E1 protein of bovine papillomavirus type 1 interferes with E2 protein-mediated tethering of the viral DNA to mitotic chromosomes

Abstract

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal plasmids. It is therefore of vital importance for viruses to ensure nuclear retention and proper segregation of their viral DNA. The bovine papillomavirus (BPV) E2 enhancer protein plays a key role in these processes by tethering the viral DNA to the host cell chromosomes. Viral genomes that harbor phosphorylation mutations in the E2 gene are transformation defective, and for these mutant genomes, neither the viral DNA nor the E2 protein is detected on mitotic chromosomes, while other key functions of E2 in transcription and replication were wild type. Moreover, secondary mutations in both the E2 and E1 proteins lead to suppression of the phosphorylation mutant phenotype and resulted in reattachment of the viral DNA and the E2 protein onto mitotic chromosomes, suggesting that E1 also plays a role in viral genome partitioning. The E1 protein was cytologically always excluded from mitotic chromatin, either as a suppressor allele or as the wild type. In the absence of other viral proteins, an E2 protein containing alanine substitutions for phosphorylation substrates in the hinge region (E2-A4) was detected as wild-type on mitotic chromosomes. However, when wild-type E1 protein levels were increased in cells expressing either the A4 mutant E2 proteins or wild-type E2, the E2-A4 protein was much more sensitive to chromosomal dislocation than was the wild-type protein. In contrast, suppressor alleles of E1 were not capable of such abrogation of E2 binding (A4 or wild-type) to chromosomes. These results suggest that wild-type E1 can be a negative regulator of the chromosomal attachment of E2.

FIG. 1.

E2 protein is localized to mitotic chromosomes in suppressor mutant-transformed cells. Cells stably transformed with BPV-1 genomes of different genotypes were obtained via coselection with a Neo^r marker, and E2 proteins were detected by confocal microscopy using a Leica TCSNT confocal laser scanning imaging system. Green, E2 immunofluorescence with monoclonal antibody B201; red, DAPI staining of chromosomal DNA. For the confocal processing, we chose red for the DAPI signal. The upper left panel shows an anaphase mitotic cell from C127 cells transformed with the A3 mutant of BPV. The other panels show E1 suppressor mutants of the A4 genome of BPV at different stages of mitosis (E2-A4/E1 C484F, metaphase plate; E2-A4/E1 F237L, anaphase; and E2-A4/E1 324E, telophase). Mitotic figures from the nontransformed C127 cells did not stain for E2 protein (control). Bar, 10 μm. The E2-A4 mutant genome does not stably transform cells, and the localization of E2 for this allele from intact plasmids can only be assessed through transient assays and has been described previously (11); the protein in the genome context is not associated with mitotic chromosomes.

FIG. 2.

Wild-type and mutant E1 proteins do not associate with mitotic chromosomes. The transformed cell lines described in Fig. 1 were used here. Wild-type and mutant E1 proteins were detected by confocal microscopy using monoclonal antibody BPV104, followed by Cy3-conjugated Ig secondary incubation (green). Red, DAPI staining of chromosomal DNA. Bar, 10 μm. We could not detect E1 by these cytological methods in wild-type BPV-1-transformed cells. Thus, the E2-A3 variant, which transforms stably and shows no segregation defects, served as the wild-type control. Control, nontransformed C127 anaphase cell.

FIG. 3.

PAVA-E2 and PAVA-E1 expressed protein is detected in COS-7 cells by immunofluorescence. COS-7 cells were grown on cover slips and incubated with 5 MOI of virus for 24 h. After fixation, E2 and E1 were detected by immunofluorescence with a Zeiss axioplan fluorescence microscope using monoclonal antibodies B201 and BPV104, respectively, and the DNA was stained with DAPI. As a negative control, mock-infected cells were incubated with B201 or BPV104, followed by the Cy3-conjugated secondary antibody. Bar, 20 μm.

FIG. 4.

E2-A4 protein by itself colocalizes with mitotic chromosomes. COS-7 cells were infected with the recombinant SV40 virus PAVAE2-A4, and after 24 h the E2-A4 protein was detected as described for Fig. 3. An infected anaphase cell is shown by fluorescence microscopy, using a Zeiss axioplan fluorescence microscope. Lower panel, E2-A4 immunofluorescence (yellow); upper panel, DAPI staining (blue). Bar, 10 μm.

FIG. 5.

E1 expression is sufficient for E2 delocalization from mitotic chromosomes. (A) COS-7 cells were grown on cover slips and infected with a constant amount of PAVA-E2 or PAVA-A4 (1 MOI) and increasing amounts of PAVA-E1 (0 to 20 MOI). After 24 h, the cells were fixed, and E2 was detected by immunofluorescence. The panels show representative fields of E2 distribution for the outlined E1/E2 ratios. Right side, E2 immunostaining (yellow); left side, DAPI staining (blue). Bar, 20 μm. (B) Immunoblot analysis of PAVA-E2- and PAVA-E1-infected cell extracts. For detection, both the E1- and E2-specific monoclonal antibodies were used. Shown are extracts derived from COS-7 cell infections using constant amounts of PAVA-E2 (1 MOI) and increasing amounts of PAVA-E1 (0, 5, 10, and 20 MOI) (lanes 1 to 4, respectively), as well as mock-infected extracts (lane 5). At the levels of expression used for these and subsequent experiments, no differences in E2-A4 or wild-type levels were detected (data not shown). Lane 6 shows the level of the E1F237L mutant protein (10 MOI) and E2 (1 MOI). The points at which mutant and wild-type E1 accumulate to the same level as E2 are equivalent. Lanes 7 through 9 show the level of E1 that accumulated at an MOI of 10 for the wild-type, E1-F237L, and E1-K235E alleles in side-by-side infections from total cell extracts. The positions of the E1 and E2 protein bands are indicated on the left side of the panel. (C) Quantitative evaluation of the dislocation of the E2 proteins by wild-type and mutant E1 proteins. For each ratio of E1- and E2-encoding viruses, approximately 60 mitotic cells that were positive for E2 protein were examined for E2 localization. The percentage of mitotic cells that had chromosomally located E2 was plotted against the ratio of the two viruses. Shown are averages and the standard deviation for three experiments for each E1-E2 combination. ⧫, wild-type E1/wild-type E2; ▴, E1-F237L/wild-type E2; ▪, wild-type E1/E2-A4; ∗, E1-K324E/E2-A4; ×, E1-F237L/E2-A4).

FIG. 5.

E1 expression is sufficient for E2 delocalization from mitotic chromosomes. (A) COS-7 cells were grown on cover slips and infected with a constant amount of PAVA-E2 or PAVA-A4 (1 MOI) and increasing amounts of PAVA-E1 (0 to 20 MOI). After 24 h, the cells were fixed, and E2 was detected by immunofluorescence. The panels show representative fields of E2 distribution for the outlined E1/E2 ratios. Right side, E2 immunostaining (yellow); left side, DAPI staining (blue). Bar, 20 μm. (B) Immunoblot analysis of PAVA-E2- and PAVA-E1-infected cell extracts. For detection, both the E1- and E2-specific monoclonal antibodies were used. Shown are extracts derived from COS-7 cell infections using constant amounts of PAVA-E2 (1 MOI) and increasing amounts of PAVA-E1 (0, 5, 10, and 20 MOI) (lanes 1 to 4, respectively), as well as mock-infected extracts (lane 5). At the levels of expression used for these and subsequent experiments, no differences in E2-A4 or wild-type levels were detected (data not shown). Lane 6 shows the level of the E1F237L mutant protein (10 MOI) and E2 (1 MOI). The points at which mutant and wild-type E1 accumulate to the same level as E2 are equivalent. Lanes 7 through 9 show the level of E1 that accumulated at an MOI of 10 for the wild-type, E1-F237L, and E1-K235E alleles in side-by-side infections from total cell extracts. The positions of the E1 and E2 protein bands are indicated on the left side of the panel. (C) Quantitative evaluation of the dislocation of the E2 proteins by wild-type and mutant E1 proteins. For each ratio of E1- and E2-encoding viruses, approximately 60 mitotic cells that were positive for E2 protein were examined for E2 localization. The percentage of mitotic cells that had chromosomally located E2 was plotted against the ratio of the two viruses. Shown are averages and the standard deviation for three experiments for each E1-E2 combination. ⧫, wild-type E1/wild-type E2; ▴, E1-F237L/wild-type E2; ▪, wild-type E1/E2-A4; ∗, E1-K324E/E2-A4; ×, E1-F237L/E2-A4).

FIG. 6.

Coimmunoprecipitation of E2 proteins with E1. Sf9 cells were infected with 1 MOI of baculoviruses encoding either E1 or mutant E1F237L proteins and increasing MOIs of baculoviruses encoding either wild-type E2 or E2-A4 protein. At 48 h postinfection, the cells were lysed, and the extracts were incubated with monoclonal antibody BPV104 coupled to protein G-Sepharose. After three wash cycles, the E2 protein was eluted with 0.4% N-lauroylsarcosine and detected by Western blotting. After quantitation of the bands, the values were normalized for equal E1 protein concentration and plotted against the E1/E2 MOI ratio, with the value of the highest wild-type E1/E2 ratio set at 100%.

FIG. 7.

E1 and E2 are expressed at comparable levels in stably transformed cells. Shown is an immunoblot analysis of nuclear extracts derived from C127 cells that were transformed with the A3 or the E1-suppressor mutant K324E. The location of the E1 E2 proteins is indicated on the left. Lane1: A3-transformed cells; lane 2: E1 suppressor mutant K324E transformed cells; lanes 3-6: increasing amounts (1, 3, 5, 10 ng) of E. coli produced E1; lanes 7-10: increasing amounts (1, 3, 5, 10 ng) of E. coli produced E2.

FIG. 8.

Model for the function of the E1 and E2 proteins in viral replication and nuclear retention of the viral genome during mitosis. E1 (green circles) and E2 (red circle/square) interaction facilitates the origin-specific binding of E1 to the viral DNA. It is believed that E2 must be removed in order for E1 to form double hexamers and perform its unwinding functions on viral origins for replication of the viral genome to occur (19). We propose that a second pool of E1-E2 complexes is present in the nucleus, especially when E1 levels are higher, as in the mutants. An unknown pathway that includes phosphorylation modification of the hinge region of E2 helps to disassemble such complexes. E2 can then bind to the pool of viral DNA that accumulates after S phase and, through interaction of the transactivation domain with a chromosomal receptor, associate with mitotic chromosomes. Phosphorylation of E2 proteins peaks in G₂/M phase (15a). However, we do not know if chromosomally bound E2 is indeed phosphorylated. As the cellular chromosomes separate in mitosis, the tethered viral DNA is also segregated, ensuring that each daughter cell contains a nuclear copy of the viral genome following cell division.