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. 2012 Jan;86(1):492-503.
doi: 10.1128/JVI.05897-11. Epub 2011 Oct 19.

Herpes simplex virus requires poly(ADP-ribose) polymerase activity for efficient replication and induces extracellular signal-related kinase-dependent phosphorylation and ICP0-dependent nuclear localization of tankyrase 1

Zhuan Li  1 , Yohei Yamauchi, Maki Kamakura, Tsugiya Murayama, Fumi Goshima, Hiroshi Kimura, Yukihiro Nishiyama
Affiliations

Herpes simplex virus requires poly(ADP-ribose) polymerase activity for efficient replication and induces extracellular signal-related kinase-dependent phosphorylation and ICP0-dependent nuclear localization of tankyrase 1

Zhuan Li et al. J Virol. 2012 Jan.

Abstract

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) which localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Poly(ADP-ribosyl)ation of the nuclear mitotic apparatus (NuMA) protein by tankyrase 1 during mitosis is essential for sister telomere resolution and mitotic spindle pole formation. In interphase cells, tankyrase 1 resides in the cytoplasm, and its role therein is not well understood. In this study, we found that herpes simplex virus (HSV) infection induced extensive modification of tankyrase 1 but not tankyrase 2. This modification was dependent on extracellular signal-regulated kinase (ERK) activity triggered by HSV infection. Following HSV-1 infection, tankyrase 1 was recruited to the nucleus. In the early phase of infection, tankyrase 1 colocalized with ICP0 and thereafter localized within the HSV replication compartment, which was blocked in cells infected with the HSV-1 ICP0-null mutant R7910. In the absence of infection, ICP0 interacted with tankyrase 1 and efficiently promoted its nuclear localization. HSV did not replicate efficiently in cells depleted of both tankyrases 1 and 2. Moreover, XAV939, an inhibitor of tankyrase PARP activity, decreased viral titers to 2 to 5% of control values. We concluded that HSV targets tankyrase 1 in an ICP0- and ERK-dependent manner to facilitate its replication.

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Figures

Fig 1
Fig 1
ERK-dependent phosphorylation of tankyrase 1 during HSV infection is modified by inhibition of viral DNA synthesis. (A and B) Extensive modification of tankyrase 1 (TNKS1) during HSV infection. HEp-2 cells were mock infected (M) or infected with HSV-1 F (A) or HSV-2 186 (B) at an MOI of 3 PFU/cell. Cell lysates were collected at 5, 10, or 24 hpi and analyzed by Western blotting. Mock-infected tankyrase 1 is indicated by black arrowheads, and the faster-migrating band observed in lysates prepared from HSV-infected cells is indicated by white arrowheads. (C) Proteasome-dependent degradation involved in reduction of tankyrase 1. HEp-2 cells were mock infected (M) or infected with HSV-1 F or HSV-2 186 in the absence or presence of MG132 (50 μM). Cell lysates were collected at 24 hpi and analyzed by Western blotting. (D) Phosphorylation is involved in tankyrase 1 modification. Uninfected HEp-2 cells (M) or HEp-2 cells infected for 14 h with HSV-1 at an MOI of 3 PFU/cell were harvested and dephosphorylated with Lambda PP (ppase) (+) or incubated without it (−). (E) Tankyrase 1 phosphorylation is ERK dependent and affected by the inhibition of viral DNA synthesis. HEp-2 cells were mock infected or infected with HSV-1 strain F at an MOI of 3 PFU/cell in the absence or presence of PAA (300 μg/ml). An equivalent amount of DMSO was used as a control. For U0126 treatment, cells were pretreated with 30 μM U0126 4 h before infection, and cells were infected with HSV-1 strain F at an MOI of 3 PFU/cell with U0126. Cell lysates were collected 24 h after infection and analyzed by Western blotting. The black arrowhead indicates the tankyrase 1 band detected in lysates prepared from mock-infected cells. HSV infection induced a faster-migrating band, and mitotic tankyrase 1 is indicated by white arrowheads.
Fig 2
Fig 2
Nuclear translocation of tankyrase 1 in HSV-1-infected cells. (A) Tankyrase 1 is recruited into the nucleus and localized in the replication compartment in HSV-1-infected cells. The intracellular localization of tankyrase 1 in HSV-1-infected cells (MOI of 1) was analyzed by indirect immunofluorescence using antibodies against tankyrase 1 (N-20) (green) and a marker of the HSV replication compartment (ICP8) (red). Arrows indicate infected cells, and arrowheads indicate uninfected cells. (B) Nuclear localization of tankyrase 1 was not due to channel overlap. HEp-2 cells were infected with the F strain for 6 h and subjected to indirect immunofluorescence analysis using anti-tankyrase 1 (N-20) (green) or anti-ICP8 (red) antibody. (C) Subcellular localization of tankyrase 1 in infected cells (MOI of 3). Nuclear and cytoplasmic extracts of infected cells were analyzed by Western blotting using the indicated antibodies. (D) Effect of infection on tankyrase 2 localization. Intracellular localization of tankyrase 2 in HSV-1-infected cells (MOI of 1) was analyzed by indirect immunofluorescence using antibodies against tankyrase 2 (green) and ICP8 (red). Imaging was performed using a Zeiss LSM 510 Meta confocal microscope with a 64× objective lens. Images were acquired at a 2× or 4× digital zoom.
Fig 3
Fig 3
Tankyrase 1 phosphorylation is not essential for nuclear translocation in HSV-1-infected cells. (A) Subcellular localization of ERK2 and active ERK (p-ERK) in infected cells. HEp-2 cells were mock infected (M) or infected with HSV-1 strain F at an MOI of 1 PFU/cell, and the intracellular localization of ERK2 and p-ERK was analyzed by indirect immunofluorescence using antibodies against ICP8 (red) and ERK2 or p-ERK (green) at the indicated times. (B) ERK activity and tankyrase 1 phosphorylation are inhibited by U0126. HEp-2 cells were treated with 30 μM U0126 4 h before infection, and cells were then infected with HSV-1 strain F at an MOI of 3 PFU/cell with U0126. Cell lysates were collected at the indicated times after infection and were analyzed by Western blotting. (C) Phosphorylation is not required for tankyrase 1 nuclear translocation during HSV-1 infection. HEp-2 cells were treated with 30 μM U0126 4 h before infection, and cells were then infected with HSV-1 strain F at an MOI of 1 PFU/cell with U0126 for 8 h. Cells were then fixed, and localization of tankyrase 1 was determined by indirect immunofluorescence using anti-tankyrase 1 (N-20) (green) and anti-ICP8 (red) antibodies. The arrow indicates infected cells, and imaging was performed using a Zeiss LSM 510 Meta confocal microscope with a 64× objective lens. Images were collected at a 2× digital zoom.
Fig 4
Fig 4
Localization of tankyrase 1 in cells treated with PAA or infected with the UV-F strain. (A and B) Viral DNA synthesis is not essential for tankyrase 1 translocation in HSV-infected cells. (A) HEp-2 cells were infected with HSV-1 strain F at an MOI of 3 PFU/cell in the absence or presence of PAA (300 μg/ml). An equivalent amount of DMSO was used as a control. Cell lysates were collected 15 h after infection, and inhibition of viral DNA synthesis was monitored by Western blotting. (B) HEp-2 cells were mock infected (M) or infected with the F strain at an MOI of 1 PFU/cell in the presence of PAA (300 μg/ml) or DMSO. Cells were fixed 8 h after infection and stained for ICP8 (red) and tankyrase 1 (green). (C and D) Tankyrase 1 shows no modification or nuclear redistribution in UV-F-infected cells. (C) HEp-2 cells were mock infected (M) or infected with the UV-F strain at an MOI of 10 PFU/cell, and cell lysates were analyzed by Western blotting at the indicated times. (D) HEp-2 cells were mock infected (M) or infected with the UV-F strain at an MOI of 10 PFU/cell and fixed for indirect immunofluorescence using tankyrase 1 (green) and ICP8 antibodies at 12 hpi. Active ERK1/2 was stained at 30 min postinfection, using p-ERK1/2 antibody. Imaging was performed using a Zeiss LSM 510 Meta confocal microscope with a 64× objective lens. Images were collected at a 2× digital zoom.
Fig 5
Fig 5
Tankyrase 1 colocalizes with ICP0 during the early phase of HSV-1 infection. (A) HEp-2 cells were mock infected (M) or infected with HSV-1 strain F at an MOI of 1 PFU/cell. Intracellular localization of tankyrase 1 and ICP0 in HSV-1-infected cells was analyzed using indirect immunofluorescence with tankyrase 1 (green) and ICP0 (red) antibodies. Imaging was performed using a Zeiss LSM 510 Meta confocal microscope with a 64× objective lens. Images were collected at a 3× digital zoom. (B) Percentages of ICP0 foci colocalized with tankyrase 1 in HSV-1-infected cells. HEp-2 cells were infected with strain F and fixed for immunofluorescence at various time points between 2 and 8 h postinfection. Infected cells were stained with anti-ICP0 monoclonal antibody and anti-tankyrase 1 polyclonal antibody. Over 50 cells for each time point were counted, and results are shown as means ± standard deviations.
Fig 6
Fig 6
ICP0 interacts with tankyrase 1 and is sufficient to redistribute tankyrase 1 in ICP0-transfected cells. (A) HEp-2 cells were mock transfected (control) or transfected with plasmids carrying the ICP0 gene (HSV-1 or HSV-2). Cells were obtained using tankyrase 1 (green) and anti-Myc (red) antibodies. Merged images of the transfected cells are shown in the right panels. Imaging was performed using a Zeiss LSM 510 Meta confocal microscope with a 64× objective lens. Images were collected at a 3× digital zoom. (B) Western blotting. Transfected cells were collected at 12, 24, and 36 h posttransfection and analyzed by Western blotting using the indicated antibodies. (C) Coimmunoprecipitation assay. Transfected cells were collected at 24 h posttransfection, and whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Myc antibodies or subjected directly to Western blotting with the indicated antibodies. Normal rabbit IgG was used as a negative control for immunoprecipitation. Immunoprecipitated ICP0 was Western blotted using anti-Myc and HSV-1 ICP0 antibodies.
Fig 7
Fig 7
ICP0 is essential for tankyrase 1 redistribution and phosphorylation in HSV-1-infected cells. (A) HEp-2 cells were mock infected (M) or infected with R7910 at an MOI of 3 PFU/cell. Cell lysates were collected 5, 10, or 24 h after infection and analyzed by Western blotting using the indicated antibodies. (B) HEp-2 cells were infected with HSV-1 strain F (left) or the mutant virus R7910 (right) at an MOI of 3 or 1 PFU/cell, respectively. Cells were fixed at the indicated times, and tankyrase 1 localization was analyzed using indirect immunofluorescence with tankyrase 1 (green) and ICP8 (red) antibodies. Imaging was performed using a 64× objective lens and a Zeiss LSM 510 Meta confocal microscope. Images were acquired at a 3× digital zoom.
Fig 8
Fig 8
siRNA-mediated tankyrase knockdown inhibits HSV growth. (A) siCON-, siTNKS1-, siTNKS2-, or siTNKS1-plus-siTNKS2 (siTNKS)-transfected HEp-2 cells were analyzed at 3 days posttransfection for protein expression. Tankyrase levels were greatly decreased in siRNA-treated cells, whereas actin levels were constant. (B) Analysis of viral growth in siRNA-treated cells. HEp-2 cells transfected with siCON, siTNKS1, siTNKS2, and siTNKS were infected with F at an MOI of 3, cells were collected at 24 hpi and lysed by a freeze-thaw cycle, and progeny viruses were titrated on Vero cells. Results for three independent experiments are shown as means with standard deviations.
Fig 9
Fig 9
Inhibition of tankyrase PARP activity significantly impairs viral replication. (A) Plaque size depends on XAV939 concentration. Vero cell monolayers were infected with approximately 100 PFU of HSV-1 in the presence of the indicated concentration of XAV939 or DMSO (control). (B) The cells were fixed at 24 hpi, and plaque size and number were determined to assess viral spread. Results for three independent experiments are shown as means ± standard deviations, and P values were determined by the two-tailed t test. (C) Inhibition of tankyrase 1 PARP activity results in defective viral protein expression. HEp-2 cells were infected with HSV-1 strain F at an MOI of 3 PFU/cell in the absence (DMSO) or presence of XAV939 (1 μM), and then lysates were analyzed by Western blotting. (D) Multistep growth curve of HSV-1 in XAV939-treated HEp-2 cells. The results shown are representative of 3 independent experiments. After similar treatment with XAV939 (1 μM) or DMSO, HEp-2 cells were infected with HSV-1 at an MOI of 0.001 PFU/cell. The virus replication kinetics was significantly diminished when PARP activity was inhibited.
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