An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

PLoS Pathogens

Upamanyu Basu¹  Kazunori Koide¹  Robert K. Bressin¹  Yoshihiro Izumiya²  Toshie Sakuma³  Sara J. Holditch³  Claire A. Schreiber³  Raymond D. Hickey³  Yasuhiro Ikeda³  Aravind Asokan⁴ 

[1] Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America;Department of Dermatology, UC Davis School of Medicine, Sacramento, California, United States of America;Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America;Gene Therapy Center and Department of Genetics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America

关键词: HeLa cells;    Small interfering RNAs;    Capsids;    Vector-borne diseases;    Luciferase;    Infectious disease control;    Library screening;    Adenoviruses;   

DOI  :  10.1371/journal.ppat.1005082

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.

【 授权许可】

CC BY