Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins

Abstract

The replication (rep) gene of adeno-associated virus (AAV) is involved in AAV DNA replication, gene regulation, and inhibition of cellular transformation induced by various oncogenes. To study the rep gene's antiproliferative eflects, we have developed cell lines which express the replication proteins under the control of an inducible mouse metallothionein transcription promoter. The Rep78 protein produced in these cell lines binds to the AAV terminal repeat sequences in vitro and supports AAV DNA replication and trans activation of the AAV p40 transcription promoter in vivo. These cell lines are capable of assembling infectious viruses containing a mutant rep gene or a vector bearing a heterologous gene. Growth rate and colony formation efficiency assays indicated that rep gene expression substantially altered cellular proliferation. Long-term induction of the cell lines followed by removal of the inducing agent suggested that constitutive expression of the Rep proteins does not necessarily result in cell death and that the cells can recover from the cytostatic effects. Flow cytometry analysis indicated that the presence of the Rep proteins increased the population of cells in the S phase of the cell cycle. Thus the rep gene's antiproliferative effects may be realized by interference with cellular DNA replication.

High-level expression of adeno-associated virus (AAV) Rep78 or Rep68 protein is sufficient for infectious-particle formation by a rep-negative AAV mutant

“…The use of expression constructs for the separate expression of Rep proteins made it feasible to clearly distinguish between Rep78 and Rep68, which had not been possible by conventional genetic analyses of AAV because of the strong overlapping of the Rep protein coding sequences. The availability of stably transfected cell lines inducibly expressing rep (11,34) allowed us to confirm A and B) The cells were infected with Ad2, and subsequently Rep78 expression was induced with 10 Ϫ7 M dexamethasone (Dex) as indicated. Low-molecular-weight DNA was extracted by a modified Hirt extraction protocol at 54 h p.i.…”

Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery

“…Based on the high expression of GFP protein, the addition of sodium butyrate or butyric acid would also have produced Rep proteins in excess. The Rep proteins have a toxic effect on HEK293 cells (Chang and Shenk, 1990;Chang et al, 1989;Clark et al, 1995;Li et al, 1997;Yang et al, 1994;Zhou and Trempe, 1999); therefore, the low AAV yield in the presence of sodium butyrate or butyric acid could be due to accumulation of other toxic proteins in the cell culture environment. Lentivirus production by HEK293 cells has been increased by the use of sodium butyrate (Ansorge et al, 2009;Karolewski et al, 2003) and caffeine (Ellis et al, 2011).…”

The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection

“…The control of AAV transcription must be sufficiently flexible to accommodate both the latent and productive phases of viral infection. In the absence of Ad, expression of the rep gene has been shown to be cytostatic (49), and thus survival of the virus probably requires a mechanism for reducing transcription from the two rep promoters, p5 and p19, as well as the capsid promoter, p40. Indeed, with the exception of 293 cells, which constitutively express the Ad E1 genes, expression of the AAV genes is usually undetectable during latent infection or in the absence of Ad coinfection (27,28) in human cells.…”