Conditional mutations in the mitotic chromosome binding function of the bovine papillomavirus type 1 E2 protein

Abstract

The papillomavirus E2 protein is required for viral transcriptional regulation, DNA replication and genome segregation. We have previously shown that the E2 transactivator protein and BPV1 genomes are associated with mitotic chromosomes; E2 links the genomes to cellular chromosomes to ensure efficient segregation to daughter nuclei. The transactivation domain of the E2 protein is necessary and sufficient for association of the E2 protein with mitotic chromosomes. To determine which residues of this 200-amino-acid domain are important for chromosomal interaction, E2 proteins with amino acid substitutions in each conserved residue of the transactivation domain were tested for their ability to associate with mitotic chromosomes. Chromatin binding was assessed by using immunofluorescence on both spread and directly fixed mitotic chromosomes. E2 proteins defective in the transactivation and replication functions were unable to associate with chromosomes, and those that were competent in these functions were attached to mitotic chromosomes. However, several mutated proteins that were defective for chromosomal interaction could associate with chromosomes after treatment with agents that promote protein folding or when cells were incubated at lower temperatures. These results indicate that precise folding of the E2 transactivation domain is crucial for its interaction with mitotic chromosomes and that this association can be modulated.

FIG. 1.

(A) Amino acid substitutions in the BPV1 E2 transactivation domain. The phenotype is color coded as described for panel B. (B) The BPV1 E2 transactivation domain was homology modeled with the pdb structure of the HPV16 E2 domain (4). The mutations shown in 1A are indicated on the four views of the domain. Group 1 mutations are shown in red and are completely defective. Group 2 mutations are somewhat functional and are shown in green. The mutations shown in orange have a conditional phenotype as described in the text.

FIG.2.

(A) Comparison of direct-fixation and hypotonic-cytospin fixation methods. (B) The effect of the order of the hypotonic treatment and cytospin centrifugation and length of hypotonic treatment on cellular mitotic chromosomes is shown. HB-1 is composed of 10 mM Tris-HCl, 10 mM NaCl, and 5 mM MgCl₂; HB-2 is composed of 25% PBS. (C) E2 expression was induced and cells fixed by the method indicated. E2 proteins were detected by indirect immunofluorescence with B201 E2-specific antibody. Cellular DNA was detected by propidium iodide staining (PI/DNA), and fluorescein isothiocyanate-labeled E2 protein was detected in the same field of cells (FITC/E2).

FIG. 3.

E2-TA forms speckles on all mitotic chromosomes. The image shown is of the E2-TA protein (green) on mitotic chromosomes (red), as detected by the hypotonic-cytospin immunofluorescence method.

FIG. 4.

Immunofluorescence was carried out as described in Fig. 2C. (A) Group 1 mutated E2 proteins are excluded from mitotic chromosomes with both fixation methods. (B) Group 2 mutated E2 proteins are associated with mitotic chromosomes with both fixation methods. (C) The association of mutated E2 proteins in group 3 with mitotic chromosomes is dependent on the fixation method.

FIG. 5.

(A) Hypotonic treatment induces group 3 E2 proteins to bind mitotic chromosomes. Chromosomal localization of group 3 mutated E2 proteins is related to the time of hypotonic treatment, as shown. (B) W33F E2 could be induced to bind mitotic chromosomes at 32°C or after 30 min of culture in 0.5 M glycerol.

FIG. 6.

(A) Group 3 E2 proteins are temperature sensitive for transactivation. CV-1 cells were electroporated with E2 expression plasmids indicated and an E2-responsive luciferase plasmid and cultured at either 34 or 37°C. Transcriptional activation is shown as a percentage of the wild-type E2 protein activity. (B) Mutated E2 proteins in group 3 are temperature sensitive for replication. Cells were electroporated with replicon DNA, E1 expression vector, and either wild-type E2 expression vector (pC59 vector), mutated E2 expression vector (in pC59 background), or empty C59 vector, as indicated, and then cultured at either 37 or 32°C. Low-molecular-weight, DpnI-resistant replicon DNA was detected by Southern blotting.