characterization of the adenovirus e3 protein that down-regulates

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267, No. 19, Issue of July 5. PP , 13480-13487,1992 Printed in U. S. A.

Characterization of the Adenovirus E3 Protein That Down-regulates the Epidermal Growth Factor Receptor EVIDENCE FOR INTERMOLECULAR DISULFIDE BONDING AND PLASMA MEMBRANE LOCALIZATION*

(Received for publication, September 11,1991)

Patricia Hoffman, Michael B. Yaffes, Brian L. Hoffman, SoonPin YeiBlI, William S. M. Wold#, and Cathleen Carlinll From the Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 and the Slnstitute for Molecular Virology, St. Louis University Medical School, St. Louis, Missouri 63110

We have characterized the biosynthesis and processing of a 91 amino acid hydrophobic integral membrane protein encoded by human group C adenoviruses which down-regulates the EGF receptor (Carlin, C. R., Tol- lefson, A. E., Brady, H. A., Hoffman, B. L., and Wold, W. S . M. (1989) Cell 57, 135-144). Previous studies have shown that two immunologically related proteins are produced in vivo, a 13.7-kDa protein encoded by E3 message f and a 11.3-kDa protein derived from 13.7 kDa by proteolysis (Hoffman, B. L., Ullrich, A., Wold, W. S. M., and Carlin, C. R. (1990) Mol. Cell. Biol. 10,5521-5524; Tollefson, A. E., Krajcsi, P., Yei, S. , Carlin, C. R., and Wold, W. S . M. (1990) J. Virol. 64,794-801). We report here that the 13.7- and 11.3- kDa proteins form intermolecular disulfide bonds cotranslationally at Cys-31 and tend to migrate as high molecular weight aggregates under nonreducing conditions. Both proteins are also present at the cell surface, as evidenced by specific immunoprecipitation from intact monolayers enzymatically labeled with

I. Moreover, an antiserum specific for a putative extracellular epitope recognizes the same viral proteins as antibodies directed against a C-terminal synthetic 15-mer. The 13.7- and 11.3-kDa proteins are detected at early time points during pulse-chase radi- olabeling of infected cells, do not undergo any further changes in molecular weight, and focus at their predicted isoelectric points (7.4 and 7.2, respectively). Identical results are obtained in stable transfectants constitutively expressing only 13.7 and 11.3 kDa, suggesting that biosynthesis and processing is not dependent on other viral proteins. These results have been incorporated into a computer-based model to predict the orientation of 13.7 and 11.3 kDa in the lipid bilayer. This model provides a basis for testing predictions regarding the topology of the viral proteins, as well as putative interactions with heterologous proteins in the microenvironment of the plasma membrane that cause down-regulation of the epidermal growth factor receptor.

126

* This work was supported by Grant CA-49540 from the National Institutes of Health and Grant 334-90 from the Diabetes Association of Greater Cleveland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by National Institutes of Health Postdoctoral Train- ing Grant HL07653.

ll Present address: Dept. of Health and Human Services, United States Public Health Service, National Institutes of Health, Bldg. 29, Rm. 304, Bethesda, MD 20892.

)I To whom correspondence should be addressed.

Adenoviruses establish and sustain latent infections in hu- mans, resulting in a variety of diseases. Although much has been discovered about the molecular biology of viral replication and transcription, relatively little is known about host- virus interactions. The adenovirus genome is a linear DNA approximately 36 kb’ in size. We have recently identified a gene product encoded by the E3 early transcription unit of group C adenoviruses that causes rapid internalization and degradation of EGF-R (1). Introduction of the coding sequence for this protein by retroviral-mediated gene transfer elicits the same response (2). Although the E3 gene is nones- sential for viral replication (3), it is highly conserved in all serotypes that have been sequenced (4-8; see Ref. 9 for compilation). Moreover, group C adenoviruses primarily at- tack epithelial cells lining the upper respiratory tract which express EGF-R (10). Taken together, these observations suggest that EGF-R down-regulation is important during host infections, although the benefit afforded the virus remains unknown.

Other viral proteins have been identified that interact with EGF-R. Vaccinia virus, for example, encodes a secreted growth factor called vaccinia growth factor that is structurally related to EGF, and recognizes the EGF-R ligand-binding domain (11). Another EGF-like molecule produced by many retrovirally transformed cells (12), as well as certain normal cells (e.g. Ref. 13), is TGF-a. Both molecules are mitogenic and cause anchorage-independent growth in cooperation with TGF-,3 (14, 15). The membrane-associated BPV E5 transforming protein also activates several protein tyrosine kinase receptors including EGF-R, but without attenuating the signal through down-regulation (16, 17). In contrast to vaccinia growth factor and TGF-a, the adenovirus E3 protein is not structurally related to EGF (1) and acts intracellularly (18). Moreover, although BPV E5 and the adenovirus protein are both hydrophobic, the biological consequences associated with the action of each molecule are presumably very different.

Two protein products of the E3 gene have been previously identified in adenovirus-infected cells (9) and following retrovirus-mediated gene transfer (2). The protein encoded by this E3 open reading frame has a molecular mass of 13.7 kDa; a second species, with a molecular mass of 11.3 kDa, is derived by proteolysis and lacks the N-terminal22 amino acids found in the 13.7-kDa protein (19). Both proteins are detected in

’ The abbreviations used are: kb, kilobase(s); BPV, bovine papii- loma virus; DTT, dithiotbreitol; EGF-R, epidermal growth factor receptor; ER, endoplasmic reticulum; MEM, minimal essential medium; NEPHGE, nonequilibrium pH gradient electrophoresis; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TGF-a, transforming growth factor-a; TGF-6, transforming growth factor-(3; EGTA, [ethylenebis(oxyethylenenitrilo) Jtetraacetic acid.

13480

Characterization of Adenovirus Protein That Down-regulates EGF-R 13481

immunoblots from whole cell lysates (9), or by immunoprecipitation from radiolabeled lysates (2, 9), using antibodies directed against a synthetic peptide corresponding to the C- terminal 15-mer. In order to better understand the mecha- nisms of action of these E3 proteins, we have undertaken a thorough study of their biosynthesis, processing, and intracellular localization in adenovirus-infected cells, as well as in transfectants with stable expression of 13.7 and 11.3 kDa. The hydrophobic nature of 13.7 and 11.3 kDa suggests that these molecules may act directly in the plasma membrane to modulate EGF-R expression, and as such are the first proteins with this structure identified that regulate receptor traffick- ing. Ultimately, by molecular analysis of the E3 proteins, we also hope to discover whether there is a link between EGF-R down-regulation and adenovirus pathogenesis.

EXPERIMENTAL PROCEDURES

Cells and Viruses-Human hepatocellular carcinoma-derived N- PLC/PRF/5 cells (20) were maintained in MEM, supplemented with 10% fetal bovine serum and 2 mM glutamine. $-crip-10.4K cells were derived by transfecting retroviral packaging $-crip cells (21) with a plasmid containing sequences encoding the adenovirus E3 13.7-kDal 11.3-kDa proteins (called 10.4) ligated into the Moloney murine leukemia virus-based DOL plasmid (22), with constitutive expression driven from the 5' long terminal repeat (Fig. IA; see Ref. 2 for cloning strategy). $-crip-DOL5 cells are transfectants containing the DOL vector without a 10.4 gene insert. Stable transfectants were selected using 0.4 mg/ml G418 (Geneticin; GIBCO). Expression of viral proteins was confirmed using immunoblot analysis of whole cell lysates (Fig. lB), demonstrating an apparently equal ratio of 13.7 and 11.3 kDa; and by indirect immunofluorescence (Fig. IC).

Virus stocks were prepared in suspension cultures of KB cells maintained in Joklik's MEM supplemented with 5% horse serum. Propagation and plaque purification was performed as described elsewhere (23). H5/2rec700 (rec700) is an Ad5-Ad2-Ad5 recombinant virus with group C wild-type phenotype; description of this virus as well as mutants that delete sequences in the 10.4 gene (e.g. dl753), or overproduce the 13.7- and 11.3-kDa proteins (e.g. in724), are provided elsewhere (24, 25). Mutant viruses with individual cysteine residues converted to serine2 were generated by oligonucleotide site-directed mutagenesis (26) and sequenced by the dideoxy chain termination method (27). Most acute infections were carried out in the presence of arabinofuranosyl-cytosine (20 pg/ml) to enhance early viral protein synthesis.

Cell Labeling-For pulse-chase and metabolic labeling, cells were preincubated for 1 h in cysteine-free medium, then incubated with 100-200 pCi of ~-[~~S]cysteine/ml ( S O 0 Ci/mmol, Du Pont-New England Nuclear) for the lengths of time indicated in the figure legends. Cell surface labeling with 1251 using the lactoperoxidase method was carried out as described previously (28). Labeling with '"P (1 mCi/ml; 900-1100 mCi/mmol, Du Pont-New England Nuclear) after a 1-h preincubation in phosphate- and serum-free medium, was for 2 h. In some experiments, cells were labeled using [9,10-3H] palmitic acid (400 pCi/ml; 30-60 Ci/mmol, Du Pont-New England Nuclear) for 16 h.

For analysis of viral protein expression, cell lysates were prepared using 1% (w/v) Nonidet P-40,0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS in 50 mM Tris, pH 8.0, 150 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 pg/ml leupeptin, and 0.7 pg/ml pepstatin A. For analysis of EGF-R, cell lysates were made using 1% (w/v) Nonidet P-40 in 0.1 M Tris, pH 6.8, supplemented 15% (w/w) glycerol, 2 mM EDTA, 1 mM EGTA, and the same protease inhibitors listed above. Phosphatase inhibitors (30 mM sodium pyrophosphate, 50 mM sodium fluoride, and 100 PM sodium orthovanadate) were also added when cells were labeled with 32P.

Immunoprecipitation and Gel Electrophoresis-Immunoprecipita- tions were carried out using antibodies adsorbed onto protein A- Sepharose CL-4B beads (Sigma). A 13.7-kDa/11.3-kDa-specific antiserum was raised in rabbits using a synthetic peptide corresponding t o the C-terminal 15-mer. The specificity of this antibody has been documented previously (2,9) and is reiterated in the immunoblot in Fig. 1B. Anti-$-crip-10.4K antibodies were generated against a pu-

s. Yei and W. s. M. Wold, unpublished results.

B

4 1 3.7 1 4 .) - 1 1 . 0

P re immune FIG. 1. Construction of cell lines that constitutively express

t h e 13.7- and 11.3-kDa proteins. A, diagram of the plasmid pDOL-10.4K, which contains the E3 sequences encoding the 13.7- kDa/ll.3-kDa proteins ligated to the DOL plasmid (2). B, immunoblot analysis (62) of viral proteins following extraction of cells infected with a virus mutant that overproduces the 13.7- and 11.3-kDa proteins (in724), and two cell lines selected following stable integra- tion of pDOL-10.4K in $-crip cells (clone l and clone 5). Blots were incubated in 10 mM Tris, pH 7.4, supplemented with 0.5% (w/w) Tween-20,0.9% NaCl, 0.01% NaN3, and 1 mM EDTA for 16 h a t 4 "C to block nonspecific binding sites. Primary and secondary incubations were carried out using viral protein-specific antiserum (1:50), and 1251-protein G (10' cpm/ml), respectively. C, indirect immunofluorescence analysis of clone 1 cells fixed with 3.7% paraformaldehyde and permeabilized with 0.2% Triton X-100. Primary staining was with either a peptide rabbit antiserum specific for the viral proteins (1:50), or preimmune serum (1:50); secondary staining was with fluorescein- conjugated goat anti-rabbit IgG antiserum (1:250; Cappel).

tative extracellular epitope of the 13.7- and 11.3-kDa viral proteins by a total of seven, biweekly, intraperitoneal injections of intact $- crip-l0.4K cells into the same strain of mice from which the mouse parental cells were derived (i.e. Swiss 3T3). This method of developing antibodies, described in detail in Ref. 29, has been used successfully to make reagents specific for human antigens encoded by chromosome 6 and 7 expressed in human-mouse cell hybrids (29, 30). EGF-R1 is an EGF-R-specific monoclonal antibody directed against an exocy- tosolic peptide core epitope (31,32). EGF-R and transferrin receptor were immunoprecipitated as control phosphoproteins (32) and palmitoylated proteins (33), respectively, using EGF-R1 or the transferrin receptor-specific monoclonal antibody OKT-9 (34). Proteins were eluted from beads by boiling in Laemmli buffer, and separated by SDS-PAGE (35); in some experiments, DTT was omitted from the Laemmli buffer.

Details of two-dimensional nonreducing/reducing SDS-PAGE analysis are described in Ref. 36. Briefly, lanes from an SDS-PAGE

buffer containing DTT, and sealed to the top of a second SDS-PAGE gel run under nonreducing conditions were excised, equilibrated in

gel using equilibration buffer supplemented with 0.1% (w/v) agarose and bromphenol blue (0.2 mg/ml). NEPHGE was carried out by the

13482 Characterization of Adenovirus Protein That Down-regulates EGF-R method of O'Farrell et al. (371, using isoelectric gels prepared with pH 3-10 ampholytes. NEPHGE pH gradients were determined by measuring the pH of 1-cm gel slices soaked in water overnight.

RESULTS

Adenovirus E3 Proteins Form Internolecular Disulfide Bonds in Vivo Which Are Composed Entirely of the 13.7- and 11.3-kDa Proteins-We have shown previously that cells pro- duce 13.7- and 11.3-kDa proteins encoded by a single tran- script following infection with group c adenoviruses (9)) or retroviral-mediated transfer of the intact E3 gene (2), that can be immunoprecipitated using an antiserum specific for a C-terminal synthetic 15-mer. Both proteins are also seen in immunoblots using the same antiserum to probe whole cell lysates (9; see also Fig. lB), arguing that the 11.3-kDa protein is an authentic product, rather than a derivative of experimental manipulation. When specific immunoprecipitates from cells infected with a virus mutant engineered to overproduce 13.7 and 11.3 kDa (24) were analyzed under nonreducing conditions, proteins with molecular weights consistent with the formation of intermolecular disulfide bonds were detected a prominent band with a molecular mass of 23.4 kDa (Fig. 223) and a second protein that is more apparent in experiments shown below, with a molecular mass of 21 kDa. This modification was further characterized by determining whether it was detected when cell extracts were prepared using the alkylating agent iodoacetamide to block free cysteine residues. Addition of iodoacetamide had no effect on the migration of the viral proteins under reducing or nonreducing conditions (Fig. 2), suggesting that the molecule exists as a dimer in vivo.

In order to determine whether dimers were comprised only of 13.7 and 11.3 kDa, or if other viral or cellular proteins formed part of the complex, the viral proteins were analyzed using two-dimensional nonreducing/reducing gel electrophoresis. In addition to analyzing adenovirus-infected cells, we also examined proteins expressed by +crip-10.4 cells, which were transfected with a plasmid containing the 13.7-kDa/ 11.3-kDa coding sequence under regulation of a retroviral long terminal repeat (see "Experimental Procedures'' and Fig. 1). These analyses clearly showed that the 13.7- and 11.3-kDa proteins were the only molecules detected following reduction of the 23.4- and 21-kDa species in transfectants (Fig. 3A), as well as infected cells (Fig. 323).

Cysteine 31 Is Essential for Dimerization of the 13.7- and 11.3-kDa Proteins-Nucleotide sequencing predicts that the 13.7-kDa protein encoded by group C adenovirus-2 has 5 cysteine residues (see Fig. 1OA). In order to determine which cysteine residue(s) is(are) involved in dimerization of the 13.7-

21.6 0

I x 14.41 '23.4 81 3.7 '1 1.3

-I 0.1 M dlthlothreltol + + - -

4 mM lodoacetarnlde - + - + FIG. 2. Analysis of viral proteins resolved by SDS-PAGE

under reducing and nonreducing conditions. N-PLC/PRF/5 cells were infected with a virus mutant which overproduces the viral proteins (in724) and metabolically labeled from 12 to 16 h postinfection. Cell extracts were prepared in the absence (-) or presence (+) of iodoacetamide, and immunoprecipitated using an antiserum specific for the C-terminal 15-mer. Immunoprecipitates were analyzed by SDS-PAGE in the presence ( A ) or absence ( B ) of DTT. Molecular mass determinations were made by linear regression analysis; see legend to Fig. 5 for equation. Molecular mass standards: lysozyme, 14.4 kDa; soybean trypsin inhibitor, 21.5 kDa; carbonic anhydrase, 31 kDa; ovalbumin, 45 kDa; bovine serum albumin, 66.2 kDa.

Non-reducing j

Reducing

L

B

rc13.1 - Cl1.3 FIG. 3. Two-dimensional (nonreducing/reducing) SDS-

PAGE analysis of intermolecular disulfide bond formation. Extracts from metabolically labeled cells were immunoprecipitated using the antiserum specific for the viral proteins, and immunoprecipitates were analyzed under nonreducing conditions in the first dimension, and reducing conditions in the second dimension. A, +- crip-10.4K clone I cells. Reduction of the dimeric species (23.4 kDa/ 21 kDa) was incomplete in this experiment. B, N-PLC/PRF/S cells infected with a virus mutant that overproduces the 13.7- and 11.3- kDa proteins (in724).

13.7-b 11.34

FIG. 4. SDS-PAGE analysis of viral protein expression in N-PLC/PRF/B cells following infection with virus mutants with individually altered cysteine residues. Infected cells were metabolically labeled from 12 to 16 h postinfection; genotypes of virus mutants are indicated. Extracts were immunoprecipitated with the antiserum specific for the C-terminal 15-mer, and immunoprecipitates were analyzed by SDS-PAGE under nonreducing conditions. Sizes of viral proteins are indicated on the left.

and 11.3-kDa proteins, we analyzed virus mutants in which each of the cysteine residues was independently converted to serine by site-directed mutagenesis. Cells infected with these mutants were then screened for the ability to form the 23.4- kDa/Zl-kDa species (Fig. 4). Four of five of these mutants showed the wild-type phenotype; that is, the mobility of the viral protein was 23.4-kDa/21-kDa under nonreducing conditions. The mutant with Cys-31 converted to Ser-31, however, did not form an intermolecular disulfide bond. The additional bands detected in some mutants (i.e. Cys-16 to Ser-16, and Cys-50 to Ser-50) are probably due to instability of mutant proteins. It was concluded from these data that the cysteine residue at position 31 is necessary for the dimerization of the 13.7- and 11.3-kDa proteins.

13.7- and 11.3-kDa Proteins Are Present at the Cell Surface of Infected Cells-We have shown previously that a correlation exists between the extent of EGF-R down-regulation and the level of 13.7- and 11.3-kDa expression (18). These and other data support the hypothesis that there is a direct or indirect interaction between either 13.7- or 11.3-kDa proteins, both of which are integral membrane proteins (2, 19), and EGF-R in the plasma membrane. In order to determine whether it was possible to detect 13.7- or 11.3-kDaproteins at the cell surface,


intact monolayers of adenovirus-infected cells were enzymatically labeled with lZ5I using lactoperoxidase. As seen in Fig. 5A, both 13.7- and 11.3-kDa proteins were specifically immunoprecipitated from cells infected with a virus mutant that overproduces these proteins, but not from cells infected with a virus mutant containing an E3 gene internal deletion. In- terestingly, the 11.3-kDa protein labels to a much greater extent than the 13.7-kDa protein. Although this suggests that the 11.3-kDa protein is the predominant species at the cell surface, preferential labeling of tyrosine residues cannot be ruled out (see Fig. 11, discussed below). Proteins that migrated a t 21 and 23.4 kDa were detected when '251-labeled immunoprecipitates were analyzed under nonreducing conditions (Fig. 5B). What appear to be higher molecular mass aggregates (i.e. 40 kDa) were also detected in nonreducing gels. A tendency for disulfide-linked cell surface proteins to form aggregates has been documented for other proteins, including fibronectin (38). These results indicate that both the 13.7- and 11.3-kDa proteins exist at the cell surface as disulfide-bonded dimers that may tend to form higher molecular mass aggregates stabilized by non-covalent interactions.

Further evidence for cell surface localization was provided using an antiserum made by immunizing Swiss 3T3 mice with +-crip-10.4 transfectants. Since 13.7- and 11.3-kDa proteins were labeled with lZ5I using lactoperoxidase, we reasoned that it might be possible to use intact $-crip-10.4 cells to generate antibodies against a putative extracellular epitope of the 13.7- and 11.3-kDa viral proteins. A similar strategy was used to make antibodies against human chromosome 7 cell surface antigens including EGF-R (29, 39), and human chromosome 6 cell surface antigens (30), by immunizing mice with human- mouse somatic cell hybrids. The cell surface specificity of the anti-$-crip-10.4 antiserum was verified by radioimmunoassay (data not shown). The anti-$-crip-10.4 antiserum specifically immunoprecipitated a protein species from adenovirus-infected cells that comigrated with the 23.4-kDa protein seen in nonreducing gels of immunoprecipitates made with the antibody directed against the C-terminal 15-mer (Fig. 6). Interestingly, the patterns of higher molecular weight complexes seen with these two antisera were different, suggesting that extracellular and C-terminal epitopes may not be equally accessible in the aggregates.

13.7- and 11.3-kDa Proteins Both Appear Rapidly in Pulse- chase Labeling Experiments-Biosynthesis and processing of

t40

66- 46-

0 7

x 31-

3 21.6- r .23.4 -2 1

FIG. 5. Immunoprecipitation of the 13.7- and 11.3-kDa proteins from infected cells labeled by surface iodination. N- PLC/PRF/5 cells were infected with a virus mutant that overproduces (in724) or deletes (dl753) the 13.7- and 11.3-kDa proteins using cyclohexamide from 3 to 7 h postinjection to enhance early viral protein synthesis (63). Monolayers were labeled enzymatically with '"1 at 18 h postinfection, and cell lysates were immunoprecipitated using the antiserum specific for the C-terminal 15-mer. Immunopre- cipitates were analyzed by SDS-PAGE under reducing ( A ) and nonreducing ( B ) conditions. Molecular masses were determined by linear regression analysis, using the equation y = a + ( b ) ( x ) , where a = 4.96077 and b = -0.02465.

A B

66.2- 'Ft 45-

31- -

FIG. 6. Comparison of specificity of antisera directed against different viral protein epitopes. N-PLC/PRF/S cells infected with the virus mutant in724 were metabolically labeled with ~-["S]cysteine. Lysates were immunoprecipitated using a rabbit antiserum directed against the C-terminall5-mer of the viral protein ( A ) or an antiserum from Swiss 3T3 mice immunized with intact rC.-crip- 10.4 cells ( B ) . Immunoprecipitates were analyzed by SDS-PAGE under nonreducing conditions.

C h a s e (min) 0 15 30 60 120 240

66.21 I 451 1 31-

2 1.5-

14.4- - 13.7 I -1 1.3

0, , .>:m*, ... "I

X

e 1 3 . 7 -11.3

N/PLC/PRF/5 cells infected with the virus mutant in724 (A), FIG. 7. Pulse-chase analysis of viral protein biosynthesis in

and $-crip-10.4K clone 1 cells ( B ) . Cells were pulse-labeled with ~-["S]cysteine for 10 min; pulse-labeling of virally infected cells was begun at 12 h postinfection. Cells were then incubated with complete medium supplemented with 500 mM cystine for the periods of time indicated in the figure legend. Lysates were immunoprecipitated with the C-terminal 15-mer-specific antiserum, and proteins were separated by SDS-PAGE using 18% acrylamide under reducing conditions.

the E3-encoded proteins was compared in virally infected cells (Fig. 7A), and $-crip-lO.4 cells (Fig. 7B). Pulse-chase labeling with ~-[~~S]cysteine showed that both proteins were detectable at early time points, with predominant labeling of the 13.7-kDa protein. However, relative abundance cannot be concluded from this analysis, since the 13.7-kDa protein has 5 cysteine residues uersus 3 in the 11.3-kDa protein (see Fig. 1OA). In fact, immunoblot analysis of whole cell lysates indicate that both species are equally abundant (9; see also Fig. 1B). Except for differences in the relative abundance of the two proteins, the pulse-chase patterns seen in transfectants and virally infected cells were identical; the fact that the ratio of 13.7- to 11.3-kDa proteins is greater in the transfectants than in infected cells probably reflects differences in promoter efficiency (i.e. retroviral long terminal repeat in $-crip-10.4 cells versus" E3 promoter in adenovirus-infected cells). Inter- estingly, the 13.7- and 11.3-kDa proteins did not exhibit a conventional precursor-product relation in either infected cells or transfectants, since neither species was entirely converted to the other after a 4- (Fig. 7) or 12-h (data not shown) chase.

These data also showed that neither of the viral proteins

13484 Characterization of Adenovirus Protein That Down-regulates EGF-R

underwent any further change in molecular mass. Similarly, when pulse-chase products were analyzed on nonreducing gels, molecular masses of the 23.4- and 21-kDa proteins, which formed cotranslationally, did not change with time (data not shown). In support of this, the protein has no sites for cotranslational N-linked glycosylation (4-8). Moreover, computer analysis using the PCGENE program revealed a consensus sequence for only one posttranslational modification: Thr-83, which is near the C terminus of the protein (see Fig. lOA), is a potential site for casein kinase 11. Our analyses suggested that this site is not utilized, however, since neither protein was detected when infected cells were labeled with 32P (data not shown). It was also possible that the 13.7- or 11.3-kDa protein was palmitoylated, since some membrane-associated proteins are modified in this manner as early as the ER (reviewed in Ref. 40); increases in molecular mass due to this modification are therefore difficult to detect in pulse-chase analysis. Neither protein, however, could be detected by immunoprecipitation following cell labeling with [9,10-3H]palmitic acid, although the transferrin receptor, known to be palmitoylated (33), yielded positive results (data not shown). It therefore appears that these E3 proteins are not modified co- or posttranslationally, except for disulfide bonding.

Both Viral Proteins Are Synthesized in the Presence of Brefeldin A-Data from pulse chase experiments (Fig. 7) suggested that both forms of the viral protein originate in an early biosynthetic compartment. Presumably, the 11.3-kDa protein is derived cotranslationally from the 13.7-kDa protein, since it is generated by in vitro translation using dog pancreatic microsomes, but not rabbit reticulocyte lysate (9). To provide further evidence that the 13.7- and 11.3-kDa proteins are both derived in the ER, cells were treated with the fungal metabolite brefeldin A, which impedes protein transport from the ER (41) and causes retrograde transport of early Golgi markers (42). We first showed that brefeldin A blocks protein transport from the ER during adenovirus infection by analyzing EGF-R biosynthesis in cells infected with a deletion mutant lacking sequences in the 13.7-kDa-encoding gene. The EGF-R contains 7 to 9 N-linked oligosaccharides (43), and the molecular mass of the receptor shifts from 160 to 170 kDa after oligosaccharide modification upon passage through the Golgi complex (32). Whereas the 170-kDa species was detected in cells following labeling in the absence of brefeldin A, the 160-kDa precursor was the only receptor molecule seen in cells treated with brefeldin A (Fig. 8A). A similar analysis using cells infected with a virus mutant overexpressing the 13.7- and 11.3-kDa proteins showed that brefeldin A did not block expression of either viral protein (Fig. 8B). These results are therefore consistent with the rapid appearance of both proteins in pulse-chase experiments.

13.7- and 11.3-kDa Proteins Focus a t Their Predicted PI Values-The apparent PIS of the 13.7- and 11.3-kDa proteins were determined using NEPHGE. The 13.7-kDa species fo- cused at a PI of approximately 7.4, and the 11.3-kDa protein at a PI of approximately 7.2 (Fig. 9). The spots representing 13.7 and 11.3 kDa are very faint since extraction was carried out using Nonidet P-40 only. Isoelectric focussing using a pH gradient of 6-8 gave identical results (data not shown). These values are in agreement with PIS predicted by the PCGENE program for amino acids 1 through 91 (i.e. 13.7 kDa), and amino acids 23 through 91 (i.e. 11.3 kDa), which were 7.5 and 7.2, respectively. The value derived for the 11.3-kDa protein is therefore consistent with N-terminal sequencing. Since predicted and actual values are virtually identical, these data also support the finding that neither species is modified by addition of charged groups.

P

B 0- 0 4 3 . 7 - - -1.3

FIG. 8. Effect of brefeldin A on biosynthesis of the EGF-R ( A ) and the 13.7- and 11.3-kDa proteins ( B ) . N/PLC/PRF/5 cells were infected with a virus mutant that deletes sequences in the viral gene ( A ) or a virus mutant that overproduces the 13.7- and 11.3- kDa proteins ( B ) . Cells were incubated in cysteine-free MEM supplemented with brefeldin A (5 pg/pl) from 8 to 9 h postinfection. Cells were pulse-labeled with ~-[:''S]cysteine for 30 min and then incubated for an additional 2 h in complete medium supplemented with brefeldin A (5 pg/pl), and 500 mM cystine. Experiments with the same concentration of EtOH used to reconstitute brefeldin A, as well as control experiments with no additions, were carried out in parallel. Cell extracts were immunoprecipitated using EGF-R1 ( A ) or the C-terminal 15-mer antiserum ( B ) ; immunoprecipitates were separated by SDS-PAGE. In A , the species denoted 160K is the EGF- R biosynthetic precursor, and the I70K species is the fully processed EGF-R.

451

3 l 5 21.5

14-41 11.3, ,13.7

FIG. 9. Two-dimensional (NEPHGE/SDS-PAGE) analysis of the 13.7- and 11.3-kDa proteins. fi-crip-lO.4K clone 1 cells were metabolically labeled with ~-["S]cysteine for 4 h. Extracts made using Nonidet P-40 only were immunoprecipitated using the C- terminal 15-mer antiserum. Immunoprecipitates were resolved by NEPHGE in the first dimension and by SDS-PAGE using 15% acrylamide in the second dimension. The pH gradient established during NEPHGE, which was linear from 4.2 to 8.0, is indicated at the top of the figure.

DISCUSSION

Adenovirus-infected cells and cells expressing the E3 gene after transfection or retrovirus-mediated gene transfer syn- thesize a protein referred to as 13.7 kDa from a single open- reading frame. Results from N-terminal sequencing suggest that a second protein, 11.3 kDa, is derived from 13.7 kDa proteolytically by cleavage between residues 22 and 23 (19). This probably occurs cotranslationally, since pulse-chase analysis and experiments with brefeldin A show that the 11.3- kDa protein originates in an early biosynthetic compartment. Moreover, 11.3 kDa is translated in vitro by dog pancreatic microsomes, but not by rabbit reticulocyte lysate (9). It is possible that the N terminus of the 13.7-kDa protein is a signal-anchor sequence that is cleaved by signal peptidase. Although the precise basis of substrate specificity recognition by signal peptidase is unclear, certain structural features of signal peptides do influence the fidelity and efficiency of the enzyme. In general, cleaved signals can be divided into three regions, a charged N-terminal domain of variable length, a central hydrophobic core 12-20 amino acids in length, and a more polar C-terminal region that governs the cleavage site (44, 45). Additionally, the last amino acid preceding the


cleavage is always small and uncharged (46). Examination of the sequence (Fig. 1OA) and hydrophobicity plot (Fig. 10B) for the N-terminal region of the 13.7-kDa protein reveals that it fulfills these requirements, including the presence of a small neutral residue (Ala-22) N-terminal to the site of cleavage. If the 13.7 kDa protein is processed by signal peptidase, it is unclear why cleavage is incomplete. One possibility is that certain structural factors of the viral protein have a negative influence on signal peptidase efficacy (47); alternatively, the viral protein may serve a dual function with independent structural requirements.

Prior experiments have shown that proteins of 13.7 and 11.3 kDa are integral membrane proteins. For example, both proteins partition into the detergent phase following extraction with Triton X-114 (2; also see the inset in Fig. 1OB); moreover, the 11.3- and 13.7-kDa proteins remain membrane- associated after alkaline extraction with Na2C03 to remove peripheral and lumenal proteins (19). Hydrophobicity analysis reveals that there are two strongly hydrophobic regions of sufficient length to span the membrane (Fig. 10B). In addition, three algorithms (48-50) predict independently that the 13.7-kDa protein has two a-helical transmembrane spanning regions, one extending from residues 5 to 25, and a second from residues 40 to 60; the 11.3 kDa species contains only the second a-helix. A model showing the predicted membrane orientation of these helices is shown in Fig. 11. The loop model for signal sequence insertion in the ER predicts that the N terminus of the putative signal-anchor sequence (i.e. H1 in Fig. 1lA) is cytosolic (51-53). The cleavage site that generates 11.3 kDa would therefore face the lumen of the ER, making it accessible for cotranslational processing by signal peptidase (54). In addition, a rule developed by correlating membrane orientation of known eukaryotic proteins with the

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Amino acld number

FIG. 10. A, predicted amino acid sequence for the 13.7-kDa protein encoded by group C adenovirus 2. The putative cleavage site that generates the 11.3-kDa protein is shown by an arrow, and cysteine residues are indicated in oversized type. The residues underlined by dashed lines are homologous with motifs in receptor PTK transmembrane domains, and residues underlined with a solid line show sequence similarity to EGF-R (see text for discussion). B, hydropathy plot for the 13.7-kDa protein encoded by adenovirus 2. The software package PCGENE was used to generate the plot by the Kyte-Doolittle algorithm (64), with computations based on intervals of 9 amino acids. The N terminus is located at amino acid position 1, and the C terminus at amino acid position 91. The inset shows immunoprecipitation of 13.7- and 11.3-kDa proteins from cells infected with wild- type virus and extracted by solubilization with 1% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS, or by partition into a 1% Triton X-114 detergent phase (2).

FIG. 11. A model depicting predicted secondary structure and orientation of the 13.7 kDa ( A ) and 11.3 kDa ( B ) proteins in the lipid bilayer. The cylinders labeled H1 and H2 represent membrane-spanning &-helices. Positions of charged residues (+ and -) that flank H1, Cys-31 (S), and all tyrosine residues ( Y ) are also indicated. Hatched areas show the relative locations of regions homologous with motifs in receptor tyrosine kinase transmembrane domains. Solid areas, C-terminal region of similarity shared with the juxtamembrane domain of EGF-R. The insets show the predicted orientation of two molecules linked by a disulfide intermolecular bond as viewed from above the lipid bilayer. Arrows show positions of putative cleavage sites utilized to generate the 11.3-kDa species. Computer-based predictions of secondary structure were performed using the software package PCGENE.

charge differences of the 15 amino acids flanking the first internal signal-anchor sequence states that proteins with a more negative charge on the C-terminal side relative to the N-terminal side of the signal-anchor sequence will have the orientation depicted in Fig. 1lA (55). The remainder of the C-terminal portion of the molecule is devoid of significant stretches of regular secondary structure, and faces the cytosol (Fig. 11, A and B ) . This aspect of the model is supported by results showing reduced size and loss of C-terminal antigenic determinants following proteinase K digestion of 13.7 kDa expressed in dog pancreatic microsomes (19).

Evidence presented here demonstrates for the first time that both viral species are transported to the plasma membrane, as shown by specific immunoprecipitation following enzymatic labeling of intact monolayers with lZ5I. Interest- ingly, this method preferentially labels the 11.3-kDa protein, whereas immunoblotting of the viral proteins from whole cell lysates indicates that the 11.3- and 13.7-kDa species are equally abundant. Taken together, these results suggest that relatively more of the 11.3-kDa protein is transported to the cell surface, although it is possible that the 2 tyrosine residues in the hydrophilic loop connecting the H1 and H2 helices are exposed to a greater extent at the cell surface in the cleaved molecule (Fig. 11B). Further evidence for plasma membrane localization is provided by the fact that an antiserum produced against intact cells expressing the viral proteins recognizes the same proteins as a C-terminal-specific antibody. Presum- ably the antiserum against intact cells recognizes an epitope in the hydrophilic loop which constitutes the N terminus of 11.3 kDa (Fig. l lB) , and connects H1 and H2 in 13.7 kDa (Fig. 11A). We have also shown that intermolecular disulfide bonds form cotranslationally at Cys-31, giving rise to dimeric species of molecular masses of 21 and 23.4 kDa. The cleavage site that generates the 11.3 kDa protein is to the N-terminal side of Cys-31 (Fig. lU), which explains the fact that both viral proteins are linked by disulfide bonds. Although the precise composition of the multimeric species are unknown, it can be inferred that the 11.3-kDa protein is a component of 23.4 and 21 kDa, since both proteins label to an equivalent extent with '"I, even though 21 kDa is a minor species after labeling with ~-[~~S]cysteine. The apparent molecular mass of a third protein seen in nonreduced gels (i.e. 40 kDa) is consistent with aggregation of disulfide-linked viral proteins. This may be similar to what has been reported for fibronectin,

13486 Characterization of Adenovirus Protein That Down-regulates EGF-R

where high molecular mass complexes are held together by disulfide bonds and noncovalent interactions (38). The composition of these aggregates is currently under investigation.

Interestingly, viral mutants with Cys-31 converted to Ser- 31 still down-regulate EGF-R.* Although this indicates that covalent linkage of viral proteins is not essential for function, it does not rule out the possibility that intermolecular disulfide bridges stabilize multimeric structures formed by noncovalent associations. The transferrin receptor, for example, is normally expressed as a disulfide-linked dimer at the cell surface (33, 34). This modification is not crucial for intermolecular associations, however, since receptors with mutated cysteine residues still form dimers on sucrose density gradients after ligand binding (56). Whether intermolecular interactions between viral proteins are important for down- regulating EGF-R is an interesting question that we are now addressing.

One of the initial events observed during EGF stimulation is formation of receptor oligomeric complexes. We have hy- pothesized that formation of hetero-oligomers between the E3 proteins and EGF-R might account for adenovirus-mediated down-regulation, if there is sufficient structural similarity between the E3 proteins and EGF-R to mimic receptor intermolecular interactions induced by ligand (1, 18). In this regard, it is interesting that a structural motif found in transmembrane domains of a number of receptor tyrosine kinases, including EGF-R, appears twice in the 13.7-kDa protein, extending from residues 19 to 23 and from residues 54 to 58 (marked with dashed lines in Fig. lOA, and shown as hatched boxes in Fig. 1lA); the motif occurs once in the 11.3- kDa species (Fig. 11B). It has been proposed that this motif, which consists of amino acids with a small side chain at position 1 and aliphatic side chains at position 4, and glycine or alanine at position 5 , stabilizes interhelical interactions in ligand-induced oligomeric complexes (57). Perhaps these motifs stabilize noncovalent interactions between viral and host proteins, or alternatively, as discussed above, in high molecular mass aggregates of the E3 proteins. Ligand-mediated receptor oligomerization is probably also dependent on cyto- plasmic determinants. We have previously reported that 18 amino acids near the viral protein C terminus have limited sequence similarity with the juxtamembrane domain of the EGF-R (1; see legends to Fig. 1OA and 11). The prediction that this region is cytosolic and anchored in the plasma membrane is therefore also consistent with the hypothesis that the viral protein acts by mimicing receptor oligomerization. Alternatively, the E3 proteins may act indirectly by binding to other cellular proteins. For example, a recent report suggests that the function of the hydrophobic BPV E5 protein, which activates receptors for EGF, CSF-1 (16), and platelet- derived growth factor (17), is dependent on its association with a 16-kDa component of vacuolar H+-ATPases (58,591.

In summary, the human group C adenovirus E3 region protein that mediates down-regulation of the EGF-R exists as two species in uiuo, of 11.3 and 13.7 kDa, both of which are generated in an early biosynthetic compartment. The viral proteins form a covalent disulfide bond at Cys-31 and are transported to the plasma membrane. Currently being inves- tigated is the idea that higher molecular mass aggregates may form through noncovalent interactions of the viral protein. Further modifications by addition of groups that contribute to charge or molecular mass are not detected. We hypothesize that the N terminus of the 13.7-kDa protein is a signal-anchor sequence that is incompletely cleaved by signal peptidase. Although proper expression of signal-anchor sequences is important for membrane insertion and folding, as well as

intracellular transport (60,61), the fact this region is partially cleaved may suggest that sequences in the 11.3-kDa molecule are sufficient to down-regulate EGF-R. A direct role for the 13.7-kDa species nevertheless cannot be ruled out. Predictions regarding the topology of the viral proteins, as well as putative sites of interaction with other cellular proteins including EGF-R, can now be tested using a molecular genetic approach.

Acknowledgments-We thank our colleagues in the laboratory for many helpful discussions. Premeela Rajakumar and Eric Kuivinen provided excellent technical assistance.

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characterization of the adenovirus e3 protein that down-regulates

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