Association of Viral Origin (ori) DNA with Spindles in the Presence of 17b -Estradiol as Revealed by Tyramide-Coupled Fluorescence in Situ Hybridization (T-FISH). If the association of human papillomavirus (HPV) E2 proteins with mitotic spindles provides the mechanism for viral chromosome partitioning during mitosis, the expectation would be that E2 should recruit the viral DNA to the spindles. To test this hypothesis, we transfected COS7 cells with pMTX-H11URR EE-E1/E2, an HPV-11 replicon which contains the 1-kb upstream regulatory region (URR) spanning the ori and the E1/E2 operon under the control of the adenovirus major promoter. A glu-rich epitope-tagged E1 protein is translated from an unspliced transcript, whereas the E2 protein is generated from a message with an E1 intragenic splice (1). Tyramide-coupled T-FISH revealed a colocalization of HPV-11 ori DNA (FITC) with the spindles (Cy-3) (Fig. 6A). In contrast, pMTX-H11URR-EEE1, a plasmid which contains the ori and expresses the EE-E1 protein but not the E2 protein, exhibited no association with the spindles (Fig. 6B).

Methods. pH11URR-Gal4BS containing HPV-11 URR and 40 copies of the Gal4 binding site were prepared as follows: The fragment containing 5 binding sites (BS) of the Gal4 protein was excised from plasmid pB/SGAL4 (2) by digestions with SacI and EcoRI and inserted into the pDsRed2-N1 vector (Clontech). The Gal4BS fragment was reexcised by digestions with SalI and XhoI and polymerized by repeated ligation and recutting with these two enzymes. The digestion products were separated in an agarose gel, and a band of >1 kb was isolated and cloned into the pEGFP-C1 vector at the SalI site (pEGFP-C1-Gal4BS). A clone containing an insertion of 1.3 kb with 40 copies of Gal4BS was selected. pMTX-H11URR-EEE1-E2 was then digested with BamHI to remove the expression cassette of EEE1 and E2 and the SV40 ori. The BamHI vector fragment containing the HPV-11 URR was then ligated to the 1.3-kb Gal4BSD fragment excised with BglII and BamHI digestions from pEGFP-C1-Gal4BS. All clones and mutations were confirmed by restriction digestions or DNA sequence analysis and protein expression by Western blots (data not shown).

pMTX-H11URR-EEE1 and pMTX-H11URR-EEE1-E2 were based on pMTX-H11EE-E1, which contains the EE-epitope tagged HPV-11 E1 (3) in pMTX (4), a derivative of the eukaryotic expression vector pMT2. The HPV11 URR fragment spanning nucleotides 7073–7933/1–99 was excised from the HPV11 ori-containing plasmid pUR23-3 (5) by HindIII, blunt-ended by the Klenow fragment of DNA polymerase I, and then inserted into pMTX-H11EEE1 and pMT2-E2 at StuI to construct pMTX-11URR-EEE1 and pMT2-11URR-E2. To construct pMTX-H11URR-EEE1-E2, we ligated the following three DNA fragments: the BglII–SpeI fragment derived from pMT2-11URR-E2, spanning a C-terminal portion of HPV11 E2, the vector, the HPV-11 URR, the SV40 ori, and the adenovirus major late promoter; the BglII–SphI fragment derived from pMTX-H11EE-E1, spanning an N-terminal portion of HPV-11 EEE1; and the SphI–SpeI fragment of the HPV-11 genome, spanning a contiguous portion E1 and E2 to restore the E1 and E2 genes. The functionality of these URR-containing clones was confirmed by transient replication in 293 cells. The plasmid pMTX-H11URR-EEE1-E2 functions as an autonomous replicon, whereas the pMTX-H11URR-EEE1 replicated only in the presence of a plasmid which supplied the E2 protein (data not shown).

Detection of HPV Ori-Containing Plasmid DNA by T-FISH. Probes were prepared by nick translation of the plasmid pMT2-H11URR-EEE1 by using a biotin Nick Translation Kit (Perkin-Elmer). Transfected cells in chamber slides were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton in 1´ PBS at 4°C, and then treated with 3% hydrogen peroxide in 1´ PBS to quench endogenous peroxidases. Slides were treated with RNase [100 m g/ml RNase A (Sigma), 2´ SSC (1´ SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7)] for 1 h at 37°C and then dehydrated in ethanol, denatured in 70% formamide, 2´ SSC, pH 7, at 72°C for 2 min, again ethanol-dehydrated, and hybridized with the probe in Hybridsol VII (Qbiogene, Carlsbad, CA). After an overnight incubation at 37°C, slides were washed extensively. Slides were next incubated with a 1:100 dilution of streptavidin-horseradish peroxidase in 4´ SSC at 37°C for 30 min and washed. The signals were detected by using a FITC-tyramide (1:100) for 10 min with the amplification diluent according to the supplier’s instructions (Perkin-Elmer). Cells were stained with 4',6-diamidino-2-phenylindole and mounted with Antifade.

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