria and in vitro (data not shown). The E6 transforming function for rat cells was impaired by the substitutions for any of the Cys tested (Table 1). Previously, we reported that the E7 gene
KANDA
542
alone is sufficient for focal transformation of rat 3Yl cells, but could not find any function of the E6 for 3Yl cells when the HPV genes were expressed under the control of the SV40 promoter. Replacement of the SV40 promoter with the SRa promoter (Takebe et al., 1988) has greatly enhanced transcription and expression of the HPV genes in 3Y 1 cells and made cooperative transformation by E6 and E7 apparent (details will be described elsewhere). Use of this system enabled us to examine the effect of the substitutions on the E6 transforming function. The lowered E6 transforming activities could be ascribed to instability, change of subcellular localization, and conformational changes of the protein. Whether the E6 transforming function for rat cells is related to its binding to ~53 also remains to be investigated. In this study we characterized the E6 protein with Gly substitution for Cys-66 or Cys-136. The Cys-66 mutation reduced zinc binding, nuclear localization, stability, and transforming capacity, and the Cys-136 mutation reduced stability and transforming capacity. These changes caused by the substitutions may have resulted primarily from the conformational changes of the E6 protein without complete sets of the metal-binding motif. Previously, we reported that the changes of Cys within the two metal-binding motifs in the HPV 16 E7 protein cause the loss of stability and transforming functions and that the functional domain of the E7 protein appears to be in the N-terminal half not containing the metal-binding motifs (Edmonds and Vousden, 1989; Watanabe eta/., 1990). Further genetic analyses of the E6 protein are needed to clarify the functional domains that possibly interact with cellular proteins. ACKNOWLEDGMENTS This work was supported by a grant-in-aid from the Ministry of Health and Welfare for the Comprehensive lo-Year Strategy for Cancer Control, by a grant from the Japan Health Sciences Foundation, by a research grant for aging and health, the Ministry of Health and Welfare, and by cancer research grants from the Ministry of Education, Science, and Culture.
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Papillomaviruses the role of viruses
in anogenital cancer as a in human cancers. Cancer
