Promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects

Abstract

Herpes simplex virus (HSV) 1 disaggregates the nuclear domain 10 (ND10) nuclear structures and disperses its organizing promyelocytic leukemia protein (PML). An earlier report showed that ectopic overexpression of PML precludes the disaggregation of ND10 but has no effect on viral replication. PML has been reported to mediate the effects of interferon (IFN) and viral mutants lacking ICP0 (Delta alpha 0 mutants). To test the hypothesis that HSV disaggregates ND10 structures and disperses PML to preclude IFN-mediated antiviral effects, we tested the accumulation of viral proteins and virus yields from murine PML(+/+) and PML(-/-) cells mock treated or exposed to IFN-alpha, IFN-gamma, or both and infected with the wild-type or Delta alpha 0 mutant virus. We report the following results. (i) The levels of growth of wild-type and mutant viruses and of accumulation of viral proteins were not significantly different in untreated PML(+/+) and PML(-/-) cells. (ii) Major effects of IFN-alpha and -gamma were observed in PML(+/+) cells infected with the Delta alpha 0 mutant virus, and more minor effects were observed in cells infected with the wild-type virus. The effects of the IFNs on either wild-type or the mutant virus in PML(-/-) cells were minimal. (iii) The mixture of IFN-alpha and -gamma was more effective than either IFN alone, but again, the effect was more drastic in PML(+/+) cells than in PML(-/-) cells. We concluded that the anti-HSV state induced by exogenous IFN is mediated by PML and that the virus targets the ND10 structures and disseminates PML in order to preclude the establishment of the antiviral state induced by IFNs.

FIG. 1.

Effects of IFN-α and IFN-γ on the accumulation of viral proteins encoded by α4, U_L38, and U_S11 open reading frames. (Top) Experimental design; (bottom) immunoblots of electrophoretically separated lysates of murine PML^+/+ or PML^−/− infected cells. At The University of Chicago, the PML^−/− and PML^+/+ cells were initially maintained in culture for approximately 20 serial passages and were then frozen. Prior to the initiation of these studies, the absence of PML from PML^−/− cells was verified for both untreated and IFN-treated cells (data not shown). In all experiments, thawed cells were maintained in culture for less than 15 serial passages. HSV-1(F), an isolate that can be passaged a limited number of times, is the prototype HSV-1 strain used in this laboratory (6). The construction and phenotypic properties of the recombinant virus R7910, lacking both copies of the α0 gene, were described elsewhere (15). The cells were harvested 24 h after infection, rinsed with phosphate-buffered saline (PBS), resuspended in PBS* (PBS containing 1% NP-40, 1% deoxycholate, and the Roche complete protease inhibitor cocktail), lysed for 30 min in ice, and centrifuged at 11,000 × g for 15 min to remove the insoluble fraction. Protein concentrations were determined by the Bradford assay (Bio-Rad). Protein lysates (100 μg) were denatured by boiling them for 5 min in disruption buffer (2% sodium dodecyl sulfate, 50 mM Tris [pH 7.0], 2.75% sucrose, 5% β-mercaptoethanol [final concentrations], and bromophenol blue), electrophoretically separated on a denaturing 12% polyacrylamide (acrylamide-DATD) gel, and transferred to nitrocellulose membranes. The nitrocellulose sheets were blocked for 1 h with 5% milk buffer, probed with the appropriate primary antibody for 2 h at room temperature or overnight at 4°C, rinsed, and then reacted with secondary antibodies conjugated with alkaline phosphatase (Bio-Rad) diluted 1:3,000 in PBS containing 0.05% Tween 20 and 1% bovine serum albumin for 1 h. The mouse monoclonal antibodies to ICP4 and U_S11 were reported elsewhere (1, 28) and were used at dilutions of 1:1,000 and 1:2,000, respectively. The polyclonal antibody to the U_L38 protein (33) was used at a 1:500 dilution. Alkaline phosphatase-conjugated antibodies were detected in AP buffer (100 mM Tris [pH 9.5], 100 mM NaCl, and 5 mM MgCl₂) containing 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium.

FIG. 2.

The effects of IFN-α and IFN-γ on the accumulation of viral proteins encoded by α4, U_L38, and U_S11 open reading frames. (Top) Experimental design; (bottom) immunoblots of electrophoretically separated lysates of murine PML^+/+ or PML^−/− infected cells. The procedures were as described in the legend to Fig. 1.