Differential effects of nerve growth factor and dexamethasone on herpes simplex virus type 1 oriL- and oriS-dependent DNA replication in PC12 cells

Abstract

The herpes simplex virus type 1 (HSV-1) genome contains three origins of DNA replication, one copy of oriL and two copies of oriS. Although oriL and oriS are structurally different, they have extensive nucleotide sequence similarity and can substitute for each other to initiate viral DNA replication. A fundamental question that remains to be answered is why the HSV-1 genome contains two types of origin. We have recently identified a novel glucocorticoid response element (GRE) within oriL that is not present in oriS and have shown by gel mobility shift assays that purified glucocorticoid receptor (GR), as well as GR present in cellular extracts, can bind to the GRE in oriL. To determine whether glucocorticoids and the GRE affect the efficiency of oriL-dependent DNA replication, we performed transient DNA replication assays in the presence and absence of dexamethasone (DEX). Because HSV-1 is a neurotropic virus and establishes latency in cells of neural origin, these tests were conducted in PC12 cells, which assume the properties of sympathetic neurons when differentiated with nerve growth factor (NGF). In NGF-differentiated PC12 cells, oriL-dependent DNA replication was enhanced 5-fold by DEX, whereas in undifferentiated cells, DEX enhanced replication ϳ2-fold. Notably, the enhancement of oriL function by DEX was abolished when the GRE was mutated. NGF-induced differentiation alone had no effect. In contrast to oriL, oriS-dependent DNA replication was reduced ϳ5-fold in NGF-differentiated PC12 cells and an additional 4-fold in differentiated cells treated with DEX. In undifferentiated PC12 cells, DEX had only a minor inhibitory effect (ϳ2-fold) on oriS function. Although the cis-acting elements that mediate the NGF-and DEX-specific repression of oriS-dependent DNA replication are unknown, a functional GRE is critical for the DEX-induced enhancement of oriL function in NGF-differentiated PC12 cells. The enhancement of oriL-dependent DNA replication by DEX in differentiated PC12 cells suggests the possibility that glucocorticoids, agents long recognized to enhance reactivation of latent herpesvirus infections, act through the GRE in oriL to stimulate viral DNA replication and reactivation in terminally differentiated neurons in vivo.

The Glucocorticoid Receptor (GR) Stimulates Herpes Simplex Virus 1 Productive Infection, in Part Because the Infected Cell Protein 0 (ICP0) Promoter Is Cooperatively Transactivated by the GR and Krüppel-Like Transcription Factor 15

“…Furthermore, the activated GR can bind a subset of GREs in silent chromatin (29,30) and induce a nuclease-hypersensitive site, which culminates in transcription (9), all properties consistent with the GR being coined as a "pioneer transcription factor" (31). One of the GREs in the viral genome is within the HSV-1 origin of replication (oriL), but not oriS, and this GRE stimulates replication of a plasmid in neuronal-like cells (32). Interestingly, mutagenesis of the GRE in oriL reduces viral pathogenesis during acute infection and impairs reactivation from latency (33).…”

Phosphorylation of the Herpes Simplex Virus Type 1 Origin Binding Protein

“…Vero cells (ATCC CCL-81) were propagated and maintained as described previously (10). PC12 cells (a gift of John Wagner, Cornell University, Medical College, New York, N.Y.) were grown and differentiated for 7 days with nerve growth factor (NGF) as described previously (16). HSV-1 wild-type strain KOS was used throughout these studies.…”

“…Gel mobility shift assay and phosphatase treatment. Nuclear extracts of HSVinfected Vero or NGF-differentiated PC12 cells were prepared as previously described (16). Total protein concentrations were measured by the method of Bradford (Bio-Rad, Hercules, Calif.) using a standard curve generated with bovine serum albumin as the protein source.…”

Herpes Virus Replication

“…Nevertheless, in cell culture, these origins appear to have similar origin activity and are redundant since mutant viruses with a single class of origin show no defect in replication (8,9). oriL has been shown to possess a functional glucocorticoid response element that increases origin activity in neuronal cells which may be related to enhanced reactivation of latent virus by glucocorticoid treatment (10). However, it should be noted that oriL is not essential for reactivation (8).…”

“…For example, the recent discovery that stress proteins modulate the originbinding activity of the HSV-1 initiator protein implicates their participation following reactivation due to stress (61,62). Similarly, the glucocorticoid response element in oriL may play a role in activating this origin during reactivation from latency (10).…”