Human immunodeficiency virus type 2 lentiviral vectors: packaging signal and splice donor in expression and encapsidation
D’Costa, Jenice and Brown, Heidi M. and Kundra, Priya and Davis-Warren, Alberta and Arya, Suresh K.,, 82, 425-434 (2001),
doi = https://doi.org/10.1099/0022-1317-82-2-425,
publicationName = Microbiology Society,
issn = 0022-1317,
abstract= Retroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)–Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 105 transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1–5×107  TU/ml.,
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