Co-localization of gammaretroviral RNAs at their transcription site favours co-packaging

Abstract

A retroviral vector-rescue system in which co-packaging of the two co-expressed vectors is required for transduction of one of the vectors has been established previously. By using this rescue system, two distinct packaging-cell populations have been generated. One cell population expressed retroviral RNA from co-localized transcription sites, resulting in local and overlapping accumulation of both RNA transcripts. In the other cell population, the two transcription cassettes were introduced separately, leading to distinct transcription sites of the two RNAs and no significant co-localization of the RNAs. Titre measurements from the two distinct cell populations showed large differences in rescue titre, which is an indirect measure of co-packaging efficiency. Thus, the cell populations with overlapping RNA accumulation gave rise to 15-80-fold-higher rescue titres than cell populations with non-overlapping RNA accumulation. These data show that the spatial position of proviral transcription sites affects the level of retroviral RNA co-packaging and suggest that there is already a linkage of RNAs for co-packaging at the transcription site. It is hypothesized that this linkage is due to RNA dimerization taking place at the transcription site.