H2AX phosphorylation is important for LANA-mediated Kaposi's sarcoma-associated herpesvirus episome persistence

Abstract

The DNA damage response (DDR) of host cells is utilized by a number of viruses to establish and propagate their genomes in the infected cells. We examined the expression of the DDR genes during Kaposi's sarcoma-associated herpesvirus (KSHV) infection of human peripheral blood mononuclear cells (PBMCs). The genes were mostly downregulated, except H2AX, which was upregulated during infection. H2AX is important for gammaherpesvirus infectivity, and its phosphorylation at serine 139 is crucial for maintenance of latency during mouse gamma-herpesvirus 68 (MHV-68) infection. We now also observed phosphorylation of H2AX at serine 139 during KSHV infection. H2AX is a histone H2A isoform shown to interact with the latency-associated nuclear antigen (LANA) encoded by KSHV. Here, we show that LANA directly interacted with H2AX through domains at both its N and C termini. The phosphorylated form of H2AX (γH2AX) was shown to colocalize with LANA. Chromatin immunoprecipitation (ChIP) assays showed that a reduction in H2AX levels resulted in reduced binding of LANA with KSHV terminal repeats (TRs). Binding preferences of H2AX and γH2AX along the KSHV episome were examined by whole-episome ChIP analysis. We showed that γH2AX had a higher relative binding activity along the TR regions than that of the long unique region (LUR), which highlighted the importance of H2AX phosphorylation during KSHV infection. Furthermore, knockdown of H2AX resulted in decreased KSHV episome copy number. Notably, the C terminus of LANA contributed to phosphorylation of H2AX. However, phosphorylation was not dependent on the ability of LANA to drive KSHV-infected cells into S-phase. Thus, H2AX contributes to association of LANA with the TRs, and phosphorylation of H2AX is likely important for its increased density at the TRs.

Fig 1

KSHV infection modulates expression and phosphorylation of H2AX. (A) Expression of H2AX and/or other DDR genes was examined by real-time PCR in KSHV-infected PBMCs; (B) KSHV infection of PBMCs was confirmed by probing LANA by immunofluorescence assays; (C) H2AX transcript levels were also examined in KSHV-positive cell lines by real-time PCR; (D) phosphorylation of H2AX in KSHV-positive cell lines and KSHV-infected PBMCs was examined by Western blotting with specific antibodies. Bar diagrams show densitometry-based relative quantification of H2AX or γH2AX normalized against GAPDH.

Fig 2

LANA interacts with H2AX and γH2AX. (A, C) Coimmunoprecipitation of LANA with γH2AX was examined with endogenous proteins in JSC-1 and BCBL1 cells; (B, D) coimmunoprecipitation of γH2AX with LANA was examined with endogenous proteins in JSC-1 and BCBL1 cells; (E, F) coimmunoprecipitation of LANA with H2AX was examined with endogenous proteins in JSC-1 and BCBL1 cells; (G) coimmunoprecipitation of H2AX with LANA was examined with endogenous proteins in JSC-1 and BCBL1 cells. In panels A to G, nonspecific IgG control antibody was used before specific antibody binding. (H) Coimmunoprecipitation of H2AX with LANA was examined with exogenously expressed proteins in HEK-293 cells. (I) GST pulldown assay shows binding of H2AX with the N (aa 1 to 340) and C (aa 930 to 1162) termini of LANA. GST-N-LANA and GST-C-LANA bound to glutathione-Sepharose beads were used to pull down H2AX from pA3M-H2AX-transfected 293 cell lysate. (J) GST pulldown assay to show relative binding of the full-length N terminus (aa 1 to 340) and C terminus (aa 930 to 1162) of LANA with H2AX. GST-H2AX bound to glutathione-Sepharose beads was used to pull down LANA and its truncations expressed in HEK-293 cells. Ten percent of input samples and 10% of elution fraction were loaded. Bar diagrams show densitometry-based relative quantification of LANA and its N and C terminus polypeptide.

Fig 3

(A) Role of H2AX in LANA binding with TR. HEK-293 cells were transfected with pA3FL-LANA, pBS-puro-TR, and pGIPz-shCtrl or pGIPz-shH2AX constructs. After 24 h posttransfection, cells were fixed and ChIP was carried out using M2 antibody. Levels of TRs were compared in the different samples. Data have been normalized using an amplicon in the Amp^r region of pBS as a reference sequence. (B) Immunofluorescent in situ hybridization (immuno-FISH) was carried out to examine the localization of KSHV TR and γH2AX in HEK-293–BAC36–KSHV cells. Cells were hybridized with the biotinylated KSHV TR probe, followed by incubation with specific antibody against γ-H2AX. The staining was detected by incubation with Alexa Fluor 594 anti-mouse IgG and Alexa Fluor 680 streptavidin conjugate. Alexa Flour 680 staining is shown as a green pseudocolor. Cells were also counterstained with DAPI.

Fig 4

Relative abundance of H2AX and γH2AX along the KSHV episome. In BCBL1 cells, ChIP assay was carried out using specific antibodies against γH2AX and H2AX. The KSHV episome was examined at a resolution of approximately 1.6 kb to evaluate the binding of H2AX and γH2AX. Relative amplification was calculated by subtracting the amplification with control antibodies.

Fig 5

H2AX is crucial for KSHV episome persistence. (A) Experimental scheme for episome retention experiment; (B) HEK-293–BAC36–KSHV cells were transfected with pGIPz-shCtrl or pGIPz-shH2AX plasmids. Episome copies have been quantified by real-time PCR on the 2nd, 5th, and 15th days posttransfection while keeping cells under puromycin selection. (C) Levels of LANA examined in the HEK-293–BAC36–KSHV cell line, stably knocked down for H2AX. Decrease in LANA level by day 15 may be indirect evidence of a decrease in the episome copies as a result of H2AX depletion. (D) Reporter assays showing that LANA promoter activity is unchanged by H2AX knockdown. Rta, a positive activator of LANA promoter, is transfected to serve as a positive control for promoter activity. Values shown in bar graphs are relative/normalized to a no-LANA negative control. (E) Colony formation assay carried out under dual antibiotic selection (puromycin and hygromycin) using the HEK-293–BAC36–KSHV cell line, stably expressing shRNA constructs for H2AX and control sequences. A significant decrease in the colony number shows that loss of H2AX also promotes loss of the KSHV episome.

Fig 6

H2AX phosphorylation is increased in response to LANA expression. (A) Immunofluorescence for examining colocalization of LANA and γH2AX in KSHV-positive and -negative cell lines. LANA was probed using anti-LANA mouse monoclonal antibody and Alexa Fluor 594-conjugated secondary antibody, whereas FITC-labeled monoclonal antibody against γH2AX was used to probe γH2AX (used after completion of secondary antibody treatment in LANA staining). (B) Binding of LANA with γH2AX in the absence of the KSHV episome was examined by transfecting HEK-293 cells with RFP-LANA-expressing plasmid. RFP-LANA shows a diffused staining pattern. The image contrast was set to high to visualize LANA-rich regions. (C) H2AX levels as affected by LANA expression. HEK-293 cells and ECV304 cells were transfected with increasing amounts of pA3M-LANA plasmid. H2AX levels were examined by real-time PCR 24 h posttransfection. (D) H2AX phosphorylation was examined in response to LANA expression. HEK-293 cells were transfected with increasing amounts of A3M-LANA constructs. After 24 h posttransfection, cells were harvested and H2AX phosphorylation (serine 139) was examined by Western blotting. (E) H2AX phosphorylation is examined in response to expression of the LANA N terminus in a dose-dependent manner. (F) H2AX phosphorylation is examined in response to expression of the LANA C terminus in a dose-dependent manner. HEK-293 cells were transfected with increasing amounts of the indicated plasmids. H2AX and γH2AX levels were examined by Western blotting 24 h posttransfection.

Fig 7

H2AX phosphorylation by LANA was examined during various stages of the cell cycle. HEK-293 cells were transfected with 15 μg of the indicated plasmids. H2AX phosphorylation in various phases of cell cycle was determined by labeling of cells with antibodies specific for γH2AX and propidium iodide (PI) staining.

Fig 8

Schematic presents a model by which γH2AX can contribute to the persistence of the viral genome after KSHV infection. As γH2AX is known to bind/localize with C-LANA and TR, along with partially assisting with the episome retention function, the model shows that γH2AX may strengthen the interaction between C-LANA and TR. This model is complementary to the classical model, where LANA by its C terminus binds with LANA binding sequences (LBSs) in the TRs. This model also presents a possible explanation for how the mammalian chromatin-associated proteins, known to bind to the C terminus of LANA, are able to contribute to LANA-mediated KSHV episome persistence after infection.