E1 empty set E4 protein of human papillomavirus type 16 associates with mitochondria

Abstract

The human papillomavirus (HPV) E1 empty set E4 protein is the most abundantly expressed viral protein in HPV-infected epithelia. It possesses diverse activities, including the ability to bind to the cytokeratin network and to DEAD-box proteins, and in some cases induces the collapse of the former. E1 empty set E4 is also able to prevent the progression of cells into mitosis by arresting them in the G(2) phase of the cell cycle. In spite of these intriguing properties, the role of this protein in the life cycle of the virus is not clear. Here we report that after binding to and collapsing the cytokeratin network, the HPV type 16 E1 empty set E4 protein binds to mitochondria. When cytokeratin is not present in the cell, E1 empty set E4 appears associated with mitochondria soon after its synthesis. The leucine cluster within the N-terminal portion of the E1 empty set E4 protein is pivotal in mediating this association. After the initial binding to mitochondria, the E1 empty set E4 protein induces the detachment of mitochondria from microtubules, causing the organelles to form a single large cluster adjacent to the nucleus. This is followed by a severe reduction in the mitochondrial membrane potential and an induction of apoptosis. HPV DNA replication and virion production occur in terminally differentiating cells which are keratin-rich, rigid squamae that exfoliate after completion of the differentiation process. Perturbation of the cytokeratin network and the eventual induction of apoptotic properties are processes that could render these unyielding cells more fragile and ease the exit of newly synthesized HPVs for subsequent rounds of infection.

FIG. 1.

Association of HPV16 E1^∧E4 protein with the cytokeratin network. (a to d) HeLa cells were transfected with the MV11E4 vector, and at various times thereafter were fixed and stained as follows: the nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole), the cytokeratin network was stained with antibodies against keratin, actin was stained with rhodamine-conjugated phalloidin, and E1^∧E4 was stained with an Alexa-conjugated anti-E4 antibody. Anti-keratin antibodies were detected with a Cy5-conjugated secondary antibody. The different fluorophores were visualized by using different filters. (e) HeLa cells were transfected with MV11E1^∧E4 and stained as described above after 3 days.

FIG. 2.

HPV16 E1^∧E4 expression in Saos-2 cells lacking cytokeratins. Saos-2 cells were infected with a recombinant adenovirus expressing the E1^∧E4 gene (rAdE1^∧E4). (a and b) Twenty hours after infection, the cells were fixed and stained for nuclei (blue), E1^∧E4 (green), actin (red), and microtubules (also red). (c) Golgi staining. Twenty-four hours after infection, cells were fed wheat germ agglutinin (Molecular Probes) at 1 μg/ml for 15 min, fixed, and processed for immunofluorescence. (d) Lysosome staining. The same procedure was used as that described for panel c, but the cells were fed Lucifer Yellow CH lithium salt (Molecular Probes) at 1 mg/ml. After 1.5 h, they were washed once with fresh medium and incubated in fresh medium without Lucifer Yellow CH for 30 min before being fixed and processed for immunofluorescence. (e) The control vector rAdbeta-galactosidase was used to infect Saos-2 cells at an multiplicity of infection of 100. After 24 h, the cells were fixed and stained with anti-HPV16 E1^∧E4 antibodies conjugated to Alexa488. The nuclei were stained with DAPI.

FIG. 3.

HPV16 E1^∧E4 protein associates with mitochondria. (a) Saos-2 cells were infected with rAdE1^∧E4, and after 24 h the cells were fed Mitotracker Red. Fifteen minutes later, the cells were fixed and stained with Alexa-conjugated anti-E4 antibodies. (b) HeLa cells were transfected with the MV11E1^∧E4 vector, and after 50 h the cells were fed Mitotracker Red and treated as described above for Saos-2 cells. (c) HeLa cells were transfected with MV11E1^∧E4. Two days later, Mitotracker Red was added to the medium for 15 min and the cells were fixed and stained for cytokeratin (pink) and E1^∧E4 (green) by using appropriate antibodies.

FIG. 4.

Colocalization of HPV16 E1^∧E4 protein with mitochondria and cytokeratin in W12 keratinocytes. (a) W12 cells were infected with rAdE1^∧E4 for 24 h, after which Mitotracker Red was added to the cells. The cells were fixed and stained with antibodies against rAdE1^∧E4 (blue) and pan-cytokeratin (green). (b) The same procedure was used as that described for panel a, but different magnifications were used. Cells were stained with antibodies against rAdE1^∧E4 (green) and with Mitotracker Red. Pictures in the bottom row are at a higher magnification than those in the top row. (c) Same as panel b, but showing granular mitochondrial and E4 staining in some W12 cells.

FIG. 5.

HPV16 E1^∧E4-associated mitochondria are displaced from microtubules. Saos-2 cells were infected with rAdE1^∧E4, fixed, and then stained with Alexa-conjugated anti-E4 antibodies 24 h (a) and 32 h (b) after infection. (c) HeLa cells were transfected with MV11E1^∧E4, fed Mitotracker Red, and then stained with Alexa-conjugated anti-E4 antibodies 48 h after transfection. (d) HeLa cells were fed Mitotracker Red for 15 min, fixed, and stained with antibodies against microtubules. Two magnifications are shown. (e) HeLa cells were treated with thymidine, etoposide, or nocodazole for 24 h, after which Mitotracker Red was added to the medium for 15 min prior to fixing and visualization.

FIG. 6.

The leucine cluster of E1^∧E4 is required to mediate its association with mitochondria. (a to e) HeLa cells were transfected with E1^∧E4 mutants in vector MV11. Approximately 60 h later, Mitotracker Red was added for 15 min and the cells fixed and stained with antibodies against keratin (revealed with Cy5-conjugated secondary antibodies) (blue) and Alexa-conjugated antibodies against E1^∧E4 (green). Mitochondria are stained red. (f) HeLa cells were transfected with the MV11LLKLLGFP vector, which expresses GFP fused to the leucine cluster sequence from HPV16 E1^∧E4. Fifty hours later, the cells were fed Mitotracker Red and fixed. LLKLLGFP is stained green and mitochondria are stained red.

FIG. 7.

E1^∧E4 reduces mitochondrial membrane potential. HeLa cells were transfected with MV11E1^∧E4. Forty-eighty hours later, the cells were stained with JC-1 for 20 min, analyzed, and photographed, after which the cells were fixed and stained with Alexa-conjugated antibodies against E4 and DAPI.

FIG. 8.

HPV16 E1^∧E4 induces apoptosis. HeLa cells were transfected with MV11E1^∧E4, and after the indicated times, they were either stained with an anti-annexin V antibody or subjected to a TUNEL assay. After photography, the cells were fixed and stained with Alexa-conjugated antibodies against E4. The results were scored as the percentages of apoptotic cells among the E1^∧E4-expressing cells or of apoptotic cells among control GFP-expressing cells.