Neurons Differentially Activate the Herpes Simplex Virus Type 1 Immediate-Early Gene ICP0 and ICP27 Promoters in Transgenic Mice

The expression of herpes simplex virus (HSV) genomes in the absence of viral regulatory proteins in sensory neurons is poorly understood. Previously, our group reported an HSV immediate early (IE) mutant (d109) unable to express any of the five IE genes and encoding a model human cytomegalovirus immediate early promoter-green fluorescent protein (GFP) transgene. In cultured cells, GFP expressed from this mutant was observed in only a subset of infected cells. The subset exhibited cell type dependence, as the fractions of GFP-expressing cells varied widely among the cell types examined. Herein, we characterize this mutant in murine embryonic trigeminal ganglion (TG) cultures. We found that d109 was nontoxic to neural cultures and persisted in the cultures throughout their life spans. Unlike with some of the cultured cell lines and strains, expression of the GFP transgene was observed in a surprisingly large subset of neurons. However, very few nonneuronal cells expressed GFP. The abilities of ICP0 and an inhibitor of histone deacetylase, trichostatin A (TSA), to activate GFP expression from nonexpressing cells were also compared. The provision of ICP0 by infection with d105 reactivated quiescent genomes in nearly every cell, whereas reactivation by TSA was much more limited and restricted to the previously nonexpressing neurons. Moreover, we found that d109, which does not express ICP0, consistently reactivated HSV type 1 (KOS) in latently infected adult TG cultures. These results suggest that the state of persisting HSV genomes in some TG neurons may be more dynamic and more easily activated than has been observed with nonneuronal cells.Infection with herpes simplex virus (HSV) in vivo occurs in three stages: primary (productive) infection, latency, and reactivation. During primary or lytic infection, the virus replicates and produces progeny in susceptible epithelial cells, an event which may or may not be accompanied by the appearance of mucocutaneous lesions, the hallmark of HSV infection (46). The primary infection almost invariably culminates in the infection of innervating sensory neurons, which then become sites of HSV latency. Throughout the lifetime of the host, HSV reactivates, both spontaneously and in response to specific reactivating stimuli, causing recurrent infections. These episodic reactivations often produce lesions at or near the primary site of infection, but are frequently asymptomatic (49,(89)(90)(91).During productive infection, viral gene expression proceeds in a temporally controlled cascade of the immediate early (IE), early (E), and late (L) genes (38, 39). Viral gene expression during latency and reactivation, both of which occur in neurons, differs from that observed during lytic infection (19,50,51). Latency is characterized by the absence of productive viral transcripts, save for the latency-associated transcripts, whose exact function in maintaining or reactivating from the latent state is still without consensus (4, 5, 20, 35, 42, 45, 52, 58, 72-74, 81, 86). Viral gene expressi...

Section: Discussionsupporting

confidence: 74%

Relaxed Repression of Herpes Simplex Virus Type 1 Genomes in Murine Trigeminal Neurons

Terry-Allison

Smith

DeLuca

2007

“…Loiacono et al employed transgenic mice expressing ␤-Gal from the ICP0 and other promoters to examine promoter function in neurons in vivo in the absence of viral gene products. Expression of ␤-Gal from the ICP0 promoter was detected in some sensory neurons in several transgenic lines (41). Very rare neurons express viral proteins during latency (21,52,59), and the differences noted between these studies may reflect differences in the probability of detecting such rare events in the various systems.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Herpes Simplex Virus ICP0 Promoter Function in Sensory Neurons during Acute Infection, Establishment of Latency, and Reactivation In Vivo

Thompson

Shieh

Sawtell

2003

Despite the extensive repression of herpes simplex virus type 1 (HSV-1) gene transcription during latency, a variety of stressful stimuli can result in the reactivation of the latent genome and reentry into lytic-phase transcription (71). How the quiescent genome exits latency is not yet known. It has been suggested that ICP0 is uniquely important for the initiation of viral reactivation (reviewed in reference 19). ICP0 is a major viral transcriptional activator important for the expression of viral genes of diverse kinetic classes (8-10, 12, 60). In addition, this protein functions to promote viral mRNA translation and regulates levels of cellular proteins through interaction with the ubiquitin-proteosome pathway (18,20,45,49). These properties suggest that ICP0 can regulate both viral and host protein levels through transcriptional, translational, and posttranslational mechanisms. In a tissue culture model of latent infection, ICP0 was implicated in the efficient establishment of latency (74). ICP0-expressing adenovirus vectors that lack all other HSV genes have been shown to induce viral replication in quiescently infected tissue cultures (28,75). Further, a viral mutant lacking functional ICP0 but containing all other viral genes did not induce viral replication in similar cultures, suggesting a specific role for ICP0 (28). However, adenovirus vectors expressing ICP4 and VP16 can also induce viral replication in primary cultures derived from latently infected ganglia (26).In animal models ICP0 mutant strains can establish latent infections and can reactivate in cocultivation assays, but the levels of efficiency for both of these processes are greatly reduced compared to that seen with the wild-type strain (7,10,27,33,37). One problem with these studies is that ICP0 null mutants establish latency inefficiently, and it is therefore difficult to distinguish between the importance of this gene for establishing latency and its importance for initiating reactivation. Recently, Halford and Schaffer infected immunosuppressed mice with ICP0 null mutants in an effort to increase the establishment of latency by the null mutants. Those authors concluded (on the basis of the total amount of HSV DNA detected in ganglia by PCR and the number of explant cultures * Corresponding author. Mailing address for Richard

“…This is especially significant when it is important to accurately quantify rare events. These same advantages are provided by whole-mount staining of reporter mutants or promoter/reporter-containing transgenic mice, and we and others have found this approach to be useful for certain latency and reactivation studies (16,23,26,37,41). However, the use of promoter reporter mutants as surrogate markers for protein expression requires demonstration that expression of the reporter and the protein coincide.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Analysis of Herpes Simplex Virus Reactivation In Vivo Demonstrates that Reactivation in the Nervous System Is Not Inhibited at Early Times Postinoculation

Sawtell

2003

Recent studies utilizing an ex vivo mouse model of herpes simplex virus (HSV) reactivation have led to the hypothesis that, under physiologic conditions inducing viral reactivation, the immune cells within the infected ganglion block the viral replication cycle and maintain the viral genome in a latent state. One prediction from the ex vivo study is that reactivation in ganglia in vivo would be inhibited at early times postinoculation, when the numbers of inflammatory cells in the ganglia are greatest. To distinguish between an effect of the immune infiltrates on (i) infectious virus produced and/or recovered in the ganglion and (ii) the number of neurons undergoing lytic transcriptional activity (reactivating), an assay to quantify the number of neurons expressing lytic viral protein in ganglia in vivo was developed. Infectious virus and HSV protein-positive neurons were quantified from days 9 through 240 postinoculation in latently infected trigeminal ganglia before and at 22 h after hyperthermic-stress-induced reactivation. Significant increases in the amount of virus and the number of positive neurons were detected poststress in ganglia at all times examined. Unexpectedly, the greatest levels of reactivation occurred at the times examined most proximal to inoculation. Acyclovir was utilized to stop residual acute-phase virus production, and this treatment did not reduce the level of reactivation on day 14. Thus, the virus measured after induction was a product of reactivation. These data indicate that, in contrast to observations in the ex vivo model, immune cells in the ganglia during the resolution of acute infection do not inhibit reactivation of the virus in ganglia in vivo.Herpes simplex virus (HSV) invades the host nervous system during infection at the body surface (7,38,40,44; for a review, see reference 8). In the nervous system, the virus proceeds through the lytic replicative cycle in some neurons, whereas in others lytic-phase transcription is either not initiated or aborted and the viral genome enters a transcriptionally repressed or latent state (see reference 31 for a recent review). These latently infected neurons serve as a lifelong reservoir of viral genetic information within the host. Periodically, in response to stressful stimuli, there is reentry into lytic-phase transcription and infectious virus is produced. This virus is amplified and shed at the surface, either asymptomatically or in association with lesions.In animal models and in humans, HSV reactivation occurs despite an activated competent immune response. Although several potential strategies for immunoevasion by HSV have been identified (discussed in references 1, 19, and 24), which, if any, of these strategies are important for recurrent disease and transmission remains unclear. There is evidence in animal models that augmenting the immune response by specific immunotherapeutic strategies can reduce peripheral disease associated with reactivation (13,14,28,30,47,48). Theoretically, immunotherapeutic strategies could function ...