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

Claire A Schreiber,Toshie Sakuma,Yoshihiro Izumiya,Aravind Asokan,Upamanyu Basu,Raymond D Hickey,Kazunori Koide,Robert K Bressin,Yasuhiro Ikeda,Sara J Holditch

doi:10.1371/journal.ppat.1005082

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.

Highlights

Viral pathogens are known to reorganize different components of the host cell machinery during the course of infection
We found PHF5A, a component of the U2 snRNP mRNA splicing factor, blocks expression from recombinant associated viruses (AAV) vectors
Genetic and pharmacological inhibition of other U2 snRNP proteins, but not spliceosome proteins involved in other splicing steps, strongly increased transgene expression from AAV vectors

Summary

Introduction

Viral pathogens are known to reorganize different components of the host cell machinery during the course of infection. Adenoviruses have been shown to induce nuclear reorganization of host splicing factors and mislocalization of the DNA damage response machinery [1]. Modest increases in accumulation of viral DNA following proteasomal inhibition cannot solely account for substantial increases in AAV transduction [12], and the underlying mechanism remains elusive. Other host factors such as the FKBP52 [13], Mre11/Rad50/Nbs complex [14,15], APOBEC3A [16] and more recently, TRIM19/promyelocytic leukemia protein (PML) [17] have been shown to inhibit AAV replication by blocking second-strand synthesis

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Conclusion