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. 2007 Feb;81(4):1736-45.
doi: 10.1128/JVI.01638-06. Epub 2006 Nov 29.

Mitotic kinesin-like protein 2 binds and colocalizes with papillomavirus E2 during mitosis

Ting Yu  1 , Yu-Cai Peng, Elliot J Androphy
Affiliations

Mitotic kinesin-like protein 2 binds and colocalizes with papillomavirus E2 during mitosis

Ting Yu et al. J Virol. 2007 Feb.

Abstract

MKlp2 is a kinesin-like motor protein of the central mitotic spindle required for completion of cytokinesis. Papillomavirus E2 is a sequence specific DNA binding protein that regulates viral transcription and replication and is responsible for partitioning viral episomes into daughter cells during cell division. We demonstrate that MKlp2 specifically associates with the E2 protein during mitosis. Using chromatin immunoprecipitation, we show viral genomes are in complex with MKlp2 only within this stage of cell cycle. By immunofluorescence, a subpopulation of papillomavirus E2 colocalizes with MKlp2 in the midbody/midplate during late mitosis. We conclude that during specific stages of mitosis, the papillomavirus E2 protein binds to MKlp2, and infer that association with this motor protein ensures viral genome partitioning during cytokinesis.

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Figures

FIG. 1.
FIG. 1.
(A) Coimmunoprecipitation of MKlp2 and BPV E2. Extracts of C33A cells cotransfected with Flag-MKlp2 and different E2 expression constructs as indicated were immunoblotted (IB) and probed with anti-E2 monoclonal B201 (panel a) or anti-Flag M2 (panel b). Immunoprecipitates with B201 were immunoblotted and probed with anti-Flag M2 (panel c). E2wt, wild type; BAP, bacterial alkaline phosphatase. (B) Coimmunoprecipitation of MKlp2 and BPV E2 mutants. Extracts of C33A cells cotransfected with Flag-MKlp2 and different expression constructs of E2 mutants as indicated were immunoblotted and probed with anti-E2 monoclonal B201 (panel a) or anti-Flag M2 (panel b). Immunoprecipitates with B201 were immunoblotted and probed with anti-Flag M2 (panel c).
FIG. 2.
FIG. 2.
MKlp2 BPV E2-binding region mapping. Extracts of C33a cells cotransfected with BPV E2 and a series of Flag-tagged MKlp2 fragment expression constructs were immunoblotted and probed by anti-Flag M2. Immunoprecipitations were performed by using B201 or anti-EE (α-EE) monoclonal antibody as a control. HC, heavy chain; LC, light chain.
FIG. 3.
FIG. 3.
Chromatin immunoprecipitation. ID13 cells transfected with Flag-tagged full-length MKlp2 and a series of truncation constructs were used for ChIP. Anti-Flag and B201 antibodies were used as indicated. (A) ChIP PCR results. (B) Corresponding protein expression. (C) Summary of ChIP and BPV E2 binding results. −, negative; +, positive.
FIG. 4.
FIG. 4.
Characterization of anti-MKlp2 antibody. (A) Fifty micrograms of HeLa cell extracts synchronized using a double-thymidine block were blotted and probed by affinity-purified anti-MKlp2. The same cell extracts were probed for cyclin B1 (Sc-245; Santa Cruz) on the bottom panel. S, S phase; M, mitotic cell lysate. (B) Extracts of C33A cells cotransfected with Flag-tagged full-length MKlp2 plus Flag-MKlp2N (aa 1 to 499) or MKlp2C (aa 496 to 886) followed by immunoprecipitation with anti-MKlp2 antibody were blotted and probed with anti-Flag M2.
FIG. 5.
FIG. 5.
HPV E2 protein complex with MKlp2. (A) CV-1 cells with inducible Flag-HPV-11 E2 were transfected with the Flag-MKlp2 expression construct. Four hours before harvesting, expression of HPV-11 E2 was induced by 2 μM CdSO4 in complete medium. Anti-MKlp2 (αMKlp2) and antigen-preabsorbed antisera (Ctrl Ab) were used for immunoprecipitations. Immunoprecipitates were blotted and probed with anti-Flag M2 (α Flag). (B) U2OS cells with or without stably expressed HPV-16 E2 were synchronized using a double-thymidine block. Extracts were blotted and probed with TVG261 (top panel) to demonstrate E2 expression. Anti-MKlp2 and antigen-preabsorbed antisera (Ctrl Ab) were used for immunoprecipitation. Precipitates and cell extracts as input were blotted and probed with anti-MKlp2 and TVG261, respectively. S, S phase; M, mitotic cell lysate.
FIG. 6.
FIG. 6.
Indirect immunofluorescence of MKlp2 and E2 through cell cycles. RPE-1 cells transfected with BPV E2 expression construct were fixed and stained. Each panel represents the specific stage of the cell cycle as indicated. Each column shows a single fluorescent signal corresponding to DNA, α-tubulin, E2b, and MKlp2, with the overlays on the far-right column. Panels E and F show different Z-sections of the same field containing a cell in telophase/cytokinesis. Panels G and H show cells in telophase/cytokinesis with E2 mutant S181F.
FIG. 7.
FIG. 7.
PCR results of ChIP in U2OS cells with the HPV-16 genome. U2OS cells were transfected with HPV-16 genomes and synchronized. Extracts from S-phase (S) and mitotic (M) cells were immunoprecipitated with TVG261, anti-MKlp2, and anti-EE as a control antibody (Ab).
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