Herpes simplex virus 1 precludes replenishment of the short-lived receptor of tumor necrosis factor alpha by virion host shutoff-dependent degradation of its mRNA

Abstract

Cell function is tightly regulated by surface receptors. Earlier reports showed that herpes simplex virus 1 regulates by diverse mechanisms the presentation of antigenic peptides, downregulates the signaling pathways associated with receptor tyrosine kinases, and posttranslationally modifies members of the Src family of protein kinases. Here we report that the receptor for tumor necrosis factor alpha (TNF-R1) rapidly disappears from both the cell surface and total cell lysates in cells infected with wild-type virus or a variety of mutants but not in cells infected with the mutant DeltaU(L)41, which lacks the U(L)41 gene, the virion host shutoff gene. The half-life of TNF-R1 appears to be less than 30 min in both mock-infected and infected cells. The disappearance of TNF-R1 correlates with the disappearance of cytoplasmic TNF-R1 mRNA in wild-type-virus-infected cells. The results suggest that by degrading the TNFR1 mRNA, the virus precludes the replenishment of naturally decaying TNF-R1.

FIG. 1.

TNF-R1 downregulation in infected HEp-2 cells. (A) HEp-2 cells were mock infected or exposed to 10 PFU of HSV-1(F) per cell. The cells were harvested 6 h after infection and total cell lysate or extracted surface proteins were separated on 10% denaturing polyacrylamide gel, transferred to a nitrocellulose gel and reacted with anti-TNF-R1. (B) Time course of down regulation of total TNF-R1 in infected cells. HEp-2 cells were mock infected or exposed to 5 PFU of HSV-1(F), ΔICP0, ICP0 RING finger mutant ΔR-F, or ΔU_L41 per cell. Cells were harvested at 2, 4, and 8 h after infection. Total cell lysates were separated on 10% denaturing polyacrylamide gels and immunoblotted with anti-TNF-R1. A, cross-reacting cellular protein that served as a loading control.

FIG. 2.

Downregulation of TNR-R1 is cell type independent. Replicate cultures of HEp-2, U2OS, or HeLa cells were mock infected or exposed to 10 PFU of the indicated virus per cell. The cells were harvested at 6 h after infection, and total cell lysates were separated on 10% denaturing polyacrylamide gels and immunoblotted with anti-TNF-R1 antibody.

FIG. 3.

Half-life of TNF-R1 in infected HEp-2 cells. HEp-2 cells were mock infected or exposed to 10 PFU of HSV-1(F) or ΔU_L41 per cell. Cycloheximide (100 μg/ml) was added at the time of exposure of cells to virus (0 h) and maintained during the infection. The cells were harvested at 0, 0.5, 1.0, 1.5, 2.0, or 2.5 h after infection. Total cell lysates were separated on a 10% denaturing polyacrylamide gel and immunoblotted with anti-TNF-R1 antibody or antiactin antibody.

FIG. 4.

Northern blot of TNF-R1 in infected HeLa cells. HeLa cells were mock infected or exposed to 5 PFU of virus per cell. Cells were harvested at 1, 3, or 7 h after infection, and cytoplasmic RNAs were extracted and separated on a 1% agarose gel followed by Northern blot analysis with ³²P-labeled full-length TNF-R1 cDNA.