Epstein-barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies

Abstract

Although the immediate-early proteins of both herpes simplex virus (HSV) and cytomegalovirus (CMV) are known to modify promyelocytic leukemia (PML) (ND10) bodies in the nucleus of the host cell, it has been unclear whether lytic infection with gamma herpesviruses induces a similar effect. The PML protein is induced by interferon, involved in major histocompatibility complex class I presentation, and necessary for certain types of apoptosis. Therefore, it is likely that PML bodies function in an antiviral capacity. SUMO-1 modification of PML is known to be required for the formation of PML bodies. To examine whether Epstein-Barr virus (EBV) lytic replication interferes with PML bodies, we expressed the EBV immediate-early genes BZLF1 (Z) and BRLF1 (R) in EBV-positive cell lines and examined PML localization. Both Z and R expression resulted in PML dispersion in EBV-positive cells. Z but not R expression is sufficient to disrupt PML bodies in EBV-negative cell lines. We show that dispersion of PML bodies by Z requires a portion of the transcriptional activation domain of Z but not the DNA-binding function. As was previously reported for the HSV-1 ICP0 and CMV IE1 proteins, Z reduces the amount of SUMO-1-modified PML. We also found that Z itself is SUMO-1 modified (through amino acid 12) and that Z competes with PML for limiting amounts of SUMO-1. These results suggest that disruption of PML bodies is important for efficient lytic replication of EBV. Furthermore, Z may potentially alter the function of a variety of cellular proteins by inhibiting SUMO-1 modification.

FIG. 1

EBV lytic replication disrupts PML bodies. EBV-positive cells (D98/HE-R-1) were transfected with expression plasmids for either Z (A and B) or R (C and D). At 48 h posttransfection, cells were fixed and immunostained with either anti-Z (monoclonal) (A), anti-R (monoclonal) (C), or anti-PML protein (polyclonal) (B and D) antibodies. Arrows indicate cells that are positively expressing Z or R.

FIG. 2

Z but not R expression disrupts PML bodies. EBV-negative (HeLa) cells were transfected with expression plasmids for either Z (A and B) or R (C and D). At 48 h posttransfection, cells were fixed and immunostained with either anti-Z (monoclonal) (A), anti-R (monoclonal) (C), or anti-PML protein (polyclonal) (B and D) antibodies.

FIG. 3

Structures of wild-type and mutant Z proteins. The transactivation (hatched), DNA-binding (DNA; black), and dimerization (dim.; stippled) domains are indicated. The amino acid (aa) numbers are noted at the top. Χ, mutated amino acid.

FIG. 4

Mapping the region of Z required for PML body dispersion. HeLa cells were transfected with 20 μg of DNA. At 48 h posttransfection, cells were fixed and immunostained with anti-Z (polyclonal) antibody (A, C, E, G, I, K, and M) and anti-PML protein (monoclonal) antibody (B, D, F, H, J, L, and N). (A and B) wild-type Z. (C and D) Z131–245. (E and F) Z86–245. (G and H) ZΔ25–42 (I and J) Z25–245. (K and L) Z311. (M and N) Z(S186A). Arrows point to cells that are positively expressing Z.

FIG. 4

Mapping the region of Z required for PML body dispersion. HeLa cells were transfected with 20 μg of DNA. At 48 h posttransfection, cells were fixed and immunostained with anti-Z (polyclonal) antibody (A, C, E, G, I, K, and M) and anti-PML protein (monoclonal) antibody (B, D, F, H, J, L, and N). (A and B) wild-type Z. (C and D) Z131–245. (E and F) Z86–245. (G and H) ZΔ25–42 (I and J) Z25–245. (K and L) Z311. (M and N) Z(S186A). Arrows point to cells that are positively expressing Z.

FIG. 5

Z expression induces a loss of PML protein isoforms. EBV-negative A549 cells were mock infected (lane 1) or infected with an adenovirus expressing either LacZ (lane 2), Z (lane 3), or R (lane 4). Cells were harvested 48 h postinfection, and Western blot analysis was performed with anti-PML protein monoclonal antibody. PML isoforms lost after Z expression are indicated by asterisks.

FIG. 6

Z is modified by SUMO-1. (A) EBV-negative DG75 cells were transfected with vector, HA-SUMO-1 (10 μg), Z (10 μg), or Z (10 μg) plus HA-SUMO-1 (1, 5, or 10 μg) expression plasmids. Cells were harvested 48 h posttransfection using conditions which preserve SUMO-1 modification, and Western blot analysis was performed with anti-Z (monoclonal) (left panel) or anti-HA (right panel) antibodies. Arrowheads indicate HA-SUMO-1-modified Z proteins. (B) EBV-negative DG75 cells were transfected with 5 μg of HA-SUMO-1 plus 10 μg of either vector, Z, Zm12/13, Z25–245, Z131–245, ZΔ25–42, or Z86–245 expression plasmids. Zm12/13 contains mutations that alter amino acids 12 and 13 (69). Cells were harvested 48 h posttransfection using conditions which preserve SUMO-1 modification, and Western blot analysis was performed with anti-HA (monoclonal) (top panel) or anti-Z (bottom panel) antibodies.

FIG. 7

Z competes with PML protein for SUMO-1. (A) EBV-negative DG75 cells were transfected with vector, HA-SUMO-1 (10 μg), PML protein (10 μg) plus HA-SUMO-1 (2 μg), Z (10 μg) plus HA-SUMO-1 (2 μg), or Z (10 μg) plus PML protein (10 μg) plus HA-SUMO-1 (2 or 10 μg) expression plasmids. Cells were harvested 48 h posttransfection, and Western blot analysis was performed with anti-HA antibody (upper panel). The blot was reprobed with anti-PML protein monoclonal antibody to check for total PML protein levels (lower panel). (B) EBV-negative DG75 cells were transfected with vector, HA-SUMO-1 (10 μg), PML protein (5 μg), or PML protein (5 μg) plus HA-SUMO-1 (2 μg). Cells were harvested 48 h posttransfection, and Western blot analysis was performed with an anti-PML protein monoclonal antibody. (C) EBV-negative DG75 cells were transfected with HA-SUMO-1 along with either vector, PML, Z, Z plus PML protein, or Zm12/13 plus PML protein expression plasmids. Ten micrograms of each plasmid was used. Cells were harvested 48 h posttransfection, and Western blot analysis was performed with anti-HA antibody (upper panel). The blot was reprobed with anti-PML protein monoclonal antibody to check for total PML protein levels (lower panel).

FIG. 8

Z mutant that cannot be SUMO modified disperses PML bodies. HeLa cells were transfected with 20 μg of DNA. At 48 h posttransfection, cells were fixed and immunostained with anti-Z polyclonal antibody (A and C) and anti-PML protein monoclonal antibody (B and D). (A and B) Wild-type Z. (C and D) Zm12/13.

FIG. 9

Z is SUMO-1 modified in Akata cells after induction with anti-IgG cross-linking. Akata cells were induced to undergo lytic replication via anti-IgG cross-linking for 3 h. Cells were harvested using conditions which preserve SUMO-1 modification, and Western blot analysis was performed with anti-Z polyclonal antibody. The open arrowhead indicates unmodified Z; the solid arrowhead indicates SUMO-1-modified Z.