Oligomerization of the E5 protein of human papillomavirus type 16 occurs through multiple hydrophobic regions

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Virology 313 (2003) 415– 426

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Oligomerization of the E5 protein of human papillomavirus type 16 occurs through multiple hydrophobic regions Christine E. Gieswein,a Frances J. Sharom,b and Alan G. Wildemana,* a

Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada N1G 2W1 b Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received 13 September 2002; returned to author for revision 23 October 2002; accepted 25 March 2003

Abstract The high risk forms of human papillomavirus (HPV) (primarily types 16 and 18) are the leading cause of cervical cancer worldwide. Infection results in expression of three oncoproteins, E5, E6, and E7, the latter two being of predominant importance in maintaining a transformed state of the host epithelial cell. While little is known about the role(s) of the HPV E5, the bovine papillomavirus type 1 (BPV1) E5 protein has been well characterized. A study of HPV16 E5 was performed, focusing on the protein’s ability to self-interact, its ability to bind to the 16-kDa subunit of the vacuolar H⫹-ATPase (16K), and its cellular localization. As has been previously shown for BPV1 E5, we found that HPV16 E5 is also capable of self-interaction and binding to 16K. Further, we examined which portions of the HPV16 E5 protein were involved in these interactions using progressive deletions of putative transmembrane helices of the protein. All of the E5 deletion mutants tested bound to full-length E5 as well as to 16K, suggesting that these protein–protein interactions are based on hydrophobic interactions. The majority of E5 expressed in HEK 293-T7 cells was perinuclear but did not appear to localize to the cis/medial-Golgi, in contrast to previous reports for both HPV16 E5 and BPV1 E5. © 2003 Elsevier Science (USA). All rights reserved. Keywords: HPV16 E5 protein; Oligomerization; Dimerization; 16K subunit; Hydrophobic interactions; Mutations

Introduction The E5 proteins of papillomaviruses studied to date share many features including striking hydrophobicity (Bubb et al., 1988), the ability to bind to the 16-kDa subunit of the vacuolar H⫹-ATPase (16K) (Goldstein and Schlegel, 1990; Conrad et al., 1993), and the ability to stimulate signal transduction pathways (Martin et al., 1989; Crusius et al., 1997). Nevertheless, the activities of human papillomavirus type 16 (HPV16) E5 are less well understood than those of bovine papillomavirus type 1 (BPV1) E5, which is the best characterized of all the papillomavirus E5 proteins. While HPV16 E5 has been shown to cause transformation in murine keratinocytes (Leptak et al., 1991), it is not required

* Corresponding author. Office of the Vice-President (Research), University of Guelph, Guelph, Ontario, Canada N1G 2W1. Fax: ⫹1-519-8371639. E-mail address: [email protected] (A.G. Wildeman).

to maintain the transformed state (Chen et al., 1994) and has thus been labeled a “minor” oncoprotein. Of all papillomaviruses, HPV16 is among the most serious for human health, contributing significantly to the occurrence of many cancers, including those of the cervix. At 44 amino acids, BPV1 E5 is among the smallest transforming proteins known. The primary mechanism by which it induces transformation is believed to be the constitutive activation of the platelet-derived growth factor ␤-chain receptor (PDGF␤-R) in the absence of ligand (Petti et al., 1991; Goldstein et al., 1994). Necessary to this mode of action is the ability of E5 to dimerize. Dimerization is accomplished through the formation of disulfide linkages between cysteine residues in the C-terminus of the BPV1 E5 proteins (Burkhardt et al., 1987) and is assisted by hydrogen bonding between each monomer, mediated by a central glutamine (Gln17), which is the only polar residue located in the hydrophobic domain (Kulke et al., 1992). The end result is a parallel, left-handed, coiled-coil structure that spans the

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membrane (Matoon et al., 2001). When each monomer within the dimer binds a PDGF␤-R monomer, two receptors are brought into close enough contact to initiate transphosphorylation and receptor activation (Lai et al., 1998). The ability of HPV16 E5 to dimerize has been less well characterized. The evidence for dimers has come from electrophoretic mobility studies, which showed that dimer-sized complexes can exist in the presence of a reducing agent (Kell et al., 1994; Hwang et al., 1995). Therefore, disulfide bonds between cysteines (five toward the N-terminus and only one near the C-terminus) are likely not responsible for the putative homodimer. Attempts to elucidate the function of these cysteines have been unsuccessful (Chen et al., 1996; Adam et al., 2000), and simple hydrophobic interactions have been proposed as the primary force driving dimerization (Kell et al., 1994; Mayer and Meyers, 1998). HPV16 E5 can enhance the transforming activity of the PDGF␤-R, but only in the presence of PDGF-B ligand (Leechanichai et al., 1992). Only one study has been able to demonstrate an interaction between HPV16 E5 and this receptor, under conditions of overexpression of both proteins (Hwang et al., 1995). Therefore, the transforming mechanisms of the BPV1 and HPV16 E5 proteins differ in this regard. The two proteins also differ in their mode of binding to 16K. Gln17 of BPV1 E5 forms a hydrogen bond with the glutamate in the fourth transmembrane helix of 16K (TM4) (Andresson et al., 1995). Mutation of several polar residues in the HPV16 E5 sequence did not hinder associations with 16K, although, interestingly, similar to BPV1 E5, the TM4 region of 16K is required for binding to HPV16 E5 (Adam et al., 2000). As with dimerization of HPV16 E5, it was suggested that the 16K–HPV16 E5 interaction relies on the hydrophobicity of the two proteins. Despite differences in the mechanisms of interaction, the association of HPV16 E5 and BPV1 E5 with 16K is believed to have similar consequences in the cell. While the full significance of the 16K interaction is not known, E5 may disrupt V-ATPase activity, leading to changes in receptor half-life by altering trafficking through the endocytic pathway (Straight et al., 1995; Schapiro et al., 2000). Additionally, E5 has been reported to interfere with cell– cell communication by perturbing gap junction complexes (Oelze et al., 1995; Ashrafi et al., 2000), of which 16K is a component. Another reported similarity between HPV16 and BPV1 E5 is their cellular localization. Both have been reported to reside primarily in the Golgi complex, which could be significant in regards to their interactions with 16K and effect on V-ATPase activity (Adam et al., 2000) or the PDGF␤-R (Sparkowski et al., 1996). To date, evidence for dimerization of HPV16 E5 comes only from observations of electrophoretic mobility of the protein. In the present study, experiments have been done to test directly whether HPV16 E5 self-interacts. Using different epitope tags to facilitate immunoprecipitation, evidence is presented to show that E5 complexes do form and that E5

monomers must be translated within the same cell to interact. This was also true of the interaction with 16K. Analysis of the binding of E5 deletion mutants to the wild-type E5 and 16K proteins provided evidence supporting previous suggestions that HPV16 E5 interactions are mediated via hydrophobic forces. Finally, immunofluorescence data suggest that the majority of HPV16 E5 protein expressed in HEK293-T7 cells is not localized to the Golgi complex as has been shown in other studies. Based on the different localization patterns of the various mutants, protein size or degree of hydrophobicity may be a contributing factor to the protein’s localization within the cell.

Results HPV16 E5 can self-interact Two full-length E5 genes were cloned in an identical manner except for the addition of the epitope tags. An HSV-tag was appended to the N-terminus of one clone (E5-HSV), while a T7-tag was similarly appended to a second clone (E5-T7). The tags facilitated extraction and detection of E5 and allowed the differentiation of two monomers of the same protein. These tagged proteins were first tested for a known E5 function, specifically, 16K binding. The proteins were transiently expressed in HEK 293-T7 cells with or without full-length human HSV-tagged 16K (16K-HSV), or a 16K deletion mutant lacking the fourth transmembrane domain (⌬TM4-HSV). The expression of these different proteins was confirmed by Western blot analysis of transfectant lysates (Fig. 1A). E5-T7 was able to coimmunoprecipitate HSV-tagged 16K (Fig. 1B, Lane 2), and vice versa (Fig. 1B, Lane 5). Conversely, an HSV-tagged E5 could coimmunoprecipitate a T7-tagged 16K protein (not shown). The E5-T7 protein was unable to bind to ⌬TM4-HSV (Fig. 1B), consistent with previous studies showing that the fourth transmembrane helix of 16K was necessary for the interaction of E5 and 16K (Adam et al., 2000). Therefore, it was concluded that the epitope tags do not interfere with one of the primary activities of the wild-type HPV16 E5, namely 16K binding. To test for E5 self-interaction, vectors encoding the HSV- and T7-tagged E5 proteins were cotransfected into HEK 293-T7 cells, and lysates were similarly analyzed by coimmunoprecipitation. Immunoprecipitation of E5-T7 pulled down E5-HSV from cell lysates in which these two proteins were coexpressed (Fig. 2A), and the reciprocal experiment showed that E5-HSV could pull down E5-T7 (Fig. 2B). Lanes 5, 6, and 7 show that this interaction is not due to the cross-reactivity of the T7 and HSV antibodies with each other or with E5. These data provide direct evidence that HPV16 E5 self-interacts. Cells were next transfected with only E5-T7, E5-HSV, or 16K-HSV. RIPA lysates from the E5-HSV-transfected cells

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Fig. 1. T7-tagged HPV16 E5 binds HSV-tagged 16K but not the 16K deletion mutant, ⌬TM4. E5-T7, 16K-HSV, and ⌬TM4-HSV were transfected into HEK 293-T7 cells alone or together with either 16K or ⌬TM4-HSV (positive controls) and immunoprecipitated for 1 h. Complexes were recovered with Protein A-Sepharose beads, run on SDS–PAGE, and Western blotted. (A) Determination of protein expression levels in transiently transfected HEK 293-T7 cells. (B) Immunoprecipitation with anti-T7 and immunoblotting with anti-HSV (left), immunoprecipitation with anti-HSV, and immunoblotting with anti-T7 (right). IP ⫽ immunoprecipitate, IB ⫽ immunoblot.

were mixed with equal volumes of lysates from either E5T7- or 16K-T7-transfected cells and immunoprecipitations were carried out for an overnight period. Immunoprecipitations of lysates from cotransfected cells (E5-T7 and either E5-HSV or 16K-HSV) were used as positive controls (Fig. 3, Lanes 6 and 8). Figure 3 shows that E5 cannot interact with 16K or itself (Lanes 7 and 9) if the proteins are not coexpressed in the same cells (Lanes 1 through 3). It is thus doubtful that the observed interactions are in vitro artifacts. Most likely, simultaneous insertion into cellular membranes is an important step for contact between these proteins. Contact between E5 and 16K at this early stage in protein expression and trafficking provides a point at which E5 could disrupt the ATPase complex (as described in Adam et

al., 2000), that is, at the time of complex assembly in the ER. In addition, E5 multimers might be required to accomplish this. These mixing experiments have also been performed by other groups, with similar results, for the E5/16K interaction (Goldstein and Schlegel, 1990; Rodriguez et al., 2000). HPV16 E5 monomers associate via hydrophobic interactions To assess whether specific regions of HPV16 E5 are required for the self-interaction, several T7-tagged deletion mutants were produced based on Kyte–Doolittle hydropathy plots predicting three hydrophobic regions in HPV16 E5,

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Fig. 2. HPV16 E5 can self-interact. E5-T7 and E5-HSV were transfected alone as negative controls or together in HEK 293-T7 cells. Lanes 1 to 4: lysates from transfected cells were run on SDS–PAGE to show the input of protein used in the immunoprecipitation reactions in Lanes 5 through 8. The control in Lane 1 was transfected with empty XJ41 vector. Lysates were immunoprecipitated with either a T7 (A) or A HSV (B), run on 14% SDS–PAGE, transferred to a PVDF membrane, and immunoblotted with a HSV or T7 antibody, respectively. The upper band in Lanes 5 through 8 is a nonspecific band caused by the ␣T7 antibody used to immunoprecipitate. IP ⫽ immunoprecipitate, IB ⫽ immunoblot.

and mutants were made that deleted one or more of them (Fig. 4). Similar mutants have been used in other studies of E5 activity (Adam et al., 2000; Rodriguez et al., 2000). Whether these domains correspond to membrane spanning ␣-helices is as yet unknown. For example, the hydropathy plot of BPV1 E5 predicts two hydrophobic domains but Fourier transform infrared (FTIR) spectroscopy studies suggest that only one transmembrane helix is present (Surti et al., 1998). In addition, there are no obvious loop or turn

sequences between these three domains in HPV16 E5. Nevertheless, although structural predictions based on a variety of algorithms can produce different results, one structure prediction study of HPV16 E5 backed up with FITR spectroscopy of membrane-spanning peptides supported the existence of three helical regions (Ullman et al., 1994). Hydropathy plots of the mutants show that they retain the corresponding hydrophobic domains present in the wildtype protein (Fig. 4).

Fig. 3. Protein complexes containing E5-T7 are produced only when the proteins are cotransfected together into the same cell. HEK 293-T7 cells were transfected with E5-HSV, E5-T7, or 16K-T7 alone (Lanes 1, 2, and 3, respectively), or cotransfected with E5-HSV and either E5-T7 or 16K-T7 (Lanes 4 and 5, respectively). Lysates from cells transfected with E5-HSV alone were added to lysates from cells transfected with either E5-T7 or 16K-T7 alone. These mixed lysates (Lanes 7 and 9) and lysates from cotransfected cells (Lanes 6 and 8) were immunoprecipitated overnight. Complexes recovered with Protein A-Sepharose beads were analyzed by Western blot analysis. (A) Immunoprecipitations were performed with the anti-HSV antibody and blotting was carried out with anti-T7. (B) The reciprocal of A. IP ⫽ immunoprecipitation, IB ⫽ immunoblot, SEP. ⫽ transfected separately.

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Fig. 4. Construction of T7-tagged HPV16 E5 mutant clones and their corresponding Kyte–Doolittle hydropathy plots. The hydropathy plots include the sequence of the two tandem T7-tags (11 amino acids each) at the N-terminal end. The x-axis is the Kyte–Doolittle hydrophobicity value. Plots were produced using a window of nine amino acids. The numbers on the diagrams of the constructs refer specifically to the amino acid number as found in the wild-type HPV 16 E5 sequence and therefore do not match the values on the x-axis of the plot (which includes the additional 22 amino acids of the tags). The full-length T7-tagged HPV 16 E5 amino acid sequence is given at the top of the page. The hyphens separate the sequences of the three putative helices.

The mutants were each cotransfected with the full-length HSV-tagged E5 protein (E5-HSV) and their expression was confirmed by Western blot analysis (Fig. 5, Lanes 1 to 7).

Interactions were tested by coimmunoprecipitations and Western blotting. Full-length E5-HSV was able to pull down each T7-tagged mutant (Fig. 5, Lanes 10 to 13, top),

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Fig. 5. Deletion mutants of HPV16 E5 can still associate with the full-length protein. RIPA lysates were immunoprecipitated with either the HSV or the T7 antibodies and complexes were purified using Protein A-Sepharose-conjugated beads, run on a 14% SDS gel, transferred to a PVDF membrane, and immunoblotted. Lanes 1 to 7: lysates of T7-tagged mutants (A) and wild-type E5-HSV (B) showing input of protein in immunoprecipitation reactions. (A) Lane 2 shows expression of E5-T7 used as a negative control in B, Lane 8. Lanes 10-13: immunoprecipitation of T7-tagged mutants by full-length E5-HSV. (B) Lane 1 shows expression of E5-HSV used as a negative control in A, Lane 8. Lanes 10 to 13: immunoprecipitation of full-length E5-HSV by T7-tagged mutants. Lane 9 contains the positive control consisting of the full-length E5 proteins immunoprecipitating one another. IP ⫽ immunoprecipitation, IB ⫽ immunoblot.

and reciprocally each T7-tagged mutant was able to pull down full-length E5 HSV (Fig. 5, Lanes 10 to 13, bottom). Since no single helix appears to be specifically required for E5 self-interaction, it is likely that the hydrophobicity of each region contributes equally to dimer formation. HPV16 E5 can interact with 16K through hydrophobic interactions The E5 deletion mutants were also tested for their ability to interact with 16K. Each mutant was cotransfected with

16K-HSV into HEK 293-T7 cells, and protein expression was confirmed by Western blots (Fig. 6, Lanes 1 to 7). All E5 proteins were able to pull down 16K-HSV, and reciprocally, 16K was able to pull down the E5 variants (Fig. 6, Lanes 10 to 13, top and bottom, respectively). However, the deletion of TM2 appeared to diminish but not eliminate complex formation, particularly when TM3 was present (Fig. 6, Lanes 11 and 13). These results are consistent with those of Adam et al. (2000), who observed no interaction between the TM2 region of E5 and 16K. Adam et al. also showed that mutation of random, conserved hydrophobic

Fig. 6. Deletion mutants of HPV16 E5 are able to associate with the 16K subunit of the vacuolar H⫹-ATPase. RIPA lysates were immunoprecipitated with either the HSV or the T7 antibodies and complexes were purified using Protein A-Sepharose-conjugated beads, run on a 14% SDS gel, transferred to a PVDF membrane, and immunoblotted. Lanes 1 to 7: lysates of T7-tagged mutants (A) and 16K-HSV (B) showing input of protein in immunoprecipitation reactions. (A) Lane 2 shows expression of E5-T7 used as a negative control in B, Lane 8. Lanes 10-13: immunoprecipitation of T7-tagged mutants by full-length E5-HSV. (B) Lane 1 shows expression of E5-HSV used as a negative control in A, Lane 8. Lanes 10 to 13: immunoprecipitation of full-length 16K-HSV by T7-tagged mutants. Lane 9 contains the positive control consisting of the full-length E5 proteins immunoprecipitating one another. IP ⫽ immunoprecipitation, IB ⫽ immunoblot.

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sequences maintained binding, leading them to suggest that the interaction occurs nonspecifically through hydrophobic regions. Therefore, the conclusion that hydrophobicity is the critical determinant remains the same. In contrast, Rodriguez and colleagues (2000) found that amino acids 54 through 78, the majority of the TM3 region, were required for 16K binding, whereas in the present study, deletion of TM3 did not disrupt complex formation. The reasons for these differences are unclear. Although unlikely, it cannot be ruled out that the 12 amino acids found at the natural N-terminus of E5, which were included in each of our mutants to ensure consistent expression, in fact contribute to an interaction with 16K through a region that protrudes from the membrane. Finally, the glutamine residue inserted during construction of our ⌬TM1 and ⌬TM2 mutants might have contributed to protein interactions similar to the Gln17 of BPV1 E5. However, neither wild-type HPV16 E5, ⌬TM3, or ⌬TM2/3 contain this glutamine, so this seems unlikely. Localization of HPV16 E5 Since membrane localization is potentially important for E5 activity, we used immunofluorescence to look at cellular distribution of E5. The majority of studies, which used various cell lines including SF9, BALB/c, HaCaT, and A431, concluded that both BPV1 and HPV16 E5 localize to the Golgi (Burkhardt et al., 1989; Oetke et al., 2000). Surprisingly, the majority of transiently expressed T7-tagged E5 did not costain with giantin, a cis-medial Golgi marker (Fig. 7). There was only a slight amount of overlap of E5 (green) and giantin (red) immunofluorescent staining (Fig. 7). Much of the full-length wild-type T7-tagged protein was found in large perinuclear aggregates. Although the mutants showed a similar localization, the size of the aggregates decreased with the size of the expressed mutant protein. ⌬TM1 and ⌬TM3 showed a more punctate morphology as if the aggregates were broken up. ⌬TM2, which is closest in size to the wild-type protein, showed a distribution most similar to the wild-type, while the ⌬TM2/3 mutant, the smallest in size, was dispersed throughout the cytosol. HSV-tagged mutants exhibited the immunofluorescence staining pattern (not shown). Transformation by BPV1 E5 has been shown to depend upon localization of the protein to the Golgi (Sparkowski et al., 1995). The hydrophobicity of E5 necessitates that it be located within cellular membranes, but the exact structures involved, whether ER, endosomes, or another compartment, are not known.

Discussion Helical membrane proteins have evolved several mechanisms for homo- and heterointeractions. For example, GCN4 has a leucine zipper motif which uses hydrophobic packing interactions of nonpolar residues to form weak

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homodimeric associations (Zhou et al., 2000). The interaction is stabilized by a single polar residue (Asp) within the membrane helix which forms hydrogen bonds. The BPV1 E5 dimer may similarly form a leucine–zipper-like interaction stabilized by Gln17 (Mattoon et al., 2001). HPV16 E5 contains several leucines, isoleucines, and polar residues which could perform similar roles in interactions with itself and/or 16K. In addition, amino acids with small side chains can be important in van der Waal’s interactions between tightly packed helices in a membrane environment (Eilers et al., 2000), with glycophorin A and M13 coat protein being examples. HPV16 E5 contains several serines, threonines, and alanines which could form polar interactions with adjacent helical backbones. These residues, in addition to cysteines, can also induce helix formation of proteins within membranes (Gray and Matthews, 1984; Gromiha, 1999), providing a potential role for the cysteines found in the HPV16E5 sequence. Overall, the results of the present study suggest that interactions of HPV16 E5 with itself and with other proteins occur primarily through hydrophobic interactions. Hydrogen and electrostatic bonding may, similar to in BPV1 E5, play an additional stabilizing role, as there are several polar residues within the hydrophobic domains. Polar residues within membrane proteins are usually highly conserved and associated with specific functional purposes (Zhou et al., 2001), and introduction of polar residues can produce unintended interactions (Zhou et al., 2000; Smith et al., 1996). Very little structural information about HPV16 E5 is available, and the roles of various amino acids and regions of the protein can only be speculated. Our data suggest that few, if any, specific interactions are occurring. It is interesting that the fourth transmembrane domain of 16K is specifically required for the interaction with HPV16 E5 (Fig. 5) (Adam et al., 2000). Glu139 in this domain is needed for binding of 16K to BPV1 E5, but mutation of this residue did not hinder the association with HPV16 E5 (Adam et al., 2000). This amino acid is lipid-exposed and critical to the function of the entire ATPase pump (Finbow and Harrison, 1997). Although HPV16 E5 does not require Glu139 to associate with 16K, it may interact with 16K in such a way that this residue is nevertheless interfered with. The role of HPV16 E5 complexes in the cell is not known. The primary activity of the BPV1 E5 dimer is to induce ligand-independent activation of the PDGF␤-R by acting as a transmembrane scaffold that creates receptor homodimers (Lai et al., 1998). Interactions between HPV16 E5 and several growth factor receptors, including the PDGF␤-R, EGF-R, CSF1-R, and p185neu, have been observed (Hwang et al., 1995). Attempts to demonstrate that HPV16 E5 can induce ligand-independent activation of receptors have been unsuccessful, although it can enhance the activity of some of them (Pim et al., 1992; Leechanachai et al., 1992; Crusius et al., 1998). Several of the associated signaling pathways are up-regulated in the presence of E5 (Gu and Matlashewski, 1995; Crusius et al., 1997, 1999). It

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is also possible that E5 complexes have a different activity than the monomer. BPV1 E5 binds to the PDGF␤-R as a dimer, whereas it binds 16K as a monomer. Binding to 16K may not be critical to the transforming activity of HPV16 E5. BPV1 E5 mutants unable to bind 16K are still capable of transforming some cell lines (Sparkowski et al., 1995, 1996; Adduci and Schlegel, 1999; Nilson et al., 1995). Both the human and the bovine E5 proteins can activate factors downstream of receptor tyrosine kinases independently of cell-surface signaling (Suprynowicz et al., 2000; Vali et al., 2001; Crusius et al., 1997, 2000), indicating yet other possible mitogenic mechanisms of what is clearly a protein with pleiotropic activities. Recently, HPV16 E5 was found to act independently of growth factors to constitutively activate a PIP2-specific PLC-␥-1 protein, thereby affecting lipid metabolism (Crusius et al., 1999). This may represent yet another consequence of the membrane localization of E5. There is limited information on the cellular localization of HPV16 E5, and our data suggest that in HEK 293-T7 cells little of the protein is in the cis-medial Golgi. Bafilomycin A1 inhibits V-ATPases in the trans-Golgi and immature secretory granules (Schoonderwoert et al., 2000). Since HPV16 E5 is presumed to inhibit V-ATPases through its interaction with 16K, this localization could be important for that function. Sparkowski et al. (1995) suggested that the translocation of BPV1 E5-bound PDGF␤-R out of the cisGolgi/ER was required for transformation, although specific marker proteins were not used. This has been a problem in several studies claiming Golgi, ER, or nuclear membrane localization of E5 (Conrad et al., 1993; Goldstein and Schlegel, 1990; Sparkowski et al., 1996). One study using 58K as a medial-Golgi marker, similar to giantin, revealed that a significant portion of BPV1 E5 was resident in this compartment in A431 and HaCaT cells (Oetke et al., 2000). The use of different cell types could account for some observed differences in E5 localization. It is possible that overexpression of the protein in our system resulted in aberrant localization of E5 and formation of the observed aggregates. These structures were possibly released from the Golgi into the cytosol, without retaining Golgi marker proteins, to prevent “clogging up” of the membranes and to maintain the organelle’s functions. It was suggested by Vali et al. (2001) that BPV1 E5 might form inactive aggregates, since E5-induced activation of phospholipase A2 (PLA2) was lost as the level of E5 expression surpassed a threshold level. Activation of the enzyme peaked when 100 ng of DNA was transfected, but then declined with increasing amounts of DNA. Ten to 20 ␮g of HPV16 E5-containing plasmid was used in our experi-

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ments. Transfection with less than 1 ␮g of DNA did not yield detectable levels of protein, although it may have been enough to activate signaling pathways. It is interesting that the HPV11 E1ˆE4 splice variant protein, considered to be a cytoplasmic protein capable of self-association, exhibited nearly identical immunofluorescence patterns to those we observed for HPV16 E5 (Bryan et al., 1998). Mutants with a decreased ability to dimerize also formed smaller, more punctate aggregates, and it was suggested that larger complexes including cellular factors may be required for the perinuclear localization. Interactions of E5 with cellular factors could also be affected by the deletions of one or more hydrophobic domains, resulting in the formation of smaller protein complexes and a more distributed staining pattern.

Materials and methods Cloning and epitope tagging All HPV16 E5-sequence containing vectors were prepared by amplifying the cDNA (kindly provided by Dr. Daniel DiMaio) from the pMT2-H16E5KC-puro plasmid (Hwang et al., 1995). Either and HSV epitope tag (SQPELAPEDPED), derived from the herpes simplex virus glycoprotein D, or a T& epitope tag (MASMTGGQQMG), derived from the amino-terminal end of the T7 major capsid protein, was added to the N-terminus of the full-length E5. This was done by PCR amplification using an upstream primer encoding the HSV or T7 epitope sequences 5⬘ to a 15 to 18 nucleotide E5 complementary region and a complementary downstream primer. Refer to Tables 1 and 2 for specific primer and cloning information. The primary vector used for all constructs was pXJ41, which uses the human cytomegalovirus promoter to drive expression in mammalian cell lines. This vector also contains a T7 promoter. A modified pXJ41 (T7-XJ41), containing a T7 epitope sequence in the EcoRI-BamHI site, was used for the E5-T7 constructs, thereby providing a second N-terminal T7 tag in addition to the one added during PCR amplification. A second modified pXJ41 vector (XJ41-T7) used for the 16K construct contained a T7 epitope sequence in the BamHIXhoI sites provided a second C-terminal T7 signal. The expression of two juxtaposed T7-tags improved detection of both E5 and 16K. 16K cDNA template, the XJ41-T7 plasmid, and the 16K mutant construct, ⌬TM4-KKO (deletion of residues 129 to 155 of the human 16K sequence), were all kindly provided by D. Mhairi Skinner. All clones were transformed into DH5␣ chemically competent Escherichia

Fig. 7. Immunofluorescence of colocalization of HPV16 E5 and its deletion mutants with giantin. HEK 293-T7 cells were grown on glass coverslips for 24 h and transfected for an additional 24 h. Control cells were transfected with empty XJ41 vector. Cells were then fixed, permeabilized, and blocked in normal goat serum. They were next incubated with anti-T7 primary antibody followed by Alexa 488 labeled secondary antibody for detection. Images were taken on a confocal light microscope using Northern Exposure Software.

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Table 1 Construction of epitope-tagged wild-type and mutant E5 proteins E5 proteins

Upstream primer

Downstream primer

Restriction sites, methods

E5-HSV (2–83)

E5-HSV-UP

E5-DN

E5-T7 (2–83) TM1-T7 (2–12) (38–83)

E5-T7-UP E5-T7-UP TM1-UP

E5-DN TM1-DN E5-DN

TM2-T7 (2–37) (55–83) TM3-T7 (2–55) TM2/3-T7 (2–37) 16K-T7

E5-T7-UP TM2-UP E5-T7-UP E5-T7-UP 16K-UP

TM2-DN E5-DN TM3-DN TM2/3-DN 16K-DN, T7-DN

T-cloned into pGem (T-cloning kit, Promega), then subcloned into NotI and XhoI sites of XJ41 Cloned into BamHI and XhoI sites of T7-XJ41 N-terminal fragment cloned into BamHI and PstI sites of PBS, then this construct used to clone C-terminal fragment into PstI and XhoI sites, then full fragment subcloned into BamHI and XhoI sites of T7-XJ41 (a glutamine residue was inserted as a result of the addition of the PstI site needed to join the two fragments) See TM1-T7 (above)

coli by heat shock, selected for by ampicillin resistance, and stored in 50% v/v glycerol at – 80°C. Cell lines, culture, and transfections The human embryonic kidney cell line HEK 293-T7 (gift from Dr. M. Billeter) was used to express the proteins described above. The “T7” refers to a modification made to the cell line such that it constitutively expresses the T7 polymerase, which enhances the expression from pXJ41. Cells were grown in ␣-modified minimal essential media (␣-MEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C and 5% CO2. Cells were transfected using the calcium phosphate precipitation method. One milliliter of precipitate was added to a 100-mm tissue culture dish for 2 to 4 h. The cells were washed twice with phosphate-buffered saline (PBS) and then incubated overnight. All DNA used in the transfections were prepared using Qiagen Maxi-prep kits, and DNA was resuspended in 0.1 M TE (1 mM Tris, 0.1 mM EDTA, pH 8.0).

Cloned into BamHI and XhoI sites of T7-XJ41 Cloned into BamHI and XhoI sites of T7-XJ41 16K-UP/16K-DN fragment cloned into EcoRI and BamHI sites of XJ41 but no expression so this construct PCR’d with 16K-UP and T7-DN and cloned into EcoRI and BamHI sites of XJ41-T7

Immunoprecipitations and Western blotting Cells were washed in PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% w/v Nonidet P-40 detergent, 0.5% w/v deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0). They were then scraped from the dish with a rubber policeman and transferred to a microfuge tube. The lysates were cleared by centrifugation for 2 min at 19,500 g. Thirty microliters of lysate was removed for analysis by SDS–PAGE and Western blotting. The remaining lysates were brought to 1 ml with RIPA buffer and the appropriate antibody was added for immunoprecipitation. Samples were gently mixed for 1 to 2 h at 4°C. Thirty-five microliters of a 50% slurry of Protein A-conjugated Sepharose beads were added and the samples were mixed for another 30 to 45 min. The beads were recovered by centrifugation at 400 g for 2 min and then washed three times in PBS, resuspended in 2⫻ Laemmli sample buffer (2% SDS, 20% glycerol, 150 mM Tris, pH 6.8, 0.02% w/v bromphenol blue, 200 mM dithiothreitol (DTT)), and heated to 95°C

Table 2 Primer sequences Primer

Sequence

E5-HSV-UP E5-T7-UP E5-DN TM1-DN TM1-UP TM2-DN TM2-UP TM3-DN TM2/3-DN 16K-UP 16K-T7-DN T7-DN

5⬘-CGGAATCCATGAGCCAGCCAGAACTCGCCCCGGAAGACCCCGAGGATACAAATCTTGATACTGCA-3⬘ 5⬘-CGGGATCCATGGCTAGCATGACTGGTGGACAGCAAATGGGTACAAATCTCGATACTGCA-3⬘ 5⬘-CCGCTCGAGTCATTATGTAATTAAAAAGCG-3⬘ 5⬘-AACTGCAGTAATGTTGTGGA-3⬘ 5⬘-AACTGCAGACATACACATCATTA-3⬘ 5⬘-AAAACTGCAGACACAGACAAAAGCAG-3⬘ 5⬘-AAAACTGCAGCGTTTAGGTGTTTTATT-3⬘ 5⬘-CCGCTCGAGTTAAGAGGCTGCTGTTATCCA-3⬘ 5⬘-CCGCTCGAGTTAAGACACAGACAAAAGCAG-3⬘ 5⬘-CGCGAATTCATGTCCGAGTCCAAGA-3⬘ 5⬘-CGGGATCCTCAACCCATTTGCTGTCCACCAGTCATGCTAGCCATCTTTGTGGAGAGGATGAG-3⬘ 5⬘-CGGGATCCACCCATTTGCTGTCCACC-3⬘

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for 10 min. Samples were run on a 13% SDS–polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes (presoaked in methanol) for 1 h in transfer buffer (25 mM Tris base, 0.5 M glycine, 20% v/v methanol). Membranes were then blocked in 5% blocking agent (Amersham) and incubated with primary antibody with mixing for 1 h. Membranes were then incubated with horseradish peroxidase conjugated secondary antibody (Sigma) for 30 min. Five 5-min washes were carried out, and detection was facilitated with Pico Supersignal Chemiluminescence (Pierce). Immunofluorescence HEK 293-T7 cells were plated on glass coverslips and transiently transfected. Cells were then washed twice in PBS and fixed in 4% paraformaldehyde in PBS for 25 min. Another three washes were followed by permeabilization in 0.1% Triton X-100 for 30 min. After three more washes, cells were blocked in 5% v/v normal goat serum (NGS) for 1 h. The coverslips were then placed on parafilm and covered in a dilution of primary antibody (1:7500 for ␣-T7 and 1:1000 for ␣-giantin) in 2% v/v NGS and PBS for 1 to 2 h. The coverslips were washed another three times and incubation of the secondary antibody (1:100 dilution in 2% NGS and PBS) was carried out in the same manner as the primary antibody for 30 min. A final set of three washes was followed by fixing to glass microscope slides in mounting medium (60% v/v glycerol, 0.02% w/v sodium azide in PBS) and viewed under a confocal microscope. Untransfected cells treated with primary antibody served as a negative control. For double labeling, a second set of antibodies (primary and secondary) followed the first set, as described above. Antibodies The anti-T7 and anti-HSV tag monoclonal antibodies were purchased from Novagen and used for immunoprecipitations, immunoblotting, and immunofluorescence. A horseradish peroxidase conjugated rabbit anti-mouse IgG was used as the secondary antibody and purchased from Sigma. For immunofluorescence detection of T7-tagged proteins the goat anti-mouse polyclonal Alexa 488 green fluorescent secondary antibody (Molecular Probes) was used. Rabbit anti-giantin polyclonal antibody was used as a Golgi marker (Covance BabCo). A goat anti-rabbit Texas red conjugated secondary antibody (Molecular Probes) was used for detection of giantin.

Acknowledgments This work was supported by an NSERC-CIHR Collaborative Health Research Grant to A. Wildeman and F. Sharom.

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