The E5 protein of the human papillomavirus type 16 down-regulates HLA-I surface expression in calnexin-expressing but not in calnexin-deficient cells
© Gruener et al; licensee BioMed Central Ltd. 2007
Received: 07 September 2007
Accepted: 30 October 2007
Published: 30 October 2007
The human papillomavirus type 16 E5 protein (HPV16 E5) down-regulates surface expression of HLA-I molecules. The molecular mechanisms underlying this effect are so far unknown. Here we show that HPV16 E5 down-regulates HLA-I surface expression in calnexin-containing but not in calnexin-deficient cells. Immunoprecipitation experiments reveal that calnexin and HPV16E5 can be co-precipitated and that this association depends on the presence of a wild-type first hydrophobic region of E5. When an E5 mutant (M1) in which the first putative transmembrane helix had been disrupted was used for the transfections calnexin-E5 co-precipitation was strongly impaired. In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein. Besides, we demonstrate that E5 forms a ternary complex with calnexin and the heavy chain of HLA-I, which is mediated by the first hydrophobic region of the E5 protein. On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells.
Epidemiological analyses have demonstrated a close association between infection of certain human papillomavirus (HPV) species within the Alphapapillomavirus genus and malignant growth of the human cervix epithelium [1–3], as HPV sequences have been found in virtually all cervical cancers . HPV types associated to cervical cancer are phenomenologically named as "high-risk HPVes", and about 70 % of the HPV sequences isolated from cervical lesions have been identified as being HPV type 16 or 18 [5, 6]. High-risk HPV infection of the stratified epithelium occurs first in the basal cell layer, where transcription of the early genes E5, E6 and E7 takes place [7, 8]. Upon upwards migration towards more superficial layers and concomitant differentiation of the infected keratinocyte, the late genes of the virus are expressed leading to the formation of viral particles and their release upon cell death.
During evolution the arms race between papillomaviruses (PVes) and their hosts has resulted in parallel selection of cellular mechanisms aiming to clear viral infection, such as inhibition of cellular apoptosis or uncoupling of the normal proliferation/differentiation program of the epithelium on the one hand, and in selection of viral mechanisms aiming to hamper cellular reaction directed to clear infection on the other. In this context, several molecular interactions between the oncogenes HPV16 E5, E6 and E7 and different apoptotic pathways have already been identified . E6 and E7 modulate apoptosis by binding and inactivating p53 and the product of tumour suppressor gene Rb1 respectively [10, 11], thereby deregulating the cell cycle. E5 impairs ligand-mediated apoptosis by reducing the amount of surface CD95 proteins or inhibiting the formation of the DISC complex , and affects the normal functioning of a number of membrane associated proteins, probably by modifying the composition and the interactions in the cell membranes . Another mechanism evolved in certain PVes proceeds through down-modulation of the host adaptive immunoresponse. In this context it should be mentioned that whereas antibodies against E6 and against E7 have been found in blood of infected patients [14, 15], no antibodies against E5 have been so far detected [16–18].
Using cellular systems it has been shown that HPV16 E5 expression results in down-regulation of cell surface expression of HLA-I and HLA-II molecules [19–22]. This down-regulation might result in diminished antigen-presentation and decreased adaptive immunoresponse of the host. Interestingly, a reduced expression of HLA-I molecules has also been detected in squamous cell carcinomas of the cervix compared to uninfected epithelium . The decrease in HLA-I surface expression seems to be mediated by a failure in the HLA-complex transport systems to the cell membrane, which accumulate instead in the endoplasmic reticulum [22, 24]. The molecular mechanisms that lead to this impaired intracellular trafficking are unknown. Recently it has been shown that HPV16 E5 may co-precipitate with the heavy chain of HLA-I in cells over-expressing the E5 protein . Nevertheless, no biological evidence has been presented demonstrating that this association is responsible for the down-regulation of HLA-I surface expression. Thus, the intimate mechanisms responsible for the reduced amount of HLA-I molecules at the cell surface remain still elusive.
Calnexin is a chaperone that plays a major role in HLA-I maturation and surface transport [25–27]. Based on the observation that in cervical cancer lesions the expression of calnexin is deregulated , we hypothesyse that this chaperone is involved in the E5-mediated down-regulation of HLA-I surface expression. In this communication we present experimental evidence showing that HPV16 E5 down-regulates cell surface expression of HLA-I in calnexin-expressing but not in calnexin-deficient cells. We further show that E5 associates and co-localizes with calnexin and forms a ternary complex with the heavy chain of HLA-I molecules. Further, we show that E5 mutants unable to bind calnexin fail to down-regulate cell surface expression of HLA-I molecules.
Cells and recombinants
HaCaT, Hela and HEK-293T cells were grown in DMEM (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin/streptomycin. The two subclones of a human T cell leukaemia cell line CEM-C7  and the calnexin-deficient CEM-NKR [30, 31] were grown in RPMI 1640 (Gibco) with 10% heat-inactivated FCS and supplements. The coding region of HPV16 E5, an E5 alpha type protein , containing a HA-tag at the 5-end terminus and was cloned into the pCI vector (Promega) devoid of the starting methionine. Further, an AU1-tagged version of the E5 gene with codon usage adapted to the human relative synonymous codon usage preferences (Accession Number EF463082) was cloned into the pCDNA 3.1(+) vector (Invitrogen). A GFP-E5 fusion recombinant was synthesized by ligating the E5 wild-type coding region to the C-terminal end of the green fluorescence protein gene of the pEGFP vector .
Mutant recombinants were prepared by changing amino acids (QuickChange® Site-Directed Mutagenesis Kit of Stratagene) in order to disrupt the putative transmembrane helix of each of the three domains of the E5 protein [34–36] without altering the length of the protein. All PCR-generated recombinants were confirmed by sequencing. Putative transmembrane domains of the E5 protein and the mutants were analysed using the TMHMM server version 2.0 [37, 38].
Transfections and confocal microscopy
Cells were transfected with Lipofectamine (HaCaT cells) or using the calcium phosphate method (Hela, HEK-293T). CEM-C7 and CEM-NKR cell lines were electroporated using 1×107 cells in 200 μl PBS, 10 μg DNA and setting the pulser to 220 Volt and 960 μFarad (Bio-Rad Gene-Pulser). Transfected CEM-C7 and CEM-NKR clones were selected with 0.8 mg/ml G418. For microscopy, transfected HaCaT cells were grown for 24 hours after transfection and then fixed with 4 % paraformaldehyde. Permeabilized, fixed cells were incubated with anti-AU1 (1:1000, Covance) or anti-calnexin (1:100, Santa Cruz), thoroughly washed and incubated with a secondary antibody labelled either with AlexaFluor® 488 or AlexaFluor® 594 (Molecular Probes). A LEICA laser scanning microscope (LEICA TCS SP) was used in all experiments.
CEM-NKR and CEM-C7 transfectants were lysed with a modified RIPA buffer (150 mM NaCl, 1% NP-40, 0,5% sodium deoxycholate, 0,1% SDS, 1 mM EDTA, 1 mM EGTA, 50 mM Tris-HCl pH 8.0) supplemented with protease inhibitors. HEK-293T and Hela cells were transfected with the corresponding recombinants or with the empty vector. At 20–24 hours post transfection, the cells were lysed with a CHAPS buffer (0.2 M NaCl, 50 mM HEPES pH 7.5, 2% CHAPS) containing phosphatase- and proteinase-inhibitors for 20 min at 4°C. From the cell extracts 0.5 up to 1.5 mg proteins were immunoprecipitated with 2 μg of anti-AU1, anti-HA, anti-GFP or anti-calnexin. Immunoprecipitates were collected with protein G-sepharose, separated on acrylamide gels, blotted onto PVDF membranes and incubated with the appropriate antibodies. Reacting bands were revealed with the Western Lightning™ Chemiluminescence Reagent Plus (Perkin Elmer).
This assay was performed essentially as described  using the glycosylable peptide TNKTRIDGQY labeled with 125I by chloramine-T-catalyzed iodination. Cells were permeabilized with Streptolysin-O (Murex Diagnostics, Dartford, UK). 2 × 106 CEM-C7 or CEM-NKR cells were incubated with peptide and 10 mM ATP in 0.1 ml translocation buffer (130 mM KCl, 10 mM NaCl, 1 mM CaCl2, 2 mM EGTA 2 mM MgCl2, 5 mM HEPES pH 7.3) for 20 min at 37°C. Following lysis in 1% NP-40 (Sigma-Aldrich, Taufkirchen, Germany) the glycosylated peptide fraction was isolated with 30 μl concanavalin A-Sepharose slurry (Amersham-Pharmacia, Freiburg, Germany) and quantified by γ-counting. For control 5.0 mM EDTA was added instead of ATP.
Flow cytometry and antibodies
HEK-293T cells were trypsinised 20 h post-transfection and incubated for 1 h in 37°C CO2-incubator to recover molecules expressed on the surface. CEM-NKR and CEM-C7 transfectants were stained with the HLA-A, B, C-reactive mAbs B9.12 . Secondary antibodies were FITC-conjugated goat anti-mouse IgG (Dianova, 1:100) or PE-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, 1:200). Incubations were performed in Eppendorf tubes for 45 min on ice in the dark, followed by two washes with ice-cold PBS/BSA. Cells were resuspended in 300 μl PBS/BSA and filtered in round-bottom polystyrene tubes (Greiner bio-one). Flow cytometry was performed with a FACSsort (Becton Dickinson).
Analysis of FACS data and Kolmogorov-Smirnov statistics were performed with CellQuest™ software (BD Bioscience). Paired data were analysed with both the Wilcoxon Matched-Pairs Signed-Ranks Test -more conservative- and with the paired Student's t-test -less conservative. Inter-group comparisons were performed with both a Kruskal-Wallis test -more conservative- and with a one-way Analysis Of Variance (ANOVA) -less conservative. Differences below p value of 0.05 were considered significant.
HPV16 E5 decreases surface expression of HLA-I molecules
HPV16 E5 expression reduces cell surface expression of HLA-I molecules in calnexin-expressing but not in calnexin-deficient cells
To test whether this effect simply reflected the presence of different total amounts of HLA-I proteins in the cells, we analysed the total amount of HLA-I molecules in CEM-NKR and CEM-C7 cells by immunoblotting. As shown in Fig. 2D, no major differences in the HLA-I content between CEM-NKR and CEM-C7 cells were found when using total cellular protein extracts from both cell lines (N = 5, pKW = 0.87, Kruskal-Wallis test, pA = 0.77, ANOVA). The E5-mediated reduction in the HLA-I amount at the cell surface was thus not mediated by a lower total cellular content of HLA-I proteins in the CEM-C7 transfectants. These results therefore strongly suggest that E5 affects surface HLA-I expression by a mechanism that involves calnexin.
HPV16 E5 does not influence the transport activity of TAP
HPV16 E5 and calnexin can be co-immunoprecipitated from cellular extracts
An intact hydrophobic region of HPV16 E5 is necessary for binding to calnexin
To analyze the differential involvement of the each of the three E5 transmembrane domains in the interaction between E5 and calnexin, we performed immunoprecipitation experiments with the three mutants M1, M2 and M3 as described above. Protein extracts from transfected cells were immunoprecipitated with antibodies against the AU1 epitope, and the precipitates were analysed for calnexin content by immunoblotting. As shown in Fig. 7B, the original codon-optimized E5 protein and the mutants M2 and M3 co-precipitated calnexin to similar extents, whereas mutant M1 precipitated clearly reduced amounts of calnexin. To discard artefacts due to different inputs of antibody, protein G-sepharose or protein, the experiments were repeated six times. As shown in Fig. 7C mutant M1 co-precipitated calnexin to only 50 % of the levels precipitated by the wild-type and mutants M2 and M3. These results could be reproduced when non-optimised viral E5-coding DNA (pEGFP-HPV16-E5 and pEGFP-M1) was used for transfection instead of the codon-adapted E5-coding DNA (Fig. 7D and 7E). Taken together, these results strongly suggest that the first hydrophobic region of E5, i.e. the first putative transmembrane domain of the protein, is involved in the interaction with calnexin.
Co-localization of HPV16 E5 and calnexin is dependent on the presence of the first hydrophobic domain of E5
The experiments described above indicate that the interacion between E5 and calnexin relies on the presence of an intact first hydrophobic region, and that this binding may be responsible for down-regulation of HLA-I expression. Should this be true, a reduction in co-localization between calnexin and mutant M1 would be expected in immunofluorescence experiments. In order to address this point, HaCaT cells were transfected with the three mutants M1, M2, and M3 and double immunofluorescence with anti-calnexin and anti tag antibodies was performed.
Calnexin, HPV16 E5 and HLA form a trimeric complex
Mutant M1 is not able to down-regulate HLA-I cell surface expression in the same extent that wild type HPV16 E5 does
Taken together, our results strongly indicate i) that E5-mediated down-regulation of HLA-I surface expression proceeds through the formation of a ternary complex between E5, calnexin and the heavy chain of HLA-I; ii) that the disruption of the first transmembrane domain of HPV16 E5 modifies the subcellular distribution of the protein; and iii) that the disruption of the first transmembrane domain of HPV16 E5 prevents the interaction, colocalisation and immunoprecipitation of the viral protein with calnexin, and also of that with the heavy chain of HLA-I.
Eukaryotic cells respond to viral infection by activating mechanisms aiming to abortion of the infection through hindering of viral protein expression, virus maturation or virus release, while viruses have developed during evolution molecular countermeasures to escape from these cellular controls. One of these viral strategies leads to a reduction in the adaptive immunoresponses of the host by reducing the exposure of the infected cells to immune surveillance. Reduced surface expression of HLA-I has been described upon expression of HPV16 E5 or HPV2 E5 proteins [22, 42], but the molecular mechanisms responsible for the decrease of HLA-I on the cell surface have not yet been elucidated. In this report we present experimental evidence demonstrating that HPV16 E5 down-regulates HLA-I surface expression by a calnexin-mediated mechanism. Using transient and stably transfected cells, we have shown that HPV16 E5 is able to reduce HLA-I surface expression in calnexin-containing cells, but not in a calnexin-deficient cell line. Published reports have described that the heavy chain of HLA-I molecules and HPV16 E5 could be co-precipitated , suggesting that this binding might be involved in HLA-I down-regulation. Nevertheless, our results point to the binding of E5 to calnexin as the critical molecular event directly involved in HLA down-regulation. Expression of E5 in CEM-C7 cells, which constitutively express calnexin, results in a decreased amount of HLA-I at the cell surface, but no down-regulation was observed in CEM-NKR cells devoid of calnexin (see Fig. 2C). Since both cell types CEM-C7 and CEM-NKR contain similar amounts of HLA-I molecules (Fig. 2D and see ) it seems unlikely that a putative binding of HPV16 E5 to the HLA-I heavy chain alone could be solely responsible for the decreased surface expression of HLA-I proteins in CEM-C7 cells.
Regarding other viruses, such as herpes simplex virus and cytomegalovirus, it has been shown that they target the transporter associated with antigen processing (TAP) in order to down-regulate HLA-I surface expression [50, 51]. In PVes it has been demonstrated that purified HPV11 E7 protein is able to inhibit ATP-dependent peptide transport into the lumen of the ER in vitro . In this context, our peptide translocation-assay results show that HPV16 E5 does not influence the transport of antigen peptides from the cytosol to the ER. Thus, the data here presented suggest that HPV16 E5 does not target the TAP transporter activity to control surface expression of HLA-I molecules.
Our co-immunoprecipitation experiments using either antibodies against different tagged versions of the E5 protein or against calnexin demonstrate that HPV16 E5 associates with calnexin in vitro. The biological significance of this interaction is further supported by the previously described intracellular co-localization of calnexin and HPV16 E5 , that we confirmed in this report.
Upon interaction between the first and the third hydrophobic segments , HPV16 E5 could be organized as a transmembrane protein with three putative transmembrane helices . In the present work we have introduced specific point mutations in this E5 gene, selectively targeting local hydrophobicity and propensity towards helix conformation in each of the three predicted transmembrane helices of the HPV16 E5 protein . These point mutations result in the selective and individual disruption of each helix without altering the overall length of the protein. Our results reveal that the first hydrophobic helix is mainly responsible for HPV16 E5 subcellular localisation and concomitantly for colocalisation between HPV16 E5 and calnexin. Mutant M1 -with the first putative transmembrane helix being disrupted- was able to bind reduced amounts of calnexin in immunoprecipitation assays, while co-localizing only weakly with calnexin in transfected cells. In addition, M1 transfectants did not down-regulate surface expression of HLA-I in the same extent than wild-type E5. Together both results suggest that i) the first putative transmembrane domain of HPV16 E5 is responsible for the HPV16 E5 localisation; ii) the interaction of HPV16 E5 and calnexin depends on the integrity of the first putative transmembrane domain; iii) the effect of HPV16 E5 on HLA-I surface expression strongly depends on the integrity of the first putative transmembrane domain and on the subsequent interaction between HPV16 E5 and calnexin.
The definitive finding presented here is the existence of a ternary protein complex of HPV16 E5, calnexin, and the heavy chain of HLA-I molecules. The formation of this complex depends on the presence of the first predicted transmembrane domain of HPV16 E5. Since the dimer calnexin-HLA is a natural step in the antigen processing route, it can be hypothesized that HPV16 E5 binds to the calnexin-HLA-I complex and that this binding blocks further trafficking of the HLA-I complex to the plasma membrane, leading instead to its accumulation in the ER/Golgi of the infected cell. A direct binding of E5 to the heavy chain of HLA-I seems under the light of our results improbable. This is further supported by our findings using calnexin-deficient cells lines. Although both cell types, calnexin-containing and calnexin-deficient, express similar amounts of heavy chain HLA-I, the E5-mediated reduction of surface HLA-I becomes evident exclusively in calnexin-containing cells.
The interaction between E5 and calnexin could be demonstrated in cells transfected with the codon-adapted version of the gene, and also in cells transfected with the wild-type gene. This association is therefore independent from the effective amount of E5 protein expressed, and cannot be due to a very large overexpression from the optimised version of the gene. This is not a trivial result, as it has been shown that codon usage optimization can lead to changes in the phenotype associated with protein expression [55, 56].
E5 mutant M1 down-regulates surface expression of HLA-I to a lesser extent than the E5 protein does.
p - value c
p - value c
IGB is the recipient of a grant from the Volkswagen Stiftung under the Thematic Impetus "Evolutionary Biology".
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