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. 2009 Feb;83(4):1700-7.
doi: 10.1128/JVI.01971-08. Epub 2008 Dec 3.

Human papillomavirus E7 protein deregulates mitosis via an association with nuclear mitotic apparatus protein 1

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Human papillomavirus E7 protein deregulates mitosis via an association with nuclear mitotic apparatus protein 1

Christine L Nguyen et al. J Virol. 2009 Feb.

Abstract

We previously observed that high-risk human papillomavirus type 16 (HPV16) E7 expression leads to the delocalization of dynein from mitotic spindles (C. L. Nguyen, M. E. McLaughlin-Drubin, and K. Munger, Cancer Res. 68:8715-8722, 2008). Here, we show that HPV16 E7 associates with nuclear mitotic apparatus protein 1 (NuMA) and that NuMA binding and the ability to induce dynein delocalization map to similar carboxyl-terminal sequences of E7. Additionally, we show that the delocalization of dynein from mitotic spindles by HPV16 E7 and the interaction between HPV16 E7 and NuMA correlate with the induction of defects in chromosome alignment during prometaphase even in cells with normal centrosome numbers. Furthermore, low-risk HPV6b and HPV11 E7s also associate with NuMA and also induce a similar mitotic defect. It is possible that the disruption of mitotic events by HPV E7, via targeting of the NuMA/dynein complex and potentially other NuMA-containing complexes, contributes to viral maintenance and propagation potentially through abrogating the differentiation program of the infected epithelium. Furthermore, in concert with activities specific to high-risk HPV E6 and E7, such as the inactivation of the p53 and pRB tumor suppressors, respectively, the disruption of the NuMA/dynein network may result in mitotic errors that would make an infected cell more prone to the accumulation of aneuploidy even in the absence of supernumerary centrosomes.

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Figures

FIG. 1.
FIG. 1.
HPV16 E7 interacts with NuMA via the C terminus of E7. (A) Western blot analysis of IPs performed with either an antibody against HPV16 E7 or an antibody against NuMA (as indicated) in HPV-negative C33a or HPV16-positive CaSki cervical cancer cells. Cells were cross-linked with DSP prior to lysis (left and middle panels), or experiments were performed at room temperature without cross-linking. (Right) IPs performed at room temperature without cross-linking. Blots were probed for NuMA, pRb (as a positive control), or HPV16 E7., nonspecific band. (B) Western blot analysis of GST pull-down experiments using the indicated GST fusion proteins and HeLa whole-cell lysates. Two hundred fifty micrograms HeLa whole-cell lysates represents 5% of the input. The blot was probed for NuMA, pRB (positive control; as expected, the HPV16 E7Δ21-24 mutant is unable to bind pRB), or GST. (C) Western blot analysis of GST pull-down experiments using the indicated GST fusion proteins and HeLa whole-cell lysates next to an anti-dynein IP also performed with HeLa whole-cell lysates. Seventy-five micrograms of HeLa whole-cell lysates represents 3.75% of the input. The blot was probed for NuMA, dynein, or GST.
FIG. 2.
FIG. 2.
HPV16 E7 expression does not alter the interphase localization of NuMA but can delocalize NuMA in mitotic cells with abnormal dynein localizations. (A) Immunofluorescent staining of NuMA (green) in stable HFF interphase cells. Expression levels and nuclear localization of NuMA remain unchanged in the presence of HPV16 E7. (B) Immunofluorescent staining of NuMA (red) and dynein (green) in HPV16 E7-expressing NIH 3T3 (left) or HFF cells (right). Merged images include DNA (blue) visualized using Hoechst 33258 dye. Examples of normal and abnormal NuMA and dynein staining are shown. Similar exposures were used for both cell lines.
FIG. 3.
FIG. 3.
HPV16 E7 expression increases the population of cells with distorted and disorganized metaphases. (A) Examples of normal metaphases in NIH 3T3 cells. Immunofluorescent staining of γ-tubulin as a centrosomal marker is shown in red. DNA was visualized using Hoechst 33258 dye (white). (B) Examples of distorted or disorganized metaphases in NIH 3T3 cells. IF was performed as described above (A). (C) Bar graphs depicting the percentage of disorganized (Disorg.) metaphases in mitotic NIH 3T3 cells stably expressing empty vector (3T3-poz), wild-type HPV16 E7 (3T3-CE7), or the HPV16 E7Δ21-24 or HPV16 E7Δ79-83 mutant (3T3-Δ21-24 or 3T3-Δ79-83); HFF cells with the stable expression of empty vector or wild-type HPV16 E7, HPV16 E7Δ21-24, or HPV16 E7Δ79-83; and NOK and NOK E7 cells. Results represent averages from two (NIH 3T3), three (NOK), or four (HFF) independent experiments where >100 cells were counted per experiment. (D) Bar graph showing the percentage of abnormal metaphases in HEK293 cells transiently transfected with the indicated HPV16 E7 expression plasmids. The results represent averages from two independent experiments where >100 cells were counted per experiment. All error bars indicate the standard errors between experiments.
FIG. 4.
FIG. 4.
Low-risk HPV E7 proteins associate with NuMA and induce disorganized metaphases. (A) Western blot analysis of GST pull-down experiments using the indicated GST fusion proteins and HeLa whole-cell lysates (WCL). One hundred fifty micrograms of HeLa whole-cell lysates represents 5% of the input. The blots were probed for NuMA, pRB (positive control; the binding pattern is as expected), or GST. (B) Bar graph showing the percentage of disorganized (Disorg.) metaphases in HEK293 cells transiently transfected with the indicated E7 expression plasmids. The results represent averages from three independent experiments where >150 cells were counted per experiment. Error bars indicate the standard errors between experiments.
FIG. 5.
FIG. 5.
Expression of HPV16 E7 results in a mitotic delay. (A) Time lapse images of a representative cell going through normal mitosis. DNA was visualized via the expression of green fluorescent protein-histone 2B. Each image was taken after a 6-min interval. (B) Time lapse images of a representative cell going through prolonged mitosis. (C) Cumulative frequency plots of the lengths of mitoses in 3T3-poz cells (diamonds) or in 3T3-CE7 cells (squares). The tables to the right show the numerical values of the cumulative frequency plot, where “minutes” signifies the time to reach anaphase.
FIG. 6.
FIG. 6.
Model of how HPV E7 may be interfering with the activities of microtubule-associated NuMA during mitosis. Association with HPV E7 may subvert mitotic NuMA by rendering it unable to bind to normal partner proteins (“inactive”), including dynein and cargoes of NuMA/dynein complexes. Because NuMA is dynamically exchanged on microtubules, “active” NuMA complexes, including NuMA/dynein complexes, would still be able to eventually associate with microtubules and function successfully, but HPV E7 expression would therefore result in a decreased efficiency of NuMA-dependent mitotic processes (see the text for details).

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