Virion-associated cofactor high-mobility group DNA-binding protein-1 facilitates transposition from the herpes simplex virus/Sleeping Beauty amplicon vector platform

Abstract

The development of the integration-competent, herpes simplex virus/Sleeping Beauty (HSV/SB) amplicon vector platform has created a means to efficiently and stably deliver therapeutic transcription units (termed "transgenons") to neurons within the mammalian brain. Furthermore, an investigation into the transposition capacity of the HSV/SB vector system revealed that the amplicon genome provides an optimal substrate for the transposition of transgenons at least 12 kb in length [de Silva, S., Mastrangelo, M.A., Lotta, L.T., Jr., Burris, C.A., Federoff, H.J., and Bowers, W.J. ( 2010 ). Gene Ther. 17, 424-431]. These results prompted an investigation into the factors that may contribute toward efficient transposition from the HSV/SB amplicon. One of the cellular cofactors known to play a key role during SB-mediated transposition is the high-mobility group DNA-binding protein-1 (HMGB1). Our present investigation into the role of HMGB1 during amplicon-based transposition revealed that transposition is not strictly dependent on the presence of cellular HMGB1, contrary to what had been previously demonstrated with plasmid-based SB transposition. We have shown for the first time that during amplicon preparation, biologically active HMGB1 derived from the packaging cell line is copackaged into amplicon vector particles. As a result, HSV/SB amplicon virions arrive prearmed with HMGB1 protein at levels sufficient for facilitating SB-mediated transposition in the transduced mammalian cell.

FIG. 1.

HMGB1 facilitates Sleeping Beauty (SB)-mediated transposition from the context of an HSV-1 amplicon plasmid. (A) Schematic representation of the colony formation assay. All assays were performed at a minimum of n = 3 per condition. (B) Western blot analysis was conducted with an anti-HMGB1 rabbit polyclonal antibody to confirm the lack of HMGB1 protein expression in the HMGB1-deficient mouse embryonic fibroblast (MEF) cell line. Wild-type MEF cells served as a positive control. (C) HMGB1-deficient MEF cells were cotransfected with pHSVT-Zeo in combination with pHSV-SB and pHSV-HMGB1 at an equivalent mass ratio. pHSVPrPUC served as a negative control and was employed to ensure that equal amounts of DNA were transfected under each condition. After a 14-day antibiotic selection period, the numbers of Zeocin-resistant colonies were enumerated via methylene blue staining. Error bars represent the standard error of the mean and statistical analysis was conducted by Student t test. Values of p are indicated.

FIG. 2.

Effect of HMGB1 overexpression on SB-mediated transposition from the HSV amplicon. (A) Detection of HMGB1 protein via Western blot analysis of cell lysates prepared from HMGB1-deficient MEF cells either nontransduced or transduced with HSV-HMGB1 amplicon at an MOI of 0.5 (top). β-Actin served as a loading control (bottom). (B) HMGB1-deficient MEF cells were cotransduced at an MOI of 1 with equal numbers of HSV-SB10 and HSVT-Zeo viral particles in combination with increasing numbers of HSV-HMGB1 viral particles [MOIs: 0.25 (+), 0.5 (++), and 1.0 (+++)]. The HSVPrPUC amplicon served as a negative control and was used as a “balance” virus to ensure that equal numbers of viral particles were transduced. Two days posttransduction, cells were placed under Zeocin selection for 14 days (n = 4 per condition), at which point Zeocin-resistant colonies were enumerated to determine the extent of transposition. Error bars represent the standard error of the mean and statistical analysis was conducted by Student t test. Values of p are indicated.

FIG. 3.

Packaging cell line-derived HMGB1 protein is associated with purified HSV amplicon viral particles. A 10-μl aliquot (equivalent to ∼5 × 10⁶ transduction units) of each HSV-1 amplicon vector packaged from the BHK cell line was lysed in RIPA buffer and subjected to Western blot analysis to determine the presence of HMGB1 protein derived from the BHK cell line (top). On HMGB1 immunoblotting, the blots were stripped and subsequently analyzed for the presence of the HSV tegument protein VP16 (bottom). Molecular masses are represented as kilodaltons (kDa).

FIG. 4.

HMGB1 protein is incorporated into the viral tegument during helper virus-free amplicon packaging and facilitates SB-mediated transposition. (A) HSVlac amplicon vector stocks separately packaged in MEF-derived HMGB1 wild-type and HMGB1-deficient cell lines (designated HSVlac^WT_MEF and HSVlac^{HMGB1–/–_MEF}, respectively) were treated with 1% NP-40 and subjected to rate zonal centrifugation in a linear sucrose gradient. Gradient fractions were analyzed by Western blotting to determine the fractions containing HMGB1 (panels 1 and 3). Subsequently, the samples were analyzed for the presence of the HSV tegument protein VP16 (panels 2 and 4). (B) HMGB1-deficient MEF cells were separately transduced with HSVlac^WT_MEF and HSVlac^{HMGB1–/–_MEF} at an MOI of 1. Four hours posttransduction, the cells were cotransfected with pHSVT-Zeo and pHSV-SB amplicon plasmids at a 1:1 mass ratio. On incubation for 48 hr, the cells were trypsinized and seeded at a 1:13 dilution on 100-mm dishes containing medium supplemented with Zeocin (100 μg/ml) (n = 3 per condition) and incubated for 14 days. Zeocin-resistant colonies were enumerated via methylene blue staining. Error bars represent the standard error of the mean and statistical analysis was conducted by Student t test. *denotes p < 0.05.