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, 85 (21), 10968-75

The Human Papillomavirus Type 16 E5 Oncoprotein Translocates Calpactin I to the Perinuclear Region

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The Human Papillomavirus Type 16 E5 Oncoprotein Translocates Calpactin I to the Perinuclear Region

Ewa Krawczyk et al. J Virol.

Abstract

The human papillomavirus type 16 (HPV-16) E5 oncoprotein is embedded in membranes of the endoplasmic reticulum and nuclear envelope with its C terminus exposed to the cytoplasm. Among other activities, E5 cooperates with the HPV E6 oncoprotein to induce koilocytosis in human cervical cells and keratinocytes in vitro. The effect of E5 on infected cells may rely on its interactions with various cellular proteins. In this study we identify calpactin I, a heterotetrameric, Ca(2+)- and phospholipid-binding protein complex that regulates membrane fusion, as a new cellular target for E5. Both the annexin A2 and p11 subunits of calpactin I coimmunoprecipitate with E5 in COS cells and in human epithelial cell lines, and an intact E5 C terminus is required for binding. Moreover, E5-expressing cells exhibit a perinuclear redistribution of annexin A2 and p11 and show increased fusion of perinuclear membrane vesicles. The C terminus of E5 is required for both the perinuclear redistribution of calpactin I and increased formation of perinuclear vacuoles. These results support the hypothesis that the E5-induced relocalization of calpactin I to the perinuclear region promotes perinuclear membrane fusion, which may underlie the development of koilocytotic vacuoles.

Figures

Fig. 1.
Fig. 1.
Association of E5 and calpactin I in COS cells. (A) A 36-kDa polypeptide that coimmunoprecipitates with E5 was excised from a Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel, digested with trypsin, and microsequenced by ProtTech, Inc. (Norristown, PA). Tryptic peptides showing complete sequence homology with human ANXA2 (NCBI protein database) are framed. (B) Association of ANXA2 with E5. Immunoprecipitates from COS cells transfected with AU1 epitope-tagged full-length E5 and E5 C-terminal deletion mutants were immunoblotted to detect associated ANXA2. The immunoblot was relabeled (lower panel) to demonstrate equivalent levels of the E5 proteins. An arrow indicates the location of ANXA2. (C) Association of E5 with ANXA2. ANXA2 immunoprecipitates from COS cells transfected with the indicated E5 constructs were immunoblotted to detect coprecipitation of E5. The immunoblot was relabeled (middle panel) to demonstrate equivalent levels of ANXA2. An ANXA2 immunoblot of proteins immunoprecipitated with a nonspecific anti-goat IgG antibody (lower panel) demonstrates the specificity of ANXA2 labeling. Arrows indicate the location of ANXA2. (D) Association of ANXA2 and p11 by immunoprecipitation and immunoblotting in COS cells. (E) Association of p11 and E5 in COS cells. Cells were transfected with E5 constructs in the pJS55 expression vector and lysed 24 h later. Molecular mass markers (in kDa) are shown on the left. IP, immunoprecipitation; IB, immunoblotting.
Fig. 2.
Fig. 2.
Association of E5 and calpactin I in HPV-16 E6/E7-immortalized HEC lines. (A) Association of ANXA2 with E5. AU1 immunoprecipitates from stable cell lines expressing AU1 epitope-tagged full-length E5 and E5 C-terminal deletion mutants were immunoblotted to detect associated ANXA2 (upper panel; arrow). The immunoblot was relabeled (lower panel) to show levels of E5 proteins in the immunoprecipitates. (B) Association of p11 with ANXA2 by immunoprecipitation and immunoblotting. (C) Association of p11 and E5 by immunoprecipitation and immunoblotting in lysates prepared using modified CHAPS buffer (23). Molecular mass markers (in kilodaltons) are indicated on the left. IP, immunoprecipitation; IB, immunoblotting.
Fig. 3.
Fig. 3.
Intracellular localization of calpactin I subunits in HPV-16 E6/E7-immortalized HFK cell lines. Immunofluorescence localization of E5 and an E5 deletion mutant lacking 20 C-terminal amino acids are indicated in the left panels (AU1; green). Immunofluorescence localization of ANXA 2 (A) and p11 (B) are shown in red. ANXA2 and p11 are predominantly perinuclear in E5-expressing HFKs, largely mirroring the distribution of E5 (merge). Scale bar, 10 μm.
Fig. 4.
Fig. 4.
E5 promotes perinuclear membrane fusion. (A) HECs that stably express HPV-16 E6 were infected with a retrovirus encoding E5 (or harboring the empty pLXSN expression vector). Three days later, the cells were labeled for 30 min at 4°C with Alexa Fluor 488 (green) or Alexa Fluor 594 (red) conjugates of CTB, washed, and fixed. Similar levels of CTB bind to the plasma membrane of E5- and LXSN-infected cells. (B) E6-HECs were infected with a retrovirus containing the empty pLXSN expression vector and, 3 days later, were pulse-labeled for 30 min (at 37°C) with Alexa Fluor 594 CTB and Alexa Fluor 488 CTB 6 h apart. Small, nonmerged vesicles labeled with Alexa Fluor 594 CTB (red) and Alexa Fluor 488 CTB (green) were present in the perinuclear area in ca. 95% of the cells (merge). Nuclei (blue) and the boundaries of cells (white lines) are indicated. (C) H&E staining of LXSN/E6-HECs reveals morphologically similar nonvacuolated cells. (D) E6-HECs were infected with a retrovirus encoding E5 and pulse-labeled as in panel B. Large perinuclear areas of complete Alexa Fluor 488 CTB and Alexa Fluor 594 CTB colocalization were present in ca. 15% of the cells (merge). (E) H&E staining of E5/E6-HECs shows morphologically similar vacuolated cells. Scale bar, 10 μm.
Fig. 5.
Fig. 5.
siRNA mediated p11 knockdown synergizes with E5 to promote perinuclear vacuole formation. (A) siRNA targeting p11 similarly decreases the level of p11 in HPV-16 E6/E7-immortalized HECs that express full-length E5 or the E5(−20) mutant. Molecular mass markers (in kilodaltons) are indicated on the left. IB, immunoblotting. (B) Measurement of perinuclear vacuolization in H&E-stained E5/E6- and E5(−20)/E6-HECs transfected with p11 or control siRNA. (C) Exclusively perinuclear localization of p11 in E5-HFKs transfected with siRNA targeting p11, but not with control siRNA (p11, red, upper panels). Predominantly diffuse cytoplasmic localization of p11 in E5(−20)-HFKs transfected with control or p11 siRNA (p11, red, lower panels). The boundaries of cells (white lines) are indicated. Scale bar, 10 μm.

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