ICP0 inhibits the decrease of HSV amplicon-mediated transgene expression

Abstract

The herpes simplex virus (HSV) amplicon vector produces an initial host response that limits transgene expression. In this study, we hypothesized that restoration of the HSV gene infected cell protein (ICP0) into the amplicon could circumvent this host response and thus overcome silencing of encoded transgenes. To test this, we constructed an amplicon vector that encodes the ICP0 under control of its native promoter (ICP0+ amplicon). Expression of ICP0 was transient and, at a multiplicity of infection (MOI) of 1, did not significantly alter interferon (IFN)-based responses against the vector or cell kinetics/apoptosis of infected cells. Chromatin immunoprecipitation (ChIP) PCR analysis revealed that conventional amplicon DNA became associated with histone deacetylase 1 (HDAC1) immediately after infection, whereas ICP0+ amplicon DNA remained relatively unbound by HDAC1 for at least 72 hours after infection. Mice administered systemic ICP0+ amplicon exhibited significantly greater and more sustained transgene expression in their livers than did those receiving conventional amplicon, likely due to increased transcriptional or post-transcriptional activity rather than increased copy numbers of vector DNA. These findings indicate that restoration of ICP0 expression may be employed within HSV amplicon constructs to decrease transgene silencing in vitro and in vivo.

Figure 1

Expression of infected cell protein 0 (ICP0) in infected mouse embryonic fibroblast cells (MEFs). (a) Schematic representation of the ICP0+ amplicon vector plasmid pHGCakg-Luc-ICP0. The plasmid contains two transgene cassettes, the enhanced green fluorescent protein (EGFP) gene driven by the herpes simplex virus (HSV) IE4/5 promoter and firefly luciferase (Luc) gene under the control of the CMV-IE/chicken β-actin/rabbit β-globin (CAG) chimeric promoter. (b) Map of pHGCag-Luc amplicon vector plasmid used for control in this study. (c) Time course induction of ICP0 mRNA in MEFs infected with ICP0+ amplicon vector was determined by real-time reverse transcriptional PCR analysis. Each value was calculated relative to that of infected MEFs after 3 hours. Data are presented as mean ± SD (n = 3). (d) Western blot analysis of ICP0. MEFs infected with ICP0+ amplicon or conventional amplicon vector were harvested at 12 and 24 hours after infection and subjected to Western blotting. M, protein size marker; Control, untreated control MEFs sample. Amp^R, ampicillin resistance gene; HSV ori_S, HSV1 origin of replication; HSV pac, HSV1 packaging signal; Kan^R, kanamycin resistance gene; pBR322 ori, Plasmid pBR322 origin of replication.

Figure 2

Time course of transgene expression in mouse embryonic fibroblast cells (MEFs). Primary cultured MEFs were infected with ICP0+ amplicon or conventional amplicon vector (multiplicity of infection = 1). Cells were harvested at various time points and subjected to (a) Luc activity assay and real-time PCR analysis of (b) Luc-encoding vector DNA as well as (c) messenger RNA transcripts. The data for cells infected with the conventional and ICP0+ amplicons are plotted as circles and diamonds, respectively (RLU, relative light units). Data are presented as mean ± SD (n = 4). ICP0, infected cell protein 0.

Figure 3

Interferon (IFN) responses to infection of ICP0+ amplicon or conventional amplicon vector in mouse embryonic fibroblast cells (MEFs). (a) Induction of IFN-β, IFN-inducible double stranded RNA protein kinase (PKR), and 2′,5′-oligoadenylate synthetase (OAS) mRNA 6 hours postinfection with ICP0+ amplicon or conventional amplicon vector (MOI = 1 or 5) were determined in MEFs by real-time RT-PCR analysis. Each value was calculated relative to that of untreated MEFs. Data are presented as mean ± SD (n = 3). Control, untreated control MEFs sample. ^*P < 0.005 comparing control to conventional amplicon and conventional amplicon to ICP0+ amplicon. (b) Western blot analysis of ICP0. MEFs infected with MOI = 1 or 5 of ICP0+ amplicon or conventional amplicon vector were harvested at 6 hours after infection and subjected to Western blotting. M, protein size marker; Control, untreated control MEFs sample. ICP0, infected cell protein 0; MOI, multiplicity of infection.

Figure 4

Histone deacetylase (HDAC) activity and chromatin modification of transgenes after amplicon infection. (a) HDAC activity of mouse embryonic fibroblast cells (MEFs) infected with conventional amplicon or ICP0+ amplicon vector (MOI = 1 or 5) were measured, 24 hours after infection. Data are presented as mean ± SD (n = 3). Control, untreated control MEFs sample. The experiments were repeated twice with similar results. (b) Ratios of vector DNA to that with input DNA associated with HDAC1 were calculated 24 hours postinfection of MEFs with conventional amplicon or ICP0+ amplicon vectors. Data are presented as mean ± SD (n = 4). ^*P < 0.02 compared with the conventional amplicon vector samples. (c) Ratios of vector DNA associated with Met-K9-H3 to that associated with Ac-K9-H3 were calculated 24 hours postinfection. Data are presented as mean ± SD (n = 3). ^**P < 0.01 and ^*P < 0.05 compared with conventional amplicon-infected cells. GFP, green fluorescent protein; ICP0, infected cell protein 0; MOI, multiplicity of infection; TSA, trichstatin A.

Figure 5

In vivo time course of transgene expression in C57Bl/6 mice. (a) Wild-type C57Bl/6 mice were systemically injected via teil vein with 1 × 10⁷ TU of ICP0+ amplicon or conventional amplicon vector and their Luc expression was monitored using a NightOWL LB981 bioluminescence imaging system. Total Luc activity in each animal was evaluated by calculating the integration of photon counts obtained from the liver over the course of 5 minutes. Total photon counts were calculated from the acquired images using the CMIR-Image program. The data for animals injected with the conventional and ICP0+ amplicons are plotted as circles and diamonds, respectively (RLU: relative light units). Data are presented as mean ± SD (n = 10). The animals were sacrificed at various time points, and the copy numbers of (b) Luc-encoding vector DNA and (c) mRNA transcripts present in their livers were determined by real-time PCR analyses. (d) The ratio of Luc mRNA copies to vector DNA copies was calculated at each time points and plotted. The data for animals injected with the conventional and ICP0+ amplicons are plotted as circles and diamonds, respectively. Data are presented as mean ± SD (n = 6). ICP0, infected cell protein 0. CMIR, Center for Molecular Imaging Research.

Figure 6

GFP transgene expression under the control of IE4/5 promoter in C57Bl/6 mice. (a) Wild-type C57Bl/6 mice were systemically injected with 1 × 10⁷ TU of ICP0+ amplicon or conventional amplicon vector. Twenty-four hours later, animals were sacrificed and their livers were harvested to evaluate GFP expression. Bar = 100 µm. Control, untreated control liver sample. (b) The animals were sacrificed at various time points and the copy number of GFP mRNA present in their liver was determined by real-time PCR analyses. The data for animals injected with the conventional and ICP0+ amplicons are plotted as circles and diamonds, respectively. Data are presented as mean ± SD (n = 6). GFP, green fluorescent protein; ICP0, infected cell protein 0.