؎ 5%, and 97% ؎ 5% of TG cell cultures, respectively (means ؎ standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% ؎ 5%, 8% ؎ 0%, 0% ؎ 0%, and 13% ؎ 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.The life cycle of herpes simplex virus type 1 (HSV-1) in humans can be divided into three phases: (i) productive replication of virus at the site of primary infection, (ii) establishment and maintenance of latency in sensory neurons, and (iii) periodic reactivation of viral infection from neuronal latency. The first phase, productive replication, is accurately reproduced in vitro in mammalian cell lines, and thus the molecular events that occur during productive HSV-1 replication have been studied extensively (44). The second and third phases of the HSV-1 life cycle, latency and reactivation, respectively, have been experimentally reproduced in animals such as mice, guinea pigs, and rabbits. These models were instrumental in identifying sensory neurons of the peripheral nervous system as the primary sites of HSV-1 latency (52), identifying and characterizing the latency-associated transcripts (LATs) (43,53), and investigating the physiological stimuli that induce HSV-1 reactivation (19,28,48). Because of the problems associated with conducting molecular studies in animals, however, it has proven difficult for investigators to move beyond descriptive and phenomenological observations. Therefore, the molecular mechanisms that control HSV-1 latency and reactivation remain to be elucidated.Primary trigeminal ganglion (TG) cell cultures were developed as an alternative model in which to study 29,36). Although HSV-1 latency is established in mice by conventional methods in this model (18,28,48), reactivation is analyzed ex vivo in dissociated cultures of latently infected TG cells. Monolayer cultures are treated transiently with acyclovir (ACV) or other antiviral drugs to repress reactivation during culture establishment (16,17,36), and latently infected, nondividing neurons are randomly distributed among dividing support cells (16). After removal of antiviral drugs, reactivation of latent HSV-1 can be induced in 70 to 95% of TG cell cultures by heat stress, and neurons have been shown

bHerpes simplex virus type 1 (HSV-1) strain KOS has been extensively used in many studies to examine HSV-1 replication, gene expression, and pathogenesis. Notably, strain KOS is known to be less pathogenic than the first sequenced genome of HSV-1, strain 17. To understand the genotypic differences between KOS and other phenotypically distinct strains of HSV-1, we sequenced the viral genome of strain KOS. When comparing strain KOS to strain 17, there are at least 1,024 small nucleotide polymorphisms (SNPs) and 172 insertions/deletions (indels). The polymorphisms observed in the KOS genome will likely provide insights into the genes, their protein products, and the cis elements that regulate the biology of this HSV-1 strain.

The herpes simplex virus type 1 (HSV-1) encoded E3 ubiquitin ligase, infected cell protein 0 (ICP0), is required for efficient lytic viral replication and regulates the switch between the lytic and latent states of HSV-1. As an E3 ubiquitin ligase, ICP0 directs the proteasomal degradation of several cellular targets, allowing the virus to counteract different cellular intrinsic and innate immune responses. In this review, we will focus on how ICP0’s E3 ubiquitin ligase activity inactivates the host intrinsic defenses, such as nuclear domain 10 (ND10), SUMO, and the DNA damage response to HSV-1 infection. In addition, we will examine ICP0’s capacity to impair the activation of interferon (innate) regulatory mediators that include IFI16 (IFN γ-inducible protein 16), MyD88 (myeloid differentiation factor 88), and Mal (MyD88 adaptor-like protein). We will also consider how ICP0 allows HSV-1 to evade activation of the NF-κB (nuclear factor kappa B) inflammatory signaling pathway. Finally, ICP0’s paradoxical relationship with USP7 (ubiquitin specific protease 7) and its roles in intrinsic and innate immune responses to HSV-1 infection will be discussed.

The cyclin-dependent kinase (cdk) inhibitor Roscovitine (Rosco) reduces transcription of herpes simplex virus early genes significantly, even in the presence of wild-type levels of immediate-early (IE) viral proteins, suggesting that the transactivating functions of IE proteins may require the activities of one or more Roscosensitive cdk (L. M. Schang, A. Rosenberg, and P. A. Schaffer, J. Virol. 73:2161-2172, 1999). Based on this observation, we sought to determine whether Rosco alters the transactivating activity and posttranslational modification state of the IE protein, infected cell protein 0 (ICP0), in KOS6␤-infected Vero cells. KOS6␤ is a KOS-derived recombinant virus containing an ICP0-inducible ICP6 promoter::lacZ cassette. To monitor ICP0's transactivating activity, KOS6␤-infected cells were released from a cycloheximide (CHX)-mediated protein synthesis block into medium with or without Rosco, and ␤-galactosidase activity was measured. Rosco inhibited the ability of ICP0 to transactivate the ICP6 promoter by 50-fold. This inhibition was shown not to be a consequence of inhibition of ICP6 basal promoter activity or aberrant nuclear localization of ICP0. Rosco also altered the electrophoretic mobility of a portion of ICP0 molecules derived from KOS-infected cells following reversal of a CHX block. Notably, however, Rosco had only a minimal effect on the phosphorylation state of ICP0. We conclude that ICP0's transactivating activity requires Rosco-sensitive cdks and hypothesize that these cdks regulate the functions of cellular enzymes which modify ICP0, and are, consequently, required for its transactivating activity. Thus, we propose that Rosco regulates ICP0's posttranslational state by mechanisms other than, or in addition to, phosphorylation.Productive infection of actively dividing epithelial cells and fibroblasts by herpes simplex virus type 1 (HSV-1) is characterized by expression of all or nearly all viral genes, leading to the production of new infectious virus and cell death (reviewed in reference 62). The activities of HSV immediate-early (IE) regulatory proteins are required for the transcriptional activation of early and late genes and for the repression of IE gene transcription during productive infection (34). In nondividing neurons, however, the activities of IE regulatory proteins are repressed such that the productive phase of viral gene expression does not occur or occurs transiently, leading to latent infection. After stress, neurons again become permissive for viral gene expression leading to the production of new infectious virus (reactivation). The fact that nondividing neurons can support latent as well as productive infection (reactivation) implicates the differential expression and/or activation of cellular proteins in determining whether neuronal infection will be latent or productive. In support of this concept, Schang and coworkers have shown that inhibitors of cyclin-dependent kinases (cdks) block productive infection and reactivation from neuronal latency, implicating cdks a...

Herpes simplex virus type 1 (HSV-1) has two distinct phases of its viral life cycle: lytic and latent. One viral immediate-early protein that is responsible for determining the balance between productive lytic replication and reactivation from latency is infected cell protein 0 (ICP0). ICP0 is a 775-amino acid really interesting new gene (RING)-finger-containing protein that possesses E3 ubiquitin ligase activity, which is required for ICP0 to activate HSV-1 gene expression, disrupt nuclear domain (ND) 10 structures, mediate the degradation of cellular proteins, and evade the host cell’s intrinsic and innate antiviral defenses. This article examines our current understanding of ICP0’s transactivating, E3 ubiquitin ligase, and antihost defense activities and their inter-relationships to one another. Lastly, we will discuss how these properties of ICP0 may be utilized as possible targets for HSV-1 antiviral therapies.