Kaposi Sarcoma Herpes Virus Latency Associated Nuclear Antigen Protein Release the G2/M Cell Cycle Blocks by Modulating ATM/ATR Mediated Checkpoint Pathway

The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA) plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole) induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block

A hypothetical model shows the putative mechanisms for the bypassing of the nocodazole induced G2/M block by LANA.

<p>Nocodazole treatment reduces the level of phosphorylated Cdc2. The viral nuclear antigen LANA binds directly to Chk2, which may result in the phosphorylation of Cdc25c and sequester it in the cytoplasm. Thus, it may be unable to regulate the phosphorylation of nuclear Cdc2 resulting the activation of cyclin B-Cdc2 and progression through the G2/M phase, releasing the nocodozole induced block.</p

Downregulation of Chk2 protein levels inhibits the ability of LANA to release the nocodazole G2/M block.

<p>(A) Western blot for Chk2 showed the specific effect of Chk2 siRNA on Chk2 protein levels. BJAB cells carrying pA3M vector (control) and pA3M-LANA were transfected with control and Chk2 siRNA for another 24 hours. The cells were harvested, whole cell lysates and subjected to western blot analysis. β-actin was used as loading control. The result shown is the representative figure of 3 independent experiments. (B) BJAB cells with pA3M vector and pA3M-LANA were treated with nocodazole and with and without Chk2 siRNA for 24 hours. The cells were then harvested, stained with PI and their cell cycle phases were determined by flow cytometry. Results in the untreated and treated cells are shown as the percentages of cells in various cell cycle phases. (C) BC3 cells transfected with control or Chk2 siRNA were treated with nocodazole for 24 hours and subjected cell cycle analysis. Results in the untreated and treated cells were shown, together with the percentages of cells in all cell cycle phases. Mean and standard deviations were derived from 3 independent experiments. +, present; −, absent.</p

LANA disrupt the nocodazole induced G2/M cell cycle block through the ATM/ATR signaling pathway.

<p>(A) The release of nocodazole induced G2/M block by LANA is inhibited by caffeine. BJAB cells (transfected with the pA3M vector or pA3M-LANA) were treated with nocodazole (200 ng/mL) with and without caffeine (5 mM) for 24 hours. The cells were then harvested, stained with PI and their DNA cell cycle profiles were determined by FACS analysis. Results shown are percentages of cells in each phase of the cell cycle. The data represent the mean of 3 separate experiments. (B, C) The release of nocodazole induced G2/M block by LANA occurs through ATR. BJAB cells (control), ATM siRNA and ATR siRNA transfected BJAB cell were treated with nocodazole (200 ng/mL) in the presence and absence of LANA. The cells were then harvested stained with PI and their DNA cell cycle phases were determined by FACS. Results are shown as the percentages of cells in all cell cycle phases. Mean and standard deviations were derived from 3 independent experiments. +, present; −, absent.</p

Cell cycle profiles of KSHV positive and negative cells treated with nocodazole showed that KSHV is capable of releasing nocodazole induced G2/M block though LANA.

<p>(A) Two KSHV positive cells (BC3 and JSC-1), and a KSHV negative cells (BJAB) were treated with or without different concentration of nocodazole (200 ng/mL, 300 ng/mL, 500 ng/mL or 1 µg/mL) for 24 hours. The cells were then harvested, stained with PI and their cell cycle profiles were determined by flow cytometry. Nocodazole treated BJAB cells showed an increase in the proportion of cells at G2/M, indicating a block, whereas BC3 and JSC-1 showed no block in G2/M phase. Results are indicated as percentages of cells in all cell cycle phases. Mean and standard deviations were derived from 3 independent experiments. (B) Western blot shows the expression of LANA in KSHV positive cells (BC-3 and JSC-1) but not in KSHV negative cells (BJAB). Histone-H3 was taken as loading control. (C) BJAB cells transfected with the pA3M vector or pA3M-LANA and were treated with nocodazole (200 ng/mL) for 24 hours. The cells were then harvested and DNA cell cycle distribution patterns were determined as above. Results in the untreated and treated cells are shown as the percentage of cells in all cell cycle phases. Mean and standard deviations were derived from 3 independent experiments. (D) Western blot shows the expression of LANA in the BJAB cells transfected with pA3M-LANA. Histone-H3 was taken as loading control. The result shown is the representative of 3 independent experiments. +, present; −, absent.</p

Nocodazole induced suppression of the Cdc2 (Tyr15) phosphorylation inhibited by the LANA expression.

<p>(A) BJAB cells transfected with the pA3M vector or pA3M-LANA were treated with nocodazole (200 ng/mL) for 24 hours. The cells were then harvested, total cell lysate were prepared and western blot was performed using antibodies against cyclin B1, Cdc2, Tyr15-phosphorylated Cdc2 [Cdc2 (Tyr15)], and Histone H3 as a protein loading control. (B) BJAB cells transfected with the pA3M vector or pA3M-LANA were grown in 6 well culture plates and treated with nocodazole and observed for colocolaization in a confocal microscope. The result shown is the representative of 3 independent experiments. +, present; −, absent.</p

LANA interacts with serine rich amino-terminal domain of Chk2 inside the nucleus.

<p>(A) BJAB and HEK-293 cells were co-transfected with constructs pCDNA3.1-HAChk2 and pA3M-LANA. Co-immunoprecipitation from the cell lysate was performed by using anti-Myc antibodies. The co-immunoprecipitates were separated by electrophoresis, transferred to a nitrocellulose membrane, and then probed with HA antibodies for Chk2. Chk2 immunoprecipitated with LANA in both cell types. (B) BJAB cells were co-transfected with the expression constructs pCDNA3.1-HAChk2 and pA3M-LANA. Following transfection, the cells were grown overnight and fixed. LANA and Chk2 were detected by using mouse monoclonal antibody against Myc-LANA and rabbit polyclonal antibody against HA- Chk2, followed by appropriate secondary antibodies conjugated to Alexa Fluor 488 (green) and Alexa Fluor 594 (red), respectively. The merged panel shows that Chk2 and LANA co-localize in the nucleus. The DAPI panel shows that both proteins are nuclear. (C) Schematic representation of full-length domains along with the different truncation constructs of Chk2. FHA: fork head association domain. (D, E) <i>In vitro</i> translated LANA or KSHV-positive BC3 cells nuclear extract were incubated with the various GST-Chk2 truncated constructs as shown in figure. The pull-down assay showed a preferential binding for the region located between amino acids 63 and 107, which includes the serine rich domain. NE, nuclear extract.</p

Kaposi Sarcoma Herpes Virus Latency Associated Nuclear Antigen Protein Release the G2/M Cell Cycle Blocks by Modulating ATM/ATR Mediated Checkpoint Pathway

The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA) plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole) induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block