PML plays both inimical and beneficial roles in HSV-1 replication

Abstract

After entry into the nucleus, herpes simplex virus (HSV) DNA is coated with repressive proteins and becomes the site of assembly of nuclear domain 10 (ND10) bodies. These small (0.1-1 μM) nuclear structures contain both constant [e.g., promyelocytic leukemia protein (PML), Sp100, death-domain associated protein (Daxx), and so forth] and variable proteins, depending on the function of the cells or the stress to which they are exposed. The amounts of PML and the number of ND10 structures increase in cells exposed to IFN-β. On initiation of HSV-1 gene expression, ICP0, a viral E3 ligase, degrades both PML and Sp100. The earlier report that IFN-β is significantly more effective in blocking viral replication in murine PML(+/+) cells than in sibling PML(-/-) cells, reproduced here with human cells, suggests that PML acts as an effector of antiviral effects of IFN-β. To define more precisely the function of PML in HSV-1 replication, we constructed a PML(-/-) human cell line. We report that in PML(-/-) cells, Sp100 degradation is delayed, possibly because colocalization and merger of ICP0 with nuclear bodies containing Sp100 and Daxx is ineffective, and that HSV-1 replicates equally well in parental HEp-2 and PML(-/-) cells infected at 5 pfu wild-type virus per cell, but poorly in PML(-/-) cells exposed to 0.1 pfu per cell. Finally, ICP0 accumulation is reduced in PML(-/-) infected at low, but not high, multiplicities of infection. In essence, the very mechanism that serves to degrade an antiviral IFN-β effector is exploited by HSV-1 to establish an efficient replication domain in the nucleus.

Keywords: Daxx; ICP0; ND10; Sp100; interferon.

Fig. 1.

Confirmation of the absence of PML in 1D2 clone. (A) Parental HEp-2 and 1D2 cell cultures were exposed to IFN-β (1,000 U/mL). After 24 h, the cells were harvested, and lysates of nuclei or of whole cells were subjected to electrophoresis in denaturing gels transferred to a nitrocellulose sheet and reacted with rabbit polyclonal antibody against PML. (B) HEp-2 or 1D2 cells grown on four-well slides were fixed and reacted with mouse monoclonal antibody to PML (a and b) or rabbit polyclonal antibody to PML (c and d). (Magnification: 100×.)

Fig. 2.

Sp100 and Daxx respond differently to exposure of HEp-2 or 1D2 cells to IFN-β. Parental HEp-2 PML^+/+ cells or PML^−/− 1D2 cell cultures were mock treated or exposed to IFN-β (1,000 U/mL). After 24 h, the cells were harvested and the relative amounts of Sp100 and Daxx were quantified, as described in Materials and Methods. mRNA levels of Sp100 and Daxx were normalized to 18S RNA, and their relative value in untreated group were set as 1. Two-tailed P values were calculated using standard t test.

Fig. 3.

Cellular localization of Sp100 and Daxx in HEp-2 and 1D2 cells. At 70% confluency, the medium of parental HEp-2 or 1D2 cells grown on four-well slides was replaced with fresh medium alone or medium supplemented with IFN-β (1,000 U/mL). After 24 h, the cells were fixed and reacted with polyclonal antibody to Sp100 (red) and either PML (green, A) or Daxx (green, B). (Magnification: 100×.)

Fig. 4.

ICP0 formed aggregates in PML^−/− cells. HEp-2 (A–C) and 1D2 cells (D–F) grown on four-well slides were treated with IFN-β, as earlier, and then exposed to 10 pfu HSV-(F) per cell. At 2 h after infection, the cells were fixed and reacted with polyclonal antibody to ICP0 (red) and monoclonal antibody to PML (green). (Magnification: 100×.)

Fig. 5.

(A–L) ICP0 and Sp100 aggregated with Daxx do not colocalize efficiently in 1D2 cells infected with wild-type or RF mutant viruses. Cells grown on four-well slide cultures and treated as earlier were exposed to 10 pfu wild-type or RF mutant virus per cell. At 3 h after infection, the cultures were fixed and reacted with monoclonal antibody to ICP0 and polyclonal antibody to Sp100. The images shown were acquired using a Zeiss confocal microscope. (Magnification: 100×.)

Fig. 6.

Degradation of Sp100 by HSV-1(F) infection is delayed in PML^−/− 1D2 cells. (A) HEp-2 and 1D2 cells were transfected with a plasmid-encoding Flag-tagged Sp100. At 48 h after transfection, the cells were exposed to 10 pfu wild-type virus per cell. At 0 (mock), 3, 5, 7, or 10 h after infection, the cells were harvested, solubilized, subjected to electrophoresis in denatured gels, and reacted with antibody to Sp100 and β-actin. (B) The relative amounts of Flag-tagged Sp100 were quantified and normalized to β-actin, and the amounts of Flag-tagged Sp100 in mock-treated cells (0 h after infection) with the aid of Image J. The degradation rates of FLAG-Sp100 were calculated as described in Materials and Methods.

Fig. 7.

Growth of HSV-1(F) in PML^−/− 1D2 cells is impaired in PML^−/− cells exposed to low ratios of virus per cell. Replicate cultures of HEp-2 or 1D2 cells were exposed to 5 or 0.1 pfu virus per cell. At times shown, the cells were harvested and virus yields were titered in Vero cell.

Fig. 8.

(A and B) Accumulation of ICP0 is reduced in PML^−/−1D2 cells exposed to low ratios of virus per cell. Replicate cultures of HEp-2 or 1D2 cells were exposed to 10 or 0.1 pfu virus per cell. Cells were harvested at indicated points and were reacted with antibody to ICP0 and β-actin.

Fig. 9.

The antiviral effects of IFN-β are diminished in cells in PML^−/− cells. HEp-2 or PML^−/− 1D2 cells were pretreated with IFN-β at 1,000 U/mL for 24 h and then exposed to 0.1 pfu virus per cell. The cells were harvested at 24 h after infection. Virus yields were titered in Vero cells. Antiviral effects of IFN-β treatment in HEp-2 or PML^−/− 1D2 cells were represented by viral titer reduction posttreatment. P value was calculated by comparison of antiviral effects of IFN-β in HEp-2 and that in PML^−/− 1D2 cells.