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

Abstract

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.

Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

“…Due to the large number of spliceosomal proteins detected in our interactome, which have been shown previously can inhibit AAV transduction 43 , we examined whether a functional relationship exists between spliceosomal genes and HDAC4. We stably knocked down HDAC4 in HEK293T cells by transducing the cells with a lentivirus expressing either a miR-RNAi control or miR-RNAi-HDAC4 sequence, and consistently observed a knockdown of at least 80% (Fig.…”

“…There have previously been two distinct approaches taken to classify the molecules mediating AAV transduction: yeast two-hybrid screens using portions of the capsid as bait 48 and siRNA library screens that assess transduction after disrupting gene expression 43 , 49 , 50 . The interactomes we identified in HEK cells (Supplemental Table 1 ) and whole mouse brain lysates (Supplemental Table 2 ) have minimal overlap with the genes recovered from the yeast two-hybrid screen using mouse liver cDNA as a library, though the authors identified nucleotide binding and intracellular trafficking as key functional categories of AAV interactors, which pathway analysis of our data also identified (Fig.…”

Spliceosomal component Sf3b1 is essential for hematopoietic differentiation in zebrafish

“…These studies lead to two non-mutually exclusive hypotheses for the differences observed: 1) different splicing events are affected in each spliceosomal mutants and 2) the diverse phenotypes could be due to non-spliceosomal functions of each splicing factor. Indeed, recent studies have found that SF3B1 has some non-splicing functions in responding to various cellular stressors 58,59 .…”

The SF3b complex: splicing and beyond

“…Recently, Schreiber et al identified an SF3b component, SF3b7, as a restriction factor for recombinant AAV [93] (Fig. 3).…”