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. 1998 Apr 14;95(8):4338-43.
doi: 10.1073/pnas.95.8.4338.

Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation

C W Lehman  1 , M R Botchan
Affiliations

Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation

C W Lehman et al. Proc Natl Acad Sci U S A. .

Abstract

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.

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Figures

Figure 1
Figure 1
Oncogenic transformation by phosphorylation site mutants of BPV. Mouse fibroblast cells (C127) were transfected with 1 μg of linear BPV DNA and fixed and stained after 2 weeks as described (11). Wt is wild-type BPV, A3 has three Ser residues mutated to Ala at phosphorylation sites in E2 (Ser-290, -298, and -301), A4 has the A3 mutations and one additional Ser → Ala change at amino acid 235, D255G and K237E/H357Q are suppressing mutations in E2 and E1 proteins, respectively, which will be discussed later, and sss is a carrier DNA control.
Figure 2
Figure 2
Loss of viral DNA from dividing cells. (A) FISH analysis of colonies of cells cotransfected with BPV DNA and the neomycin-resistance gene. After drug killing of nontransfected cells and growth of colonies for 2 weeks, BPV DNA was detected by the red precipitate and cellular DNA was detected by blue DAPI staining. Wild-type (Wt) and A3 DNAs were detected in nearly all surviving cells. The A3 example shows the edge of two neighboring colonies, all of the rest are from individual colonies. The neor control shows that colonies obtained by single transfection of the drug marker do not give a signal with the BPV probe. A4 DNA was observed in a subset of cells in a range from a few to all within the colony. These examples show sectoring of the colonies. (Bar = 100 μm.) (B) Loss of A4 DNA requires cell division. Cells were transfected, drug-selected, and grown for the same time as in A but were plated under two different conditions. In lanes labeled 4, the cells were plated densely allowing only about four doublings before contact inhibition of further growth. In lanes labeled 15, the cells were plated sparsely and split every 3 days to facilitate logarithmic-phase growth (about 15 doublings). Low molecular weight DNA was isolated from equivalent numbers of cells for each lane and uncut DNA was examined by Southern blotting with random-primed BPV DNA as probe.
Figure 3
Figure 3
Colocalization of BPV DNA with cellular metaphase chromosomes. DNA staining with DAPI revealed mouse chromosomes that showed predominantly acrocentric organization with brightly staining centromeric regions. BPV DNA was detected by FISH. The chromosomes from one mitotic plate are shown in each panel. Intensely colocalizing BPV and DAPI staining produced magenta, which was pseudocolored yellow, and lower-intensity colocalization produced pink. (Right) Magnifications ×4 of a small region of the merged images. The neor control background levels of staining were similar to those shown in Fig. 2A. (Bar = 5 μm.)
Figure 4
Figure 4
Behavior of suppressors in replication and chromosomal localization assays. (A) Graphical representation of the functional domains of E1 and E2 proteins with the suppressor mutations indicated as vertical bars. The positions of known phosphorylated Ser residues in E2 (11, 13) or Ser and Thr residues in E1 (14, 15) are indicated by Ps. (B) One microgram of the genomes containing the indicated mutation was transfected into C127 cells, low molecular weight DNA was harvested at day 6 and examined by Southern blotting using random-primed BPV as probe. The position of EcoRI-linearized, DpnI-resistant full-length BPV is indicated. (C) Suppressors restored metaphase chromosomal localization of the BPV DNA. FISH analysis was performed as in Fig. 3.
Figure 5
Figure 5
Direct evidence for the role of E2 in chromosomal attachment. E2 protein colocalized with mitotic chromosomes, whereas the A4 mutant protein did not. Cells were cotransfected with the neomycin-resistance gene and BPV genomes that do not express the repressor forms of E2 and fixed after drug selection at day 10 (at which point about half of the cells were neor and contained BPV DNA because of the time course of neomycin killing). E2 protein was detected by B201 monoclonal antibody followed by Cy3-conjugated secondary antibody, and DNA was detected by DAPI staining. The B201 antibody recognizes the wild-type and mutant forms of E2 equally, as demonstrated by the equivalent interphase signals obtained (panels labeled Inter and cells labeled with ∗). Arrows indicate the positions of condensed chromosomes in cells at the metaphase or anaphase stages of mitosis. Two metaphase and two anaphase examples are shown for the A3 and A4 samples. Quantitatively, 14 of 26 mitotic cells transfected with the A3 virus contained chromosomal staining, whereas 0 of 17 mitotic cells transfected with the A4 virus showed this colocalization. Interphase nuclei for both A3 and A4 samples stain for E2. Panels labeled Neo show that the E2 reagents do not stain interphase or anaphase nucleoprotein when BPV genomes were not cotransfected.
Figure 6
Figure 6
Proposed model for E1 and E2 function in viral replication and maintenance in the cell cycle. Hypophosphorylated E2 loads E1 monomers onto DNA in G1 phase, facilitating E1 multimerization into the active double hexameric E1 helicase and release of E2 from DNA in S phase. After replication, E1 and E2 bind to viral DNA and E2 can be phosphorylated by a G2/M kinase facilitating chromosomal binding. The DNA can then be accurately partitioned to daughter cells and retained in nuclear structures reformed around the cellular chromosomes in telophase. The oligomeric state of E1 and E2 as attached to the chromosome is hypothetical and based on the findings that show a 1:1 complex between an E2 dimer and an E1 monomer. Suppressor mutations in E1 may arise that do not require E2 phosphorylation because these new E1 proteins create a stronger binding surface for either E2 or the chromosome. To start the cycle anew, E2 can be synthesized de novo or a phosphate can be removed from E2. The E2 and E1 functions in regulating transcription are not included here.
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Comment in

  • Stability without a centromere.
    Calos MP. Calos MP. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4084-5. doi: 10.1073/pnas.95.8.4084. Proc Natl Acad Sci U S A. 1998. PMID: 9539691 Free PMC article. Review. No abstract available.

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