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.