Hexamethylene bisacetamide leads to reduced helper virus‐free HSV‐1 amplicon expression titers via suppression of ICP0

Abstract

The Herpes simplex virus (HSV)-derived amplicon vector has evolved into a promising gene transfer platform for widespread DNA delivery in gene replacement strategies and vaccine development given its ease of molecular manipulation, large transgene capacity, and transduction efficiencies of numerous cell types in vivo. The recent development of helper virus-free packaging methodologies bodes well for this vector system in its eventual implementation as a clinically viable therapeutic modality. For realization of clinical application, efforts have been made to enhance yields and quality of helper-free amplicon stocks. Hexamethylene bisacetamide (HMBA), a hybrid polar compound that exhibits stimulatory activity of HSV-1 immediate-early gene expression, has been employed as a standard reagent in helper virus-free packaging given its purported mode of action on virus gene expression kinetics. Unexpectedly, we have found that HMBA exhibits no titer-enhancing activity; in contrast, the compound enhances the proportion of amplicon virions that are non-expressive. Omission of HMBA during vector packaging led to a marked reduction in the ratios of vector genometransducing to transgene-expressing virions. This effect was neither packaging cell-specific nor amplicon promoter-dependent. Analysis of resultant vector stocks indicated amplicon genome replication/concatenation was unaffected, but the level of particle-associated ICP0 was reduced in stocks packaged in the presence of HMBA. Inclusion of a co-transfected, ICP0-expressing plasmid into the packaging process led to significant rescue of amplicon expression titers, indicating that regulation of ICP0 concentrations is critical for maintenance of the amplicon genome expressive state.

Ubiquitin-specific protease 9X in host cells interacts with herpes simplex virus 1 ICP0

“…Notably, ICP0 expression was downregulated by USP9X, in contrast to other USP9X-interacting proteins. Accordingly, we found that USP9X depletion increased viral replication, in agreement with a previous report [ 4 ] demonstrating that ICP0 enhances HSV-1 replication. These observations suggested that USP9X negatively regulates ICP0 expression to inhibit viral replication, although we cannot exclude the possibility that USP9X may suppress HSV-1 replication through other pathways.…”

Progress and prospects: Biological properties and technological advances of herpes simplex virus type 1-based amplicon vectors

“…4 As, as already quoted, one important role of ICP0 is to induce degradation of several ND10 constitutive proteins that are thought to contribute to silencing of gene expression, including PML and Sp100, these results lend further support to the hypothesis that an inherent antiviral mechanism is partially responsible for the silencing of ampliconmediated transgene expression in human fibroblasts. Lastly, the observation that the reduction of particleassociated ICP0 levels, that results from packaging the amplicon genome in the presence of the transcriptional regulator hexamethylene bisacetylamide, also results in reduced transgene expression, 7 provides further evidence for a role of ICP0 in suppressing silencing mechanisms that repress amplicon-mediated transgene expression.…”

HSV-1-derived amplicon vectors: recent technological improvements and remaining difficulties - a review

“…Furthermore, siRNA downregulation of Sp100 and Daxx, two regulatory proteins that localize to PML bodies, resulted in at least 5-fold enhancement in the number of GFP-expressing normal human fibroblasts infected with standard amplicons (Tsitoura & Epstein, unpublished observations). Lastly, the observation that the reduction of particle-associated ICP0 levels, which results from packaging the amplicon genome in the presence of the transcriptional regulator hexamethylene bisacetylamide, also results in reduced transgene expression (Burris et al 2008), provides further evidence for a role of ICP0 in suppressing silencing mechanisms that repress GFP expression.…”