PubMed · 9760518
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G D Foster. 1998. Expression library screening.. https://doi.org/10.1385/0-89603-385-6%3A287
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A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1.
Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.
A beet yellows closterovirus (BYV) variant expressing green fluorescent protein and leaves of BYV local lesion host Claytonia perfoliata were used to reveal genetic requirements for BYV cell-to-cell movement in leaf epidermis and mesophyll. A series of mutations targeting genes that are not involved in amplification of the viral positive-strand RNA was analyzed. The products of genes coding for a 6-kDa hydrophobic protein (p6) and a 64-kDa protein (p64), as well as for minor and major capsid proteins, were found to be essential for intercellular translocation of BYV. In a previous work, we have demonstrated that the BYV HSP70-homolog (HSP70h) also plays a critical role in viral movement (V. V. Peremyslov, Y. Hagiwara, and V. V. Dolja, 1999, Proc. Natl. Acad. Sci. USA, 96, 14771-14776). Altogether, a unique protein quintet including three dedicated movement proteins (p6, p64, and HSP70h) and two structural proteins is required to potentiate the cell-to-cell movement of a closterovirus. The corresponding BYV genes are clustered in a block that is conserved among diverse representatives of the family Closteroviridae.