Modulation of the cell division cycle by human papillomavirus type 18 E4

Abstract

The life cycle of human papillomaviruses (HPVs) is tightly coupled to the differentiation program of their host epithelial cells. HPV E4 gene expression is first observed in the parabasal layers of squamous epithelia, suggesting that the E4 gene product contributes to the mechanism of differentiation-dependent virus replication, although its biological function remains unclear. We analyzed the effect of HPV type 18 E4 on cell proliferation and found that E4 expression induced cell cycle arrest at the G(2)/M boundary. The functional region of E4 necessary for the growth arrest activity was located in the central portion of the molecule, and this activity was independent of the E4-mediated collapse of cytokeratin intermediate filament structures.

FIG. 1.

Growth-inhibitory effect of HPV E4. (A) HeLa cells (2 × 10⁵) were transfected with 2 μg of expression plasmid for 16E4, 18E4, or HIV-1 Vpr. A GFP expression plasmid, pGreenLantern-1 (Cr), was cotransfected to distinguish the transfected cells. GFP-positive cells were counted at the indicated times after transfection. Transfection efficiency was over 80%. The results shown are derived from an experiment performed in triplicate; error bars indicate standard deviations. (B) At 48 h after transfection, cell extracts were prepared with RIPA buffer. Both detergent-soluble (s) and non-detergent-soluble (i) fractions were analyzed by SDS-PAGE, and E4 and Vpr were detected by immunoblot analysis with an anti-FLAG antibody. The two panels represent short (left) and long (right) exposures of the same film. The positions of molecular weight markers (in thousands) are indicated on the left.

FIG. 2.

Cell cycle analysis of E4- and Vpr-expressing cells. (A) HeLa cells were transfected with 2 μg of Vpr or the indicated E4 expression plasmid in combination with pGreenLantern-1. Cells were collected at the indicated times after transfection, and the DNA contents were analyzed by flow cytometry (FACScan). Nuclei were stainedwith PI. Only the population of transfected GFP-positive cells was counted. Closed and open arrows indicate peaks corresponding to G₁ and G₂/M phases, respectively. The ratio of G₂/M to G₁ is shown in each panel. (B) CV1 and C33A cells were transfected with an 18E4 expression plasmid, and the cell cycle profiles were analyzed as described for panel A. Cr, control. (C) Induction of apoptosis by E4 or Vpr expression, as determined by Annexin V staining at 50 h after transfection. The samples were costained with PI for the detection of necrotic cells. Apoptotic cells are positive for Annexin V staining (FL1-H) and negative for PI staining (FL2-H).

FIG. 3.

Morphology of E4-expressing cells. The cells were transfected with 4 μg of an 18E4 expression plasmid, fixed with 50% acetone-methanol at 70 h after transfection, and stained with methylene blue. Nuclei were visualized by DAPI staining. Control cells were transfected with the empty expression plasmid.

FIG. 4.

Identification of the region within the 18E4 protein that is necessary for G₂/M arrest. (A) Schematic representation of the mutant 18E4 proteins tested. The truncated regions of the FLAG-tagged 18E4 protein are represented by black-gray bars. The motifs and functional regions reported for 16E4 are indicated (45). The corresponding regions in 18E4 are represented by gray boxes, and the amino acid sequence alignment of the conserved regions is presented. (B) Steady-state levels of mutant 18E4 proteins expressed in transfected cells. Both detergent-soluble (s) and non-detergent-soluble (i) fractions were analyzed by SDS-PAGE, followed by immunoblot analysis with an anti-FLAG antibody. The two panels represent short (upper) and long (lower) exposures of the same film. The positions of molecular weight markers (in thousands) are indicated on the left. (C) Cell cycle profiles of HeLa cells expressing the indicated 18E4 mutants. Cells were transfected with 4 μg of an E4 expression plasmid and pGreenLantern-1. FACScan analysis was performed as described in the legend to Fig. 2A. Cr, control; WT, wild type.

FIG. 5.

Immunofluorescence analysis of 18E4 proteins and cytokeratin 8/18 in transfected cells. Cells transfected with wild-type, NΔ20, and CΔ70 18E4 expression plasmids (5 μg) were fixed at 48 h after transfection. E4 proteins (left panels) and cytokeratin (CK) (middle panels) were detected by using anti-FLAG and anticytokeratin 8/18 antibodies, respectively. Nuclei (right panels) were visualized by DAPI staining.