The binding of histone deacetylases and the integrity of zinc finger-like motifs of the E7 protein are essential for the life cycle of human papillomavirus type 31

Abstract

The E7 oncoprotein of high-risk human papillomaviruses (HPVs) binds to and alters the action of cell cycle regulatory proteins such as members of the retinoblastoma (Rb) family of proteins as well as the histone deacetylases (HDACs). To examine the significance of the binding of E7 to HDACs in the viral life cycle, a mutational analysis of the E7 open reading frame was performed in the context of the complete HPV type 31 (HPV-31) genome. Human foreskin keratinocytes were transfected with wild-type HPV-31 genomes or HPV-31 genomes containing mutations in HDAC binding sequences as well as in the C-terminal zinc finger-like domain, and stable cell lines were isolated. All mutant genomes, except those with E7 mutations in the HDAC binding site, were found to be stably maintained extrachromosomally at an early passage following transfection. Upon further passage in culture, genomes containing mutations to the Rb binding domain as well as the zinc finger-like region quickly lost the ability to maintain episomal genomes. Genomes containing mutations abolishing E7 binding to HDACs or to Rb or mutations to the zinc finger-like motifs failed to extend the life span of transfected keratinocytes and caused cells to arrest at the same time as the untransfected keratinocytes. When induced to differentiate by suspension in methylcellulose, cells maintaining genomes with mutations in the Rb binding domain or the zinc finger-like motifs were impaired in their abilities to activate late viral functions. This study demonstrates that the interaction of E7 with HDACs and the integrity of the zinc finger-like motifs are essential for extending the life span of keratinocytes and for stable maintenance of viral genomes.

FIG. 1.

Diagram of the HPV-31 genome and the series of mutations introduced into the E7 open reading frame. The three conserved regions of E7 are indicated as follows: CR1 (light gray); CR2 (horizontal bars); zinc finger-like motif (dark gray). The individual mutations and their approximate locations in the E7 open reading frame are also indicated. The names of the corresponding plasmids containing the mutated HPV-31 genomes are indicated to the right of each mutant E7 protein. Locations of early and late HPV-31 promoters are shown in the top line.

FIG. 2.

Binding of HDAC1, HDAC2, and Rb to wild-type and mutant E7 proteins in GST pull-down assays. GST fusions of wild-type and mutant E7 proteins were incubated with HFK cell extracts, and bound proteins were isolated through the use of glutathione beads and visualized by Western analysis. Each quadrant represents a single GST pull-down experiment with Western analysis for HDAC1, HDAC2, and Rb. Equal amounts of GST fusion proteins were examined, and equal loading was confirmed by Western analysis for GST (data not shown). At the right end of each panel is a lane containing 5% of the total cell extract.

FIG. 3.

Southern analysis of HPV-31 DNA in cell lines transfected with wild-type and mutant HPV-31 genomes at passage 2 after selection. Plasmids used for transfection are indicated above each lane. Lanes designated with the letter “U” include total DNA sheared but digested only with DPN1. Lanes designated by “C” were restricted with DPN1 and XbaI, which cuts the HPV-31 genome once. In several of the “C” lanes, there was incomplete digestion of viral DNA. Copy number standards are indicated to the left of each gel. The lower panel of gels was exposed approximately four times longer than was the upper panel of gels to allow for visualization of low copy numbers of episomes for several HPV-31 mutant genomes. The four forms of HPV-31 DNA found in these analyses are present: supercoiled (I), relaxed circle (II), integrated/multimers (III), and liner (IV). Integrated copies of the L67R genomes are seen more clearly at longer exposures of this gel (data not shown).

FIG. 4.

Average growth rates of keratinocytes transfected with wild-type and mutant HPV-31 genomes after completion of drug selection. The average growth rates were determined at passage 3 following completion of the transfection and drug selection protocol as described in Materials and Methods. The plasmid constructs used in the transfections are indicated below each column. Data shown are the averages of three experiments in two different transfected cell lines.

FIG. 5.

Southern analysis of cell lines transfected with HPV-31 mutant genomes at passage 9 following selection. Data are from a single exposure of the same gel, and copy number standards are shown at the left. Plasmids used for transfection are indicated above each lane. Lanes designated with “U” indicate that total DNA was sheared but digested only with DPN1, which does not cut the genome. Lanes designated with “C” were restricted with DPN1 and XbaI, which cuts the HPV-31 genome once. In several of the “C” lanes, there was incomplete digestion of viral DNA.

FIG. 6.

Amplification of wild-type and mutant HPV-31 genomes following suspension in methylcellulose. Cells transfected with wild-type and mutant genomes at passage 3 following completion of drug selection were incubated in methylcellulose for 24 or 48 h, DNA harvested, and examined by Southern analysis for the levels of viral episomes. The fastest migrating band is supercoiled viral DNA.

FIG. 7.

Stability of wild-type and mutant E7 proteins. Expression vectors for HA-tagged wild-type and mutant E7 proteins were transfected into Cos cells and total cell extracts were harvested after 48 h. (A) Western analysis was performed using an antibody to HA. Two experiments are shown in the left and right panels. Each experiment included a mock transfection and wild-type E7 control. The Western blots were stripped and reprobed with an antibody to GAPDH to confirm equal loading. (B) Quantitation of average stabilities of wild-type and mutant E7 proteins. The average E7 protein levels seen at 72 h after transfection, obtained from two experiments, are shown. E7 protein quantitation was performed by scanning of autoradiographs of gels.