It is estimated that over five billon people are presently infected with herpes simplex virus (HSV) type 1 (HSV-1) worldwide. The perpetual HSV pandemic is due in large part to the ability of the virus to establish latent infections that persist for the life of the host. These latent infections periodically reactivate, which can lead to contagious surface lesions, stromal keratitis (an important cause of blindness), and rare but fatal encephalitis (59
During acute infection of sensory ganglia, some neurons are productively infected and die, while concomitantly in other neurons a latent infection is established (23
). After the acute stage of infection ends, viral proteins are very rarely detected during latency, and these rare positive neurons are thought to be undergoing reactivation (12
). Viral genetics, inoculation titer, and route of infection are important parameters that influence the number of neurons in which latency is established (41
). In mice, as many as 25% of the neurons in a trigeminal ganglion (TG) can become latently infected, and the individual neurons in this reservoir contain variable numbers of viral genomes ranging from 1 to >1,000 (36
). These values are in general agreement with estimates of latency in human TG (7
). The latent virus can reactivate following a variety of stressful stimuli (59
), and the percentage of neurons in which latency is established as well as the average number of viral genomes present have been shown to correlate with the frequency of reactivation in the mouse model (38
). The molecular mechanisms that regulate reactivation are not known.
A central question is how reactivation from latency is initiated in neurons in the absence of the virion proteins that are critical for initiating lytic-phase transcription from the viral genome, such as VP16. Various stressful stimuli can lead to reactivation from latency (Fig. ). These systemic stresses induce changes in the physiologic state of sensory neurons that contain the latent viral genome (42
). If stress-induced changes result in detectable viral proteins in a neuron, the viral genome has exited the latent state. Thus, the process of reactivation has been initiated in these neurons. The initiation of reactivation is highly correlated with progression to infectious virus production (reactivation) with wild-type strains of HSV-1 (39
FIG. 1. Schematic of HSV reactivation from the latent state following stress. Stressful stimuli induce changes within sensory neurons that induce virus reactivation from the latent state. Many studies have shown that infectious virus is produced in latently infected (more ...)
A favored hypothesis is that the promoter for the viral ICP0 gene responds to stress-induced signals and the ICP0 protein serves to initiate reactivation from latency (11
). ICP0 is a multifunctional protein that can regulate the levels of both viral and host proteins at the transcriptional, translational, and posttranslational levels (11
). Another potential mechanism for ICP0 altering host and perhaps viral gene expression is the interaction of ICP0 with host cell histone deacetylases (13
). Indeed, several studies have demonstrated that ICP0 null mutants reactivate less efficiently (as measured by the detection of infectious virus) than the wild type when assayed in vitro (1
Importantly, excising and explanting the ganglia result in rapid, widespread changes in the physiology of the ganglion cells, including neurons. These changes do not occur during reactivation in vivo (42
), raising the possibility that in the explant setting, the role of viral proteins such as ICP0 could be obviated or obscured. Since it is critical to unambiguously determine the viral gene(s) and its (their) promoter(s) that initiate reactivation, we examined the potential role of ICP0 for the initiation of reactivation in vivo. The long-term goal for these studies is to identify the responsive viral promoter elements to gain insight into the cellular factors and, ultimately, the signaling cascade that mediate reactivation following subjection to stressful stimuli.
Distinguishing between a requirement for ICP0 in the initiation of reactivation and one for the efficient production and/or detection (i.e., plaquing efficiency) of infectious virions has not been attempted. We have recently developed approaches that can distinguish between these possibilities. For example, while viral thymidine kinase (TK)-negative mutants do not produce infectious virus (reactivate), they do express lytic-phase viral proteins (initiate reactivation) in sensory neurons following hyperthermic stress in vivo (42
). Therefore, the number of neurons in which HSV exits latency can be quantified in the absence of infectious virus production.
Here we report the results of experiments designed to directly investigate the role of ICP0 in the initiation of reactivation from latency in vivo. The strain 17syn+ ICP0 null mutants dl1403 (50
) and FXE (10
) did not reactivate in vivo (as assayed by infectious virus production), and the KOS-based ICP0 promoter mutant, ΔTfi (9
), was severely impaired. Thus, ICP0 appears to be required for the production of detectable infectious virus during reactivation in vivo. However, the ICP0 null mutants did enter the lytic cycle as efficiently as the parental strain, as evidenced by the number of neurons expressing viral lytic-phase proteins. Further, ΔTfi promoter mutant initiated reactivation as efficiently as ΔTfiR and the parental wild-type virus. We conclude that the initiation of reactivation occurs by a mechanism that does not require ICP0 function.