Analysis of the Functions of Herpes Simplex Virus Type 1 Regulatory Protein ICP0 That Are Critical for Lytic Infection and Derepression of Quiescent Viral Genomes

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 is important for stimulating the initiation of the lytic cycle and efficient reactivation of latent or quiescent infection. Extensive investigation has suggested several potential functions for ICP0, including interference in the interferon response, disruption of functions connected with PML nuclear bodies (ND10), and inhibition of cellular histone deacetylase (HDAC) activity through an interaction with the HDAC-1 binding partner CoREST. Analysis of the significance of these potential functions and whether they are direct or indirect effects of ICP0 is complicated because HSV-1 mutants expressing mutant forms of ICP0 infect cells with widely differing efficiencies. On the other hand, transfection approaches for ICP0 expression do not allow studies of whole cell populations because of their limited efficiency. To overcome these problems, we have established a cell line in which ICP0 expression can be induced at levels pertaining during the early stages of HSV-1 infection in virtually all cells in the culture. Such cells enable 100% complementation of ICP0-null mutant HSV-1. Using cells expressing the wild type and a variety of mutant forms of ICP0, we have used this system to analyze the role of defined domains of the protein in stimulating lytic infection and derepression from quiescence. Activity in these core functions correlated well the ability of ICP0 to disrupt ND10 and inhibit the recruitment of ND10 proteins to sites closely associated with viral genomes at the onset of infection, whereas the CoREST binding region was neither sufficient nor necessary for ICP0 function in lytic and reactivating infections.Herpes simplex virus type 1 (HSV-1) is an important human pathogen that infects the majority of the population at an early age and then establishes a life-long latent infection in sensory neurones. Periodic reactivation of latent virus causes episodes of active disease characterized by epithelial lesions at the site of the original primary infection. As with all herpesviruses, the ability of HSV-1 to establish and reactivate from latency is key to its clinical importance and evolutionary success. Therefore, the molecular mechanisms that regulate these processes have been the subject of intensive research (reviewed in reference 15). HSV-1 immediate-early (IE) protein ICP0 is required for efficient reactivation from latency in both mouse models and cultured cell systems of quiescent infection (15). ICP0 is also required to stimulate lytic infection by enhancing the probability that a cell receiving a viral genome will engage in productive infection (reviewed in references 19, 20 and 42). Therefore, a full understanding of the biology of HSV-1 infection requires a definition of the functions and mode of action of ICP0.The basic phenotype of ICP0-null mutant HSV-1 is a low probability of plaque formation, particularly in human diploid fibroblasts, that causes a high particle-to-PFU ratio (reference 20 and references therein). Biochemically, ICP0 is an E3 ubiquitin ligase of the RING finger class (4) that induces the degradation of several cellular proteins, including the promyelocytic leukemia (PML) protein (23), centromere proteins including CENP-C (54, 55), and the catalytic subunit of DNA-protein kinase (53, 72). Among the consequences of these activities are the disruption of PML nuclear bodies (herein termed nuclear domain 10 [ND10]) (24, 58) and centromeres (54). ICP0 has also been reported to interact with histone deacetylase enzymes (HDACs) (56) and the CoREST repressor protein, thereby disrupting the CoREST/HDAC-1 complex (37, 39). Evidence has also been presented that expression of ICP0 correlates with increased acetylation of histones on viral chromatin (12). ICP0-null mutant viruses replicate less efficiently than the wild type (wt) in cells pretreated with interferon (IFN) (44, 66), and there is evidence that ICP0 is able to impede an IFN-independent induction of IFN-stimulated genes that arises after infection with defective HSV-1 mutants (16, 59, 60, 65, 67, 76). As a further complication, ICP0-null mutant HSV-1 replicates more efficiently in cells that have been highly stressed by a variety of treatments (5, 6, 79).On the basis of this evidence, several not necessarily mutually exclusive hypotheses have been put forward to explain the biological effects of ICP0. These include (i) that ICP0 counteracts an intrinsic cellular resistance mechanism that involves PML and other components of ND10, (ii) that ICP0 overcomes the innate cellular antiviral defense based on the IFN pathway, and (iii) that ICP0 counteracts the establishment of a repressed chromatin structure on the viral genome by interfering with histone deacetylation. The aim of this paper is to investigate some of these issues using a novel inducible expression system. The question of the effects of ICP0 on IFN pathways is considered in the companion paper (28).The brief and by no means exhaustive summary of the functions and activities attributed to ICP0, presented above, illustrates that the understanding of ICP0 is a difficult issue. It is further complicated by the difficulty of working with ICP0-null mutant viruses under tightly controlled conditions. This arises because the defect varies greatly between different cell types, is highly dependent on the multiplicity of infection (MOI), and varies in a nonlinear manner with respect to virus dose (reference 20 and references therein). Furthermore, use of ICP0 mutant viruses in cultured cell models of reactivation of quiescent HSV-1 is complicated by competition between the resident quiescent viral genome targeted for reactivation and the genomes of the superinfecting virus used to induce the reactivation (75). Therefore, it is very difficult to establish infections with wt and ICP0 mutant viruses that are truly comparable in a way that allows clear distinctions between the direct effects of ICP0 and indirect effects that are due either to expression of other viral proteins that are expressed more efficiently in the presence of ICP0 or to less specific consequences of an active infection and subsequent effects on the cell. Here, we describe a system that enables expression of ICP0 in an inducible manner at levels similar to those at the early stages of infection in almost all cells in a population. We have used this system to study wt and mutant forms of ICP0 in assays of lytic infection and derepression of quiescent viral genomes in a cultured cell model of latency. We discuss the results in terms of the requirements of specific regions of the ICP0 protein for stimulating lytic infection and derepression of quiescent genomes, the potential biological significance of ND10 disruption, recruitment of ND10 components to the sites of HSV-1 genomes at the outset of virus infection, and the interaction of ICP0 with CoREST.