Induction of Cellular Stress Overcomes the Requirement of Herpes Simplex Virus Type 1 for Immediate-Early Protein ICP0 and Reactivates Expression from Quiescent Viral Genomes

Abstract

Herpes simplex virus type 1 (HSV-1) mutants impaired in the activities of the structural protein VP16 and the immediate-early (IE) proteins ICP0 and ICP4 establish a quiescent infection in human fibroblasts, with most cells retaining an inactive, repressed viral genome for sustained periods in culture. To date, the quiescent state has been considered stable, since it has been reversed only by provision of herpesviral proteins, such as ICP0, not by alteration of the cell physiological state. We report that the interaction of HSV-1 with human fibroblasts can be altered significantly by transient treatment of cultures with sodium arsenite, an inducer of heat shock and oxidative stress, or gramicidin D, a toxin that selectively permeabilizes cell membranes, prior to infection. These regimens stimulated gene expression from IE-deficient HSV-1 mutants in a promoter sequence-independent manner and also overcame the replication defect of ICP0-null mutants. Reactivation of gene expression from quiescent HSV-1 genomes and the resumption of virus replication were observed following addition of arsenite or gramicidin D to cultures. Both agents induced reorganization of nuclear domain 10 structures, the sites of quiescent genomes, but appeared to do so through different mechanisms. The results demonstrate that the physiological state of the cell is important in determining the outcome of infection with IE-deficient HSV-1 and show novel methods for reactivating quiescent HSV-1 in fibroblasts with a high efficiency.Transcription of the herpes simplex virus type 1 (HSV-1) genome at early stages of infection is controlled by the activities of three viral proteins, VP16, ICP4, and ICP0. VP16 is a virion component that stimulates immediate-early (IE) transcription through interaction with the cellular proteins Oct-1 and HCF-1 (51, 77). ICP4 is an IE protein that is essential for early and late gene expression, acting on basal cellular transcription factors (10,64,80). The IE protein ICP0 exerts a general stimulatory effect on HSV-1 transcription and is important for initiation of the viral gene expression program (17,19,31,61,68).The VP16 and ICP4 proteins can be viewed as conventional transcription activators since they interact with cellular factors that localize to promoter regions of the viral genome. In contrast, the effects of ICP0 are not promoter specific or even restricted to the HSV-1 genome, since the protein stimulates gene expression from a variety of plasmids and viruses (3,18,30,31,48,52). One of the most striking features of ICP0 is its effect on cellular intranuclear structures known as promyelocytic leukemia (PML) bodies or nuclear domain 10 (ND10). Early in infection, ICP0 localizes to ND10 and directs the disruption of these bodies (21,22). Protein components of ND10, including the PML protein itself, are targeted for degradation due to a ubiquitin E3 ligase activity that requires the RING domain of ICP0 (6,21,25). HSV-1 mutants that lack the ICP0 coding region exhibit a multiplicity-and cell type-dependent def...

Stress‐activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory

“…The viability of the heat-treated (or sodium arsenitetreated) DC cells were tested by staining with 7-AAD (BD Biosciences, BD Europe) and no significant difference was found between treated and untreated cells. Oxidative stress was induced by treatment of immature DC with sodium arsenite [39]. Increasing concentrations of 25, 50, 75 and 100 mM sodium arsenite (Sigma, Poole, UK) were used in tissue culture medium for 90 min at 371C.…”

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

“…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.…”

A Comparative Study of Stress-mediated Immunological Functions with the Adjuvanticity of Alum

“…We selected an oxidative agent (sodium arsenite), K releasing (Gramicidin), and a heavy metal ionophore (dithiocarbamate) for their potential adjuvanticity in comparison with alum. These differ in their properties, they act as stress agents that reactivate quiescent HSV-1 viral genomes (22) and they have been used in clinical practice. Gramicidine is an antibiotic, which functions as an ionophore, penetrating cell membranes, causing K ϩ efflux (23) and is effective against Gram-positive bacteria, fungi, protozoa, and viruses (24).…”