Characterization of a nerve growth factor-inducible cellular activity that enhances herpes simplex virus type 1 gene expression and replication of an ICP0 null mutant in cells of neural lineage

Abstract

Herpes simplex virus type 1 (HSV-1) ICP0 is required for efficient viral gene expression during lytic infection, especially at low multiplicities. A series of cellular activities that can substitute for ICP0 has been identified, suggesting that when the activity of ICP0 is limiting, these activities can substitute for ICP0 to activate viral gene expression. The cellular activities may be especially important during reactivation of HSV from neuronal latency when viral gene expression is initiated in the absence of prior viral protein synthesis. Consistent with this hypothesis, we have identified an inducible activity in cells of neural lineage (PC12) that can complement the low-multiplicity growth phenotype of an ICP0 null mutant, n212. Pretreatment of PC12 cells with nerve growth factor (NGF) or fibroblast growth factor (FGF) prior to infection produced a 10- to 20-fold increase in the 24-h yield of n212 but only a 2- to 4-fold increase in the yield of wild-type virus relative to mock treatment. Slot blot analysis of nuclear DNA isolated from infected cells treated or mock treated with NGF indicated that NGF treatment does not significantly affect viral entry. The NGF-induced activity in PC12 cells was expressed transiently, with peak complementing activity observed when cells were treated with NGF 12 h prior to infection. Addition of NGF 3 h after infection had little effect on virus yield. The NGF-induced cellular activity was inhibited by pretreatment of PC12 cells with kinase inhibitors that have high specificity for kinases involved in NGF/FGF-dependent signal transduction. RNase protection assays demonstrated that the NGF-inducible PC12 cell activity, like that of ICP0, functions to increase the level of viral mRNA during low-multiplicity infection. These results suggest that activation of viral transcription by ICP0 and transcriptional activation of cellular genes by NGF and FGF utilize common signal transduction pathways in PC12 cells.

FIG. 1

Effects of growth factors on replication of n212 and KOS. PC12 cells (10⁶/35-mm-diameter dish) were treated with 100 ng of NGF, FGF, or EGF per ml or 50 mM KCl in the presence and absence of EGF for 3 h prior to infection with 0.01 PFU of KOS or n212 per cell. At 24 hpi, the cultures were harvested and viral yields were measured. Titers of KOS and n212 were determined on Vero cells and U2OS cells, respectively. The data are expressed as fold difference in titer relative to mock treatment.

FIG. 2

Growth of KOS and n212 in PC12 cells in the presence and absence of NGF. PC12 cells (10⁶/35-mm-diameter dish) were mock treated or treated with NGF at 100 ng/ml for 3 h prior to infection with KOS or n212 at 0.02 or at 2.5 PFU/cell. At the times indicated, cultures were harvested and viral yields were measured by plaque assay. Titers of KOS and n212 were determined on Vero cells and U2OS cells, respectively.

FIG. 3

NGF does not affect viral entry. PC12 cells were mock treated or treated with NGF (100 ng/ml) for 3 h prior to infection with KOS or n212. Nuclear DNA was harvested prior to the onset of viral DNA replication at 3 hpi and applied to a nylon membrane by slot blot hybridization (inf.). Cesium chloride-purified KOS DNA was applied to the membrane as indicated (std.). The blot was probed for viral DNA by using ³²P-labeled nick-translated KOS DNA. The blot was stripped and reprobed for cellular DNA by using a ³²P-labeled antisense RNA probe specific for the cellular gene GAPDH. The image was visualized by PhosphorImager analysis. The range values for the image display were set at 0 to 791 counts for the KOS probe and 0 to 258 counts for the GAPDH probe.

FIG. 4

Serine/threonine kinase inhibitors block NGF-dependent replication of n212. PC12 cells (10⁶/35-mm-diameter dish) were incubated for 30 min prior to NGF addition with serine/threonine kinase inhibitors at the following concentrations: K252a, 0.25 μM; KT5720, 0.5 μM; PD98059, 20 μM; and calphostin C, 0.5 μM. At 3 h posttreatment, the cultures were infected with n212 (0.01 PFU/cell) in the presence and absence of inhibitors and NGF. At 24 hpi, the cultures were harvested and viral yields were measured. The data are expressed as percentage of NGF-induced n212 replication, which was set at 100% to show all of the data in a single figure. MAPKinase, mitogen-activated kinase.

FIG. 5

Time course of the NGF-induced n212-complementing activity. (A) PC12 cells (10⁶/35-mm-diameter dish) were treated with NGF (100 ng/ml) 3 h prior to infection, at the time of infection, or 15 min, 30 min, 1 h, and 3 h after infection with 0.01 PFU of KOS and n212 per cell. Virus yields were measured at 24 hpi and expressed as fold increase in viral replication relative to mock-treated samples. (B) PC12 cells (10⁶/35-mm-diameter dish) were treated with NGF (100 ng/ml) for 144, 72, 48, 24, 12, 6, and 3 h prior to infection, at the time of infection, or 3 h after infection with 0.01 PFU of n212 or KOS per cell. Virus yields were measured at 24 hpi. NGF-induced n212-complementing activity is expressed as fold increase in viral titer relative to mock-treated samples.

FIG. 6

NGF induces viral mRNA accumulation in infected PC12 cells. PC12 cells (5 × 10⁶/60-mm-diameter dish) were mock treated or treated with NGF (100 ng/ml) for 12 h prior to infection with 0.1 PFU of KOS or n212 per cell. At 0, 4, 7, and 10 hpi, cytoplasmic RNA was isolated and levels of ICP4, TK, and gC mRNAs were measured by quantitative RNase protection assay. The range values for the image display were set at 0 to 2,000 (ICP4), 0 to 1,000 (TK), and 0 to 200 (gC).

FIG. 7

ICP0 does not induce c-fos expression. PC12 cells (5 × 10⁶/60-mm-diameter dish) were treated with NGF (100 ng/ml) for 0, 15, 30, and 60 min or infected with 5.0 PFU of KOS and n212 per cell for 30, 60, 90, and 120 min. Cytoplasmic RNA was isolated at each time point as described in the text. The RNA was size fractionated by denaturing gel electrophoresis and transferred to a nylon membrane by Northern blotting. The Northern blot was probed with 100 ng of ³²P-labeled antisense RNA probe (8 × 10⁵ cpM/ng) specific for the mouse c-fos message. The blot was reprobed with 150 ng of ³²P-labeled antisense RNA probe (8.8 × 10⁵ cpM/ng) specific for the rat GAPDH message. The image was visualized by PhosphorImager analysis. The range values for the image display were set at 0 to 500 counts.