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First published online 27 May 2008
doi: 10.1242/jcs.031450

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Herpes simplex virus induces extensive modification and dynamic relocalisation of the nuclear mitotic apparatus (NuMA) protein in interphase cells

Department of Virology, Graduate School of Medicine, Nagoya University, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan

View larger version (38K): [in this window] [in a new window]   Fig. 1. (A) Extensive modification of NuMA in HSV-infected cells. HEp-2 cells infected with HSV-2 or HSV-1 at a MOI of 3 PFU/cell were harvested at the indicated time points and analysed by SDS-PAGE and western blotting with a NuMA polyclonal antibody. The slower migrating band is prominent at later times post infection. (B) NuMA is phosphorylated in HSV-infected cells. Uninfected HEp-2 cells (lanes 1, 2) or HEp-2 cells infected for 17 hours with HSV-2 at a MOI of 5 PFU/cell (lanes 3, 4) were harvested and dephosphorylated with phosphatase (+) or incubated without phosphatase (–). NuMA in HSV-2-infected cells was phosphorylated (lane 3) and phosphatase treatment resulted in a faster migrating band (lane 4). (C) Blockage of viral DNA synthesis by PAA treatment reduces modification of NuMA in HSV-2-infected cells. HEp-2 cells were infected with HSV-2 at a MOI of 5 PFU/cell in the absence (lane 2) or presence (lane 3) of 300 µg/ml PAA and harvested at 17 hours post infection. Mock-infected cells were harvested before infection (lane 1) and also 17 hours later (lane 4). (D) The Cdc2 consensus sites in the C-terminal region of NuMA (Thr2000, Thr2040 and Ser2072) are not modified in HSV-2-infected cells. HEp-2 cells were transfected with pFLAG-NuMAwt and pFLAG-NuMAcdc2 and 24 hours later were mock-infected (lanes 1, 3) or infected with HSV-2 at a MOI of 3 PFU/cell for 15 hours (lanes 2, 4). The cells were harvested and analysed by SDS-PAGE and western blotting with an anti-FLAG monoclonal antibody. The motility of the bands in lanes 2 and 4 are nearly identical, suggesting that NuMA was not modified at the Cdc2 sites.

View larger version (41K): [in this window] [in a new window]   Fig. 2. (A-F) NuMA is relocalised in HSV-1-infected cells. HEp-2 cells were either mock-infected (A) or infected with HSV-1 at a MOI of 3 PFU/cell, fixed at 3 hours (B), 6 hours (C), 9 hours (D), 12 (E) and 15 hours (F) post infection, and then analysed for NuMA localisation. NuMA diminuition in the centre of the nucleus can be observed from 9 hours post infection (D). The percentage of cells exhibiting such nuclear localisation of NuMA (arrow in panel D) were quantified by examining at least 200 cells (given at bottom left within each panel).

View larger version (82K): [in this window] [in a new window]   Fig. 3. (A-I) Comparison of the localisation of NuMA and lamin A/C. In mock-infected HEp-2 cells, NuMA was diffusely distributed throughout the nucleus excluding nucleoli (A) and surrounded by the nuclear membrane protein, lamin A/C (B,C). In cells infected for 15 hours with HSV-1, NuMA formed a ring-like structure similar to that of lamin A/C (D-F). However, magnification of the inner rims of the nucleus showed that the two proteins did not necessarily colocalise (G-I) and that NuMA was located on the inner nuclear side of lamin A/C.

View larger version (95K): [in this window] [in a new window]   Fig. 4. NuMA and its relation to viral proteins and replication compartments. (A-C) HEp-2 cells were infected with 14R-VP16G at a MOI of 1 PFU/cell. Cells were fixed at 8 hours post infection and analysed for VP16-GFP and NuMA by indirect immunofluorescence. VP16-GFP (A) and NuMA (B) localised almost exclusively of each other. Note the difference in NuMA localisation between an uninfected cell (arrowhead) and an infected cell (arrow) (B). (D-G) Further analysis with triple fluorescence of VP16-GFP (D), UL17 (a tegument protein) (E) and NuMA (F) showed that, whereas the two viral proteins colocalised in the nucleus, NuMA localisation excluded these proteins. Analysis of NuMA and ICP8 localisation in mock-infected (H) and infected cells (I-K). Cells were infected with HSV-1 at a MOI of 3 PFU/cell and then fixed at 12 hours post infection. NuMA and ICP8 (a marker for replication compartments) exhibited mutually exclusive localisation.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Solubilisation of NuMA by HSV infection. HEp-2 cells (A,B) or HEp-2 cells transiently expressing NuMA-FLAG (C,D) were either mock-infected or infected with HSV-1 at a MOI of 3 PFU/cell. Twenty hours later, the cells were pre-extracted with 0.1% Triton X-100, fixed, and analysed by indirect immunofluorescence using an anti-NuMA polyclonal antibody (A,B) or anti-FLAG monoclonal antibody (C,D). In mock-infected cells, both NuMA proteins were resistant to extraction with Triton X-100 (A,C). In infected cells, the majority of NuMA and NuMA-FLAG were extracted except for that in the inner nuclear rims and subtle intranuclear foci (B,D), suggesting that NuMA was extensively solubilised. (E) NuMA is solubilised in infected cells and solubilisation is suppressed by PAA. HEp-2 cells were either mock infected (lanes 1, 2) or infected with HSV-2 at a MOI of 3 PFU/cell in the absence (lanes 3, 4) or presence (lanes 5, 6) of PAA. At 15 hours post infection, cells were fractionated, and resulting insoluble (P) and soluble (S) fractions were subjected to SDS-PAGE and western blotting. The blot was detected for NuMA, the major capsid protein VP5, lamin A/C and calnexin (loading control). The soluble fraction of NuMA and lamin A/C increased upon infection. Inhibition of viral DNA synthesis had a suppressive effect on the solubilisation of these proteins. VP5 expression was greatly reduced upon PAA treatment.

View larger version (58K): [in this window] [in a new window]   Fig. 6. Live cell, time-lapse photographs of Vero cells transiently expressing RFP-NuMA and infected with F-VP26/GFP at a MOI of 5 PFU/cell (5-12 hours post infection). Individual panels were projected from 20-25 z-axes, confocal sections to represent the total fluorescence in the cell. RFP-NuMA began to decrease in the central parts of the nucleus where the initial accumulation of VP26-GFP was observed (A,B). The solid-lined arrows in B3 and C3 show foci of potential primary enveloped capsids whose egress into the cytoplasm was accompanied by a contortion of the nucleus (C2,D2,E2: dotted-lined arrows) and a pushing-out of the nucleus into the direction of the cytoplasm (C2,D2: fine dotted-lined arrows). Replication compartments (RC, E1) formed most readily in places comparatively void of RFP-NuMA (E3,F3). At late times post infection, the overall RFP-NuMA distribution did not change and remained mostly in a ring-like pattern (G2) while VP26-GFP was dispersed in the cytoplasm (G1). At times later than 15 hours, VP26-GFP in progeny virions was found also in the extracellular space (see supplementary material Movie 1).

View larger version (57K): [in this window] [in a new window]   Fig. 7. The hollowing of NuMA is blocked by PAA. HEp-2 cells mock infected (A) or infected with HSV-1 in the absence (E) or presence (I) of PAA were pre-extracted with 0.1% Triton X-100 prior to fixation at 12 hours post infection. hollowing of NuMA was observed in normal, infected cells (arrow) (E). In cells infected in the presence of PAA, the localisation of NuMA (I) was the same as that in mock-infected cells (A), suggesting that viral DNA synthesis was important for the relocalisation and solubilisation of NuMA. In Vero cells that were transiently transfected with RFP-NuMA, infected with F-VP26/GFP, and then fixed at 7 hours post infection, the centre of the nucleus (where VP26-GFP had accumulated) lacked RFP-NuMA (F-H). In PAA-treated infected cells, however, the pattern of RFP-NuMA was similar to uninfected cells (B-D) at 7 hours post infection (data not shown). Even at 20 hours post infection, most of RFP-NuMA remained in the central parts of the nucleus (J-L).

View larger version (73K): [in this window] [in a new window]   Fig. 8. Disruption of G-actin or F-actin does not affect the exclusion of NuMA from replication compartments. HEp-2 cells were infected with HSV-1 at a MOI of 3 PFU/cell and after 1 hour, cultured in the presence of the solvent DMSO (A-D), cytochalasin D (E-H) or latrunculin A (I-L). At 10 hours post infection, the cells were fixed and analysed for NuMA or NuMA/ICP8 localisation. DMSO-treated cells showed the typical decrease in NuMA inside the nucleus as shown by the arrows (A-C) and its exclusion from replication compartments (D). Cytochalasin D, which disrupts F-actin, slightly affected nuclear morphology but exhibited the representative NuMA pattern as shown by the arrows (E-G,H). Latrunculin A, which inhibits G-actin, affected the size of both replication compartments and NuMA-void areas (I-K,L). The arrows in panel I show that decrease of NuMA fluorescence was constricted to much smaller areas. The percentage of cells exhibiting such NuMA relocalisation were quantified (in at least 200 cells) and shown in panels A,E,I.

View larger version (29K): [in this window] [in a new window]   Fig. 9. NuMA knockdown using siRNA results in an HSV growth defect. (A) siNuMA-, siCont- or mock-transfected HEp-2 cells were analysed 3 days post-transfection for protein expression. NuMA levels were greatly decreased in siNuMA-transfected cells, whereas levels of lamin B and actin were constant. (B-G) Analysis of viral cell-to-cell growth in siRNA-treated cells. HEp-2 cells transfected with siCont (B,C,D) or siNuMA (E,F,G) were infected with 14R-VP16G at a MOI of 0.001 PFU/cell, fixed at 48 hours post infection, and examined for VP16-GFP (B,E). DIC (C,F) and merged images (D,G) show that cell-to-cell viral spread was considerably affected in siNuMA-transfected cells and that this was not a consequence of low cell density. (H) Comparison of 14R-VP16G plaque diameters in siCont- and siNuMA-transfected cells. Cells were fixed at 48 hours post infection and 30 randomly chosen plaques were scanned with the confocal microscope followed by measurement of plaque diameter with the Zeiss LSM software. Error bars represent standard deviations. (I) A multi-step growth curve of HSV-1 in siRNA-treated HEp-2 cells. The results shown are representative of three independent experiments. After similar treatment with siRNA, HEp-2 cells were infected with HSV-1 at a MOI of 0.01 PFU/cell. Cells were harvested at various times post infection to obtain viral growth curves and the resulting viral yield was normalised to per 106 cell. Cells that were not transfected with siRNA showed similar growth kinetics as siCont-transfected cells (not shown). The viral growth kinetics were considerably hampered in NuMA knockdown cells.

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