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

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.Several prominent events take place after the entry of herpes simplex virus (HSV) DNA into the nucleus of newly infected cells. Thus, viral DNA becomes coated by repressive proteins, the function of which is to block viral gene expression (1–6); nuclear domain 10 (ND10) bodies colocalize with the viral DNA (7, 8); α or immediate early viral genes are expressed; and one viral protein, ICP0, degrades promyelocytic leukemia protein (PML) and Sp100, two key constituents of ND10 bodies in conjunction with the UbcH5A ubiquitin-conjugating enzyme (9–11). What is left of the ND10 bodies is infiltrated by viral proteins and becomes the viral replication compartment (12–15).ND10 bodies range between 0.1 and 1 μM in diameter. The composition of ND10 bodies varies depending on the cellular function or in response to stress, such as that resulting from virus infection (16–19). Among the constant components of ND10 are PML, Sp100, and death-domain associated protein (Daxx). PML has been reported to be critical for the recruitment of components and for the organization of the ND10 bodies (18–23). The function of ND10 bodies may vary under different cellular conditions and may also depend on their composition.A key question that remains unanswered is the function of ND10 bodies in infection, and in particular, why HSV has evolved a strategy that specifically targets PML and Sp100 for degradation. Two clues that may ultimately shed light on the function of ND10 is that exposure of cells to IFN leads to an increase in the number of ND10 bodies and an increase in PML (16, 24–26). The second clue emerged from the observation reported earlier by this laboratory is that pretreatment of murine PML^+/+ cells with IFN-β led to a drastic reduction in virus yields. In contrast, exposure of PML^−/− cells to IFN-β led to a significantly smaller decrease in virus yields (27). The results suggest PML is an antiviral effector of IFN-β, but many questions regarding the function of PML remain unanswered (28).In this study, we constructed a PML^−/− cell line (1D2) derived from HEp-2 cells. The first part of this report centers on the structure of ND10 bodies bereft of PML and the interaction of these bodies with ICP0. In the second part, we report on the replication of HSV-1 in PML^−/− cells. Here we show that HSV-1 replication and the accumulation of ICP0 are significantly reduced in PML^−/− cells exposed to low ratios of virus per cell. HSV has evolved a strategy to take advantage of PML before its degradation.